Rotenon’un İnsan Lenfositlerinde İn Vitro Genotoksisitesi

Giriş: Rotenon, bir taşıyıcıdan bağımsız olarak hücresel membranları kolayca geçen, lipofilik, geniş spektrumlu insektisit ve pisisit sınıfı bir pestisittir. Bu çalışmada Rotenon’un insan periferik kan lenfositlerinde DNA üzerine olan etkisi comet metodu ile değerlendirilmiştir.Materyal-Metot: Çalışmada 4 erkek 4 kadın toplam 8 gönüllüden alınan periferik kan lenfositleri Rotenon ile 10, 50 veya 100 µM olmak üzere üç farklı dozda ve her bir doz için 1, 2 veya 4 saat olmak üzere üç farklı sürede muamele edilmiştir. Comet metodu uygulanmış ve kuyruk DNA yüzdesi parametresi DNA hasarının göstergesi olarak negatif ve pozitif kontrol grupları ile istatiksel olarak karşılaştırılmıştır.Bulgular: Rotenon uygulamaları inkübasyon saatine ve doza bağlı olarak farklı sonuçlar ortaya koymuştur. 10 veya 50 µM Rotenon ile 1 s ve 2 s inkübasyon uygulanan gruplar negatif kontrol gruplarına kıyasla DNA hasarında artışa sebep olmuş ancak bu artış istatistiksel olarak anlamlı bulunmamıştır (p>0,05). 100 µM doz ile 1 ve 2 s inkübasyon uygulanan gruplar, kontrol gruplarına kıyasla DNA hasarında anlamlı artışa sebep olmuştur (p<0,05). 10, 50 veya 100 µM Rotenon ile 4 s inkübasyon uygulanan gruplarda negatif kontrol grubuna kıyasla DNA hasarında anlamlı seviyede artış tespit edilmiştir (p<0,05).Sonuç: Rotenon maruziyeti kısa süreli ve düşük dozlarda olduğunda DNA hasarında artış olmakla birlikte bu artış anlamlı değildir. Doz yükseldikçe, kısa maruziyet sürelerinde de anlamlı seviyede DNA hasarı oluşmaktadır. Uzun süreli Rotenon maruziyetinde ise doz bağımsız şekilde anlamlı seviyede DNA hasarı görülmektedir.

Anahtar Kelimeler:

Comet metodu, DNA hasarı, Pestisit, Rotenon

In Vitro Genotoxicity of Rotenone in Human Lymphocytes

Objective: Rotenone is a lipophilic, broad-spectrum, insecticide and piscicide class pesticide that readily crosses cellular membranes. In this study, the effect of Rotenone on DNA of human peripheral blood lymphocytes was evaluated by the comet assay.Material-Method: In the study, peripheral blood lymphocytes taken from 8 volunteers, 4 male and 4 female, were treated with Rotenone at three different doses as 10, 50 or 100 µM, and for three different time periods of 1, 2 or 4 hours for each dose. Comet assay was applied and tail DNA percentage parameter was chosen as measure of DNA damage and statistically compared with negative and positive control groups.Results: Rotenone applications showed different results depending on incubation time and dose. Groups incubated with 10 or 50 µM Rotenone for 1 and 2 h caused an increase in DNA damage compared to the negative controls, but this increase was not statistically significant (p>0,05). There was a significant increase in DNA damage in the groups that were incubated with 10, 50 or 100 µM Rotenone for 4 h compared to the negative control group.Conclusion: When Rotenone exposure is short-term and at low doses, there is an increase in DNA damage, but this increase is not statistically significant. As the dose increases, statistically significant DNA damage also occurs in short-term exposures. Long-term exposure to Rotenone, on the other hand, shows significant DNA damage regardless of dose.

Keywords:

Comet assay, DNA damage, Pesticide, Rotenone,

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[1] Koureas M, Tsezou A, Tsakalof A, Orfanidou T, Hadjichristodoulou C. Increased levels of oxidative DNA damage in pesticide sprayers in Thessaly Region (Greece). Implications of pesticide exposure. Science of the Total Environment. 2014;496:358-64.
[2] Mokarizadeh A, Faryabi MR, Rezvanfar MA, Abdollahi M. A comprehensive review of pesticides and the immune dysregulation: mechanisms, evidence and consequences. Toxicology mechanisms and methods. 2015;25(4):258-78.
[3] Lee G-H, Choi K-C. Adverse effects of pesticides on the functions of immune system. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 2020;235:108789.
[4] Isman MB. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annual Review of Entomology. 2006;51:45-66.
[5] Ramalingam M, Huh Y-J, Lee Y-I. The impairments of α-synuclein and mechanistic target of rapamycin in rotenone-induced SH-SY5Y cells and mice model of Parkinson’s disease. Frontiers in neuroscience. 2019;13:1028.
[6] Fitzmaurice AG, Bronstein JM. Pesticides and Parkinson’s disease (Chapter) in: Pesticides in The Modern World–Effects of Pesticides Exposure. 2011:307. IntechOpen; London
[7] Duty S, Jenner P. Animal models of Parkinson's disease: a source of novel treatments and clues to the cause of the disease. British journal of pharmacology. 2011;164(4):1357-91.
[8] Kitamura Y, Inden M, Miyamura A, Kakimura J-i, Taniguchi T, Shimohama S. Possible involvement of both mitochondria-and endoplasmic reticulum-dependent caspase pathways in rotenone-induced apoptosis in human neuroblastoma SH-SY5Y cells. Neuroscience letters. 2002;333(1):25-8.
[9] Janelle D-O, Francesca C. Pesticides and Parkinson’s disease (Chapter) in: Pesticides - The Impacts Of Pesticide Exposure. 2011:103. IntechOpen; London
[10] de Lima PDL, Yamada ES, da Costa ET, Pessoa CdO, Rabenhorst S, Bahia MdO, et al. Genotoxic effects of rotenone on cultured lymphocytes. Genetics and Molecular Research. 2005;4(4):822-31.
[11] Yarmohammadi F, Wallace Hayes A, Najafi N, Karimi G. The protective effect of natural compounds against rotenone-induced neurotoxicity. Journal of Biochemical and Molecular Toxicology. 2020;34(12):e22605.
[12] Franco R, Li S, Rodriguez-Rocha H, Burns M, Panayiotidis MI. Molecular mechanisms of pesticide-induced neurotoxicity: Relevance to Parkinson's disease. Chemico-biological interactions. 2010;188(2):289-300.
[13] Toğay VA, Baş FY, Çelik DA, Özçelik N, Türel GY, Calapoğlu M, et al. Increased DNA Damage of Radiology Personnel Chronically Exposed to Low Levels of Ionizing Radiations. Süleyman Demirel Üniversitesi Sağlık Bilimleri Dergisi. 2020;11(2):212-6.
[14] Bolognesi C, Holland N. The use of the lymphocyte cytokinesis-block micronucleus assay for monitoring pesticide-exposed populations. Mutation Research/Reviews in Mutation Research. 2016;770:183-203.
[15] Barrón Cuenca J, Tirado N, Barral J, Ali I, Levi M, Stenius U, et al. Increased levels of genotoxic damage in a Bolivian agricultural population exposed to mixtures of pesticides. Science of The Total Environment. 2019;695:133942.
[16] Garaj-Vrhovac V, Zeljezic D. Evaluation of DNA damage in workers occupationally exposed to pesticides using single-cell gel electrophoresis (SCGE) assay: Pesticide genotoxicity revealed by comet assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2000;469(2):279-85.
[17] Ündeğer Ü, Başaran N. Assessment of DNA damage in workers occupationally exposed to pesticide mixtures by the alkaline comet assay. Archives of Toxicology. 2002;76(7):430-6.
[18] Celik DA, Togay VA, Turel GY, Tuluceoglu EE, Kosar PA. DNA Damages of Widely Used Pesticides; A Comet Assay Report for Chlorothalonil and Glyphosate Potassium Salt. Fresenius Environmental Bulletin. 2021;30(4 A):4170-6.
[19] Yavuz Türel G, Toğay VA, Aşcı Çelik D. Genotoxicity of thiacloprid in zebrafish liver. Archives of Environmental & Occupational Health. 2022:1-6. DOI: 10.1080/19338244.2022.2118212
[20] Aşci Çelik D, Toğay VA, Karabacak P. DNA damage assessment in pneumonia patients treated in the intensive care unit. Biologia. 2022:(77): 1909 - 1913.
[21] Karabacak P, Toğay VA, Çelik DA. Lymphocyte DNA damage in sepsis and septic-shock intensive-care patients: Damage is greater in non-intubated patients. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2022;879:503516.
[22] OECD. Test No. 489: In Vivo Mammalian Alkaline Comet Assay. OECD Publishing; Paris: 2016.
[23] Gyori BM, Venkatachalam G, Thiagarajan P, Hsu D, Clement M-V. OpenComet: An automated tool for comet assay image analysis. Redox biology. 2014;2:457-65.
[24] IBM. SPSS Statistics for Windows, Version 20.0. IBM Corp, Armonk, NY: 2012.
[25] Rupa D, Reddy P, Reddi O. Analysis of sister-chromatid exchanges, cell kinetics and mitotic index in lymphocytes of smoking pesticide sprayers. Mutation Research/Genetic Toxicology. 1989;223(2):253-8.
[26] Au WW, Sierra-Torres CH, Cajas-Salazar N, Shipp BK, Legator MS. Cytogenetic effects from exposure to mixed pesticides and the influence from genetic susceptibility. Environmental health perspectives. 1999;107(6):501-5.
[27] Lerda D, Bistoni MB, Peralta N, Ychari S, Vazquez M, Bosio G. Fumonisins in foods from Cordoba (Argentina), presence and genotoxicity. Food and Chemical Toxicology. 2005;43(5):691-8.
[28] Çelik DA, Toğay VA, Türel GY, Özçelik N. Gıda Katkı Maddesi Olarak Kullanılan Sitrik Asit, Askorbik Asit Ve Sodyum Sitratın İnsan Lenfosit Hücrelerinde Genotoksisitesinin Değerlendirilmesi. SDÜ Tıp Fakültesi Dergisi. 2022;29(3):486-92.
[29] Ündeğer Ü, Başaran N. Effects of pesticides on human peripheral lymphocytes in vitro: induction of DNA damage. Archives of Toxicology. 2005;79(3):169-76.
[30] Kocaman AY, Topaktaş M. The in vitro genotoxic effects of a commercial formulation of α‐cypermethrin in human peripheral blood lymphocytes. Environmental and molecular mutagenesis. 2009;50(1):27-36.
[31] Alzahrani S, Ezzat W, Elshaer R, Abd El-Lateef A, Mohammad H, Elkazaz A, et al. Standarized Tribulus terrestris extract protects against rotenone-induced oxidative damage and nigral dopamine neuronal loss in mice. J Physiol Pharmacol. 2018;69(6):979-94.
[32] El- Shamarka MEA, Hussein AMS, N. Sayed O, S Said E, Mwaheb MA. Spirulina Ameliorates Oxidative Damage and Inflammation in Rotenone-Induced Neurotoxicity in Male Mice. International Journal of Medical Toxicology and Forensic Medicine. 2022;12(1):35583.
[33] Sun Z, Xue L, Li Y, Cui G, Sun R, Hu M, et al. Rotenone-induced necrosis in insect cells via the cytoplasmic membrane damage and mitochondrial dysfunction. Pesticide Biochemistry and Physiology. 2021;173:104801.
[34] Swarnkar S, Singh S, Goswami P, Mathur R, Patro IK, Nath C. Astrocyte Activation: A Key Step in Rotenone Induced Cytotoxicity and DNA Damage. Neurochemical Research. 2012;37(10):2178-89.
[35] Kurpik M, Zalewski P, Kujawska M, Ewertowska M, Ignatowicz E, Cielecka-Piontek J, et al. Can Cranberry Juice Protect against Rotenone-Induced Toxicity in Rats? Nutrients. 2021;13(4):1050.
[36] Guadaño A, González-Coloma A, de la Peña E. Genotoxicity of the insecticide rotenone in cultured human lymphocytes. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 1998;414(1):1-7.
[37] Syromyatnikov MY, Gureev AP, Starkova NN, Savinkova OV, Starkov AA, Lopatin AV, et al. Method for detection of mtDNA damages for evaluating of pesticides toxicity for bumblebees (Bombus terrestris L.). Pesticide Biochemistry and Physiology. 2020;169:104675. DOI:10.1016/j.pestbp.2020.104675.