Toxicological Research

Korean Society of Toxicology & Korea Environmental Mutagen Society (한국독성학회)
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Exposure to a sublethal dose of technical grade flubendiamide hampers angiogenesis in the chicken chorioallantoic membrane

  • Dhanush Danes (Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda) ;
  • Pooja Raval (Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda) ;
  • Anjali Singh (Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda) ;
  • Lakshmi Pillai (Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda) ;
  • Suresh Balakrishnan (Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda)
  • Received : 2024.02.19
  • Accepted : 2024.06.26
  • Published : 2024.10.15
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Abstract

Pesticides are commonly employed to enhance agricultural productivity to meet the demands of the expanding global populace. Their harmful impact on non-target organisms is a severe cause of concern, and hence, new, presumably safer variants are developed. Flubendiamide is one such insecticide that targets caterpillars of insect pests. To evaluate its safety, we exposed early chicken embryos to technical-grade flubendiamide. Based on a dose range analysis, a lowest observed effect concentration (LOEC) of 25 ㎍/50µL (500 ppm) was selected for further experiments. LC-MS/MS analysis confirmed the presence of flubendiamide in the treated embryos. Gross morphology of embryos on days 2, 3 and 4 revealed reduced vascular area in the chorioallantoic membrane (CAM). The CAM vessel analysis showed reduced vascular networks in treated group. Hence, we hypothesized that flubendiamide, at LOEC, alters the expression patterns of the essential signaling molecules involved in angiogenesis, leading to compromised blood vessel development in CAM. An initial in silico study of flubendiamide was conducted with proteins involved in the CAM angiogenesis pathway. The docking scores revealed fubendiamide's direct influence on the functionality of angiogenic proteins. Hence, the expression patterns of key regulators of angiogenesis were studied on days 2, 3 and 4 at the transcript and protein levels. The results revealed a significant reduction in VEGFα, AKT, KDR, PCNA, PI3K, BMP2, BMP6, SHH and WNT7A expression in treated embryos, while expression of CASPASE-3 and RHOB were upregulated. Immunolocalization of Cl. Caspase-3 reaffirmed heightened apoptosis in the CAM of day 2 embryos. The study thus confirms that flubendiamide at a sublethal dose can hamper CAM angiogenesis and reduce the vascular networks in developing chick embryos by targeting the VEGF signaling cascade. This finding points to the teratogenic potential of flubendiamide and prompts throughput screening for safety.

Keywords

Acknowledgement

This work was supported by two major projects DBT-BUILDER-Cat III (Grant number: BT/INF/22/SP41403/2021) and Gujarat State Biotechnology Mission (GSBTM) Gandhinagar, India, for financial assistance (Grant number: GSBTM/JD(R&D)/618/21-22/1224, Date: 28/12/2021).

References

  1. Troczka BJ, Williamson MS, Field LM, Davies TE (2017) Rapid selection for resistance to diamide insecticides in Plutella xylostella via specific amino acid polymorphisms in the ryanodine receptor. Neurotoxicology 60:224-233. https://doi.org/10.1016/j.neuro.2016.05.012 
  2. Nauen R, Steinbach D (2016) Resistance to diamide insecticides in lepidopteran pests. Advances in insect control and resistance management. Springer, pp 219-240. https://doi.org/10.1007/978-3-319-31800-4_12 
  3. Li W, Wei-Wei S, Guo-Hua D, Xiao-Li F, Miao-Ling Y, ZhiHua L (2014) Acute and joint toxicity of three agrochemicals to Chinese tiger frog (Hoplobatrachus chinensis) tadpoles. Zool Res 35:272. https://doi.org/10.13918/j.issn.2095-8137.2014.4.272 
  4. Sun Q, Lin J, Peng Y, Gao R, Peng Y (2018) Flubendiamide enhances adipogenesis and inhibits AMPKα in 3T3-L1 adipocytes. Molecules 23:2950. https://doi.org/10.3390/molecules23112950 
  5. Sarkar S, Dutta M, Roy S (2014) Potential toxicity of flubendiamide in Drosophila melanogaster and associated structural alterations of its compound eye. Toxicol Environ Chem 96:1075-1087. https://doi.org/10.1080/02772248.2014.997986 
  6. Sarkar S, Roy A, Roy S (2018) Flubendiamide affects visual and locomotory activities of Drosophila melanogaster for three successive generations (P, F 1 and F 2). Invertebr Neurosci 18:1-11. https://doi.org/10.1007/s10158-018-0210-x 
  7. Sarkar S, Roy S (2017) Monitoring the effects of a lepidopteran insecticide, Flubendiamide, on the biology of a non-target dipteran insect, Drosophila melanogaster. Environ Monit Assess 189:1-14. https://doi.org/10.1007/s10661-017-6287-6 
  8. Liu Z, Chen D, Lyu B, Wu Z, Li J, Zhao Y, Wu Y (2022) Occurrence of phenylpyrazole and diamide insecticides in lactating women and their health risks for infants. J Agric Food Chem 70:4467-4474. https://doi.org/10.1021/acs.jafc.2c00824 
  9. Stern CD (2018) The chick model system: a distinguished past and a great future. Int J Dev Biol 62:1-4. https://doi.org/10.1387/ijdb.170270cs 
  10. Wittig JG, Munsterberg A (2016) The early stages of heart development: insights from chicken embryos. J Cardiovasc Dev Dis 3:12. https://doi.org/10.3390/jcdd3020012 
  11. Felmeden DC, Blann AD, Lip GYH (2003) Angiogenesis: basic pathophysiology and implications for disease. Eur Heart J 24:586-603. https://doi.org/10.1016/S0195-668X(02)00635-8 
  12. Ahmed TA, Cordeiro CM, Elebute O, Hincke MT (2022) Proteomic analysis of chicken chorioallantoic membrane (CAM) during embryonic development provides functional insight. BioMed Res Int 2022:7813921. https://doi.org/10.1155/2022/7813921 
  13. Schmitd LB, Liu M, Scanlon CS, Banerjee R, D'Silva NJ (2019) The chick chorioallantoic membrane in vivo model to assess perineural invasion in head and neck cancer. JoVE (J Vis Exp) 148:e59296. https://doi.org/10.3791/59296 
  14. Hiratsuka S, Kataoka Y, Nakao K, Nakamura K, Morikawa S, Tanaka S, Shibuya M (2005) Vascular endothelial growth factor A (VEGF-A) is involved in guidance of VEGF receptor-positive cells to the anterior portion of early embryos. Mol Cell Biol 25:355-363. https://doi.org/10.1128/MCB.25.1.355-363.2005 
  15. Pulkkinen HH, Kiema M, Lappalainen JP, Toropainen A, Beter M, Tirronen A, Laakkonen JP (2021) BMP6/TAZ-Hippo signaling modulates angiogenesis and endothelial cell response to VEGF. Angiogenesis 24:129-144. https://doi.org/10.1007/s10456-020-09748-4 
  16. Guo D, Murdoch CE, Liu T, Qu J, Jiao S, Wang Y, Chen X (2018) Therapeutic angiogenesis of Chinese herbal medicines in ischemic heart disease: a review. Front Pharmacol 9:428. https://doi.org/10.3389/fphar.2018.00428 
  17. Karar J, Maity A (2011) PI3K/AKT/mTOR pathway in angiogenesis. Front Mol Neurosci 4:51. https://doi.org/10.3389/fnmol.2011.00051 
  18. Blankenship AL, Hilscherova K, Nie M, Coady KK, Villalobos SA, Kannan K, Giesy JP (2003) Mechanisms of TCDD-induced abnormalities and embryo lethality in white leghorn chickens. Comp Biochem Physiol Part C Toxicol Pharmacol 136:47-62. https://doi.org/10.1016/S1532-0456(03)00166-2 
  19. Zudaire E, Gambardella L, Kurcz C, Vermeren S (2011) A computational tool for quantitative analysis of vascular networks. PLoS One 6:e27385. https://doi.org/10.1371/journal.pone.0027385 
  20. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) Autodock4 and AutoDockTools4: automated docking with selective receptor flexiblity. J Comput Chem 16:2785-2791. https://doi.org/10.1002/jcc.21256 
  21. Biovia DS (2017) Discovery studio visualizer, vol 936. San Diego, CA 
  22. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402-408. https://doi.org/10.1006/meth.2001.1262 
  23. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254. https://doi.org/10.1016/0003-2697(76)90527-3 
  24. World Health Organization (2016) Manual on development and use of FAO and WHO specifications for pesticides. Food & Agriculture Org 
  25. Kalliora C, Mamoulakis C, Vasilopoulos E, Stamatiades GA, Kalafati L, Barouni R, Tsatsakis A (2018) Association of pesticide exposure with human congenital abnormalities. Toxicol Appl Pharmacol 346:58-75. https://doi.org/10.1016/j.taap.2018.03.025 
  26. Xiao-Ming X, Wen D, Peng L, Shu-Jin W, Min H, Li-Ying L (2010) Subchronic toxicity organophosphate insecticide-induced damages on endothelial function of vessels in rabbits by inhibiting antioxidases. Progress Biochem Biophys 37:1232-1239. https://doi.org/10.1016/j.fct.2007.06.038 
  27. Priyadarshini CS, Balaji T, Kumar JA, Subramanian M, Sundaramurthi I, Meera M (2020) Chlorpyrifos and its metabolite modulates angiogenesis in the chorioallantoic membrane of chick embryo. J Basic Clin Physiol Pharmacol 31:20190041. https://doi.org/10.1515/jbcpp-2019-0041 
  28. Strzalka W, Ziemienowicz A (2011) Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation. Ann Bot 107:1127-1140. https://doi.org/10.1093/aob/mcq243 
  29. Zavala G, Prieto CP, Villanueva AA, Palma V (2017) Sonic hedgehog (SHH) signaling improves the angiogenic potential of Wharton's jelly-derived mesenchymal stem cells (WJ-MSC). Stem Cell Res Ther 8:1-17. https://doi.org/10.1186/s13287-017-0653-8 
  30. Salybekov AA, Salybekova AK, Pola R, Asahara T (2018) Sonic hedgehog signaling pathway in endothelial progenitor cell biology for vascular medicine. Int J Mol Sci 19:3040. https://doi.org/10.3390/ijms19103040 
  31. Lei X, Zhong Y, Huang L, Li S, Fu J, Zhang L, Yu X (2020) Identification of a novel tumor angiogenesis inhibitor targeting Shh/Gli1 signaling pathway in Non-small cell lung cancer. Cell Death Dis 11:232. https://doi.org/10.1038/s41419-020-2425-0 
  32. Vanhollebeke B, Stone OA, Bostaille N, Cho C, Zhou Y, Maquet E, Stainier DY (2015) Tip cell-specific requirement for an atypical Gpr124-and Reck-dependent Wnt/β-catenin pathway during brain angiogenesis. Elife 4:e06489. https://doi.org/10.7554/eLife.06489 
  33. Olsen JJ, Pohl SOG, Deshmukh A, Visweswaran M, Ward NC, Arfuso F, Dharmarajan A (2017) The role of Wnt signalling in angiogenesis. Clin Biochem Rev 38:131 
  34. Benn A, Hiepen C, Osterland M, Schutte C, Zwijsen A, Knaus P (2017) Role of bone morphogenetic proteins in sprouting angiogenesis: differential BMP receptor-dependent signaling pathways balance stalk vs tip cell competence. FASEB J 31:4720. https://doi.org/10.1096/f.201700193RR 
  35. Howe GA, Addison CL (2012) RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA. Vascular cell 4:1-11. https://doi.org/10.1186/2045-824X-4-1 
  36. Brentnall M, Rodriguez-Menocal L, De Guevara RL, Cepero E, Boise LH (2013) Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. BMC Cell Biol 14:1-9. https://doi.org/10.1186/1471-2121-14-32