Exposure to a sublethal dose of technical grade flubendiamide hampers angiogenesis in the chicken chorioallantoic membrane

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 µg/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 flubendiamide’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.