Sublethal Exposure to a Chlorpyrifos‐Cypermethrin Mixture Disrupts Limb Development and Causes Severe Skeletal Abnormalities in Domestic Chick Embryos

This study examines the teratogenic effects of a sublethal dose (0.1 ppm) of a combination insecticide containing 50% chlorpyrifos (CP) and 5% cypermethrin (CM) on chick embryo limb development, revealing severe skeletal abnormalities and disruptions in key molecular pathways. Early-stage defects, including reduced somite numbers and abnormal somite patterning, were observed by day 2, with prominent limb deformities, such as limb shortening and twisted digits, emerging by days 10 and 21. X-ray analysis confirmed skeletal anomalies, while Alcian blue-Alizarin red staining revealed malformed cartilage and bone structures. Gene and protein expression analyses showed significant downregulation of Sonic hedgehog (SHH), Fibroblast Growth Factors (FGF10, FGF8), and WNT7A, leading to impaired digit formation and limb elongation. HOX genes (HOXA11, HOXD11) were initially upregulated but later downregulated, disrupting limb segment identity. Additionally, downregulation of Tbx4 and Pitx1 contributed to hindlimb malformations. Dysregulation of CASPASE3 impaired apoptosis, resulting in defective digit separation. Molecular docking studies indicated strong binding affinities of CP and CM to SHH, FGF10, WNT7A, and HOX proteins, suggesting interference with these signaling pathways. Notably, the disruption of the SHH-FGF-WNT axis, essential for limb patterning, was a key driver of these defects. Biochemical assays further revealed reduced hydroxyproline content, indicating impaired collagen synthesis. These findings highlight severe disruptions in SHH, FGF, WNT, HOX, Tbx4, and Pitx1 pathways, emphasizing the need for stricter pesticide regulations to prevent congenital limb malformations.