Forchlorfenuron (CPPU) causes disorganization of the cytoskeleton and dysfunction of human umbilical vein endothelial cells, and abnormal vascular development in zebrafish embryos

Forchlorfenuron (CPPU) has been used worldwide, to boost size and improve quality of various agricultural products. CPPU and its metabolites are persistent and have been detected frequently in fruits, water, sediments, and organisms in aquatic systems. Although the public became aware of CPPU through the exploding watermelon scandal of 2011 in Zhenjiang, China, little was known of its potential effects on the environment and wildlife. In this study, adverse effects of CPPU on developmental angiogenesis and vasculature, which is vulnerable to insults of persistent toxicants, were studied in vivo in zebrafish embryos (Danio rerio). Exposure to 10 mg CPPU/L impaired survival and hatching, while development was hindered by exposure to 2.5 mg CPPU/L. Developing vascular structure, including common cardinal veins (CCVs), intersegmental vessels (ISVs) and sub-intestinal vessels (SIVs), were significantly restrained by exposure to CPPU, in a dose-dependent manner. Also, CPPU caused disorganization of the cytoskeleton. In human umbilical vein endothelial cells (HUVECs), CPPU inhibited proliferation, migration and formation of tubular-like structures in vitro. Results of Western blot analyses revealed that exposure to CPPU increased phosphorylation of FLT-1, but inhibited phosphorylation of FAK and its downstream MAPK pathway in HUVECs. In summary, CPPU elicited developmental toxicity to the developing endothelial system of zebrafish and HUVECs. This was do, at least in part due to inhibition of the FAK/MAPK signaling pathway rather than direct interaction with the VEGF receptor (VEGFR).