Single-Cell Transcriptomic Profiling of Human Blood Vessel Organoids Exposed to 6‑Chloronicotinic Acid

6-Chloronicotinic acid (6-ClNA) is a major metabolite of chloropyridinyl neonicotinoids in humans; however, its toxic effect on blood vessel development remains to be understood. In this study, we utilized a humanized blood vessel organoid (hBVO) model and single-cell transcriptomics technology to investigate the impact of 6-ClNA exposure on hBVO development. Morphologically, 6-ClNA did not significantly alter the growth of hBVO throughout 20 days of culture. Interestingly, single-cell transcriptomic analyses revealed that 6-ClNA-treated hBVOs exhibited significant imbalance in cell subpopulation homeostasis, including impaired endothelial cell function and abnormal proliferation of vascular smooth muscle cells. This was echoed by the morphological changes in the aortic arch and abdominal aorta of mice after acute exposure to 6-ClNA. Enrichment analysis showed that 6-ClNA exposure affected critical biological processes in a cell-type-specific manner, such as vascular developmental signaling, cellular energy metabolism, protein homeostasis, and hemodynamic responses. In addition, trajectory analysis suggested that 6-ClNA altered the differentiation trajectory of vascular progenitors. Together, our study profiles the toxicity of 6-ClNA on blood vessel development in a humanized 3D model at single-cell resolution, which provides important and detailed evidence for understanding the potential risk of early life exposure to chloropyridinyl neonicotinoids.