Aydın RÜSTEMOĞLU, Sibel Demir ÖZTÜRK, Sadegül TUNCER, Hüsniye RÜSTEMOĞLU

The effects of endosulfan on the expression levels of DNA damage and apoptotic genes in HT22 cells: First preliminary study

Aim: The aim of this study was to determine the IC50 dose of endosulfan in the hippocampal HT22 cell line, and also to elucidate theeffect of expression of DNA-PK, Bax, Bcl2 and Casp-3 genes involved in DNA repair and apoptotic pathway.Material and Methods: Cytotoxic effect of endosulfan and IC50 dose were determined by using XTT method after 24 hours of cultureby applying endosulfan at 5 different concentrations (10, 25, 50, 75 and 100 μM) to HT22 cell lines.HT22 cells were then seeded into 6 sterile plates and treated with Endosulfan at IC50 for 12 hours. The expression changes of DNAPk, Bax, Bcl2 and Casp-3 genes, after total RNA isolation and cDNA formation, were determined by RT-PCR. Expression levels werecalculated using the comparative 2-ΔΔCt method.Results: After 24 hours of endosulfan treatment at different doses in HT22 cell lines, a signifiant loss of viability was observed in allendosulfan treated groups. It was determined by XTT test, that the IC50 dose of endosulfan was 50 μM in 24 hours treatment. After12 hours administration of IC50 endosulfan dose in HT22 cells following the examinations of DNA-PK and some apoptotic genes weobserved different amounts of increases in expression as follows; 5-fold for DNA-PK, 18-fold for Bax, and 4-fold for Casp-3. On theother hand, approximately 2-fold decrease was detected in Bcl-2 gene.Conclusion: The IC50 dose of 24-hour endosulfan administration in HT22 cell lines was found to be 50 μM. Expression changes inthe proapoptotic and antiapoptotic genes have shown that apoptosis is induced in endosulfan-administrated cells. In addition, theincrease in DNA-PK gene expression suggests that endosulfan causes DNA damage in cells and triggers DNA repair mechanisms.

PDF

___

1. Sanpera C, Ruiz X, Llorente GA, et all. Persistent organochlorine compounds in sediment and biota from the Haleji lake: a wildlife sanctuary in South Pakistan. Bull. Environ. Contam Toxicol 2002;68:237- 44.
2. Ali U, Syed JH, Malik RN, et all. Organochlorine pesticides (OCPs) in South Asian region: a review. Sci Total Environ 2014;476:705-17.
3. Usenko S, Landers DH, Appleby PG and Simonich SL. Current and historical deposition of PBDEs, pesticides, PCBs, and PAHs to Rocky Mountain National Park. Environ. SciTechnol 2007;41:7235-41.
4. Saiyed H, Dewan A, Bhatnagar V, et all. Effect of endosulfan on male reproductive development. Environ Health Perspect 2003;111:1958-62.
5. Mrema EJ, Rubino FM, Brambilla G, et all. Persistent organochlorinated pesticides and mechanisms of their toxicity. Toxicology 2013;307:74-88.
6. Kamijima M, Casida JE. Regional modifiation of [(3) H]Ethynylbicycloorthobenzoate binding in mouse brain GABA(A) receptor by endosulfan, fironil, and avermectin B(1a). Toxicol Appl Pharmacol 200;163:188-94.
7. Sebastian R, Raghavan SC. Induction of DNA damage and erroneous repair can explain genomic instability caused by endosulfan. Carcinogenesis 2016;37:929– 40.
8. van Gent DC, Hoeijmakers JH, Kanaar R. Chromosomal stability and the DNA doublestranded break connection. Nat Rev Genet 2001;2:196-206.
9. Ghosal G, Chen J. DNA damage tolerance: a doubleedged sword guarding the genome. Transl Cancer Res 2013;2:107-29.
10. Lieber MR. NHEJ and its backup pathways in chromosomal translocations. Nat Struct Mol Biol 2010;17:393-5.
11. Iyama T, Wilson DM. DNA repair mechanisms in dividing and non-dividing cells. DNA Repair 2013;12:620-36.
12. Jeong GS, Li B, Lee DS, et all. Cytoprotective and antiinflammatory effects of spinasterol via the induction of heme oxygenase-1 in murine hippocampal and microglial cell lines. Int Immunopharmacol 2010;10:1587-94.
13. Donovan L, Welford SM, Haaga J, et all. Hypoxia-- implications for pharmaceutical developments. Sleep Breath 2010;14:291-8.
14. Kaizaki A, Tanaka S, Ishige K, et all. The neuroprotective effect of heme oxygenase (HO) on oxidative stress in HO-1 siRNA-transfected HT22 cells. Brain Res 2006;1108:39-44.
15. Atlante A, Calissano P, Bobba A, et all. Glutamate neurotoxicity, oxidative stress and mitochondria. FEBS Lett 2001;497:1-5.
16. Liu J, Rasul I, Sun Y, et all. GRK5 defiiency leads to reduced hippocampal acetylcholine level via impaired presynaptic M2/M4 autoreceptor desensitization. J Biol Chem 2009;284:19564-71
17. Clement AB, Gimpl G, Behl C. Oxidative stress resistance in hippocampal cells is associated with altered membrane fluidity and enhanced nonamyloidogenic cleavage of endogenous amyloid precursor protein. Free Radic Biol Med 2010;48:1236-41.
18. Yang EJ, Lee JY, Park SH, et all. Neuroprotective effects of neolignans isolated from Magnoliae Cortex against glutamate-induced apoptotic stimuli in HT22 cells. Food Chem Toxicol 2013;56:304-12.
19. Brossaud J, Roumes H, Moisan MP, et all. Retinoids and glucocorticoids target common genes in hippocampal HT22 cells. J Neurochem 2013;125:518-31.
20. Sinha N, Adhikari N, Narayan R et al. Cytotoxic effect of endosulfan on rat Sertoli-germ cell coculture. Reprod Toxicol 1999;13:291-4.
21. Sohn HY, Kwon CS, Kwon GS, et al. Induction of oxidative stress by endosulfan and protective effect of lipidsoluble antioxidants against endosulfan-induced oxidative damage. Toxicol Lett 2004;151:357-65.
22. Xu, Y, Wang, N, Shi ZX, et all. In vitro mechanistic study of endosulfan-induced spermatogenic cell apoptosis in the mouse. Toxicol Ind Health 2016;32:1550-63.
23. Xu D, Liang D, Guo Y, et al. Endosulfan causes the alterations of DNA damage response through ATM-p53 signaling pathway in human leukemia cells. Environmental Pollution 2018;238:1048-55