6PPD induces cerebrovascular defects by triggering oxidative stress and ferroptosis in zebrafish

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), which is widely used as an antiozonant in rubber tires, has recently got much attention for its acute aquatic toxicity. However, the developmental toxicity of 6PPD in cerebrovascular network remains unknown. Here, we investigated the effects of 6PPD exposure in cerebral vascular using the larvae of zebrafish. 6PPD would not affect the body length and shape of zebrafish larvae at the concentrations ranging from 20 μg/L to 1000 μg/L. 6PPD induced developmental defects in the brain in a concentration-dependent manner. The trunk vascular development would not be affected while the cerebrovascular network was disrupted upon 6PPD exposure. 6PPD would trigger excessive Reactive Oxygen Species (ROS) in the brain, indicating abnormal oxidative stress. Mechanistically, brain-specific transcriptome analysis showed that 6PPD could potentially cause the blockage of arachidonic acid (AA) metabolism-related genes and the upregulation of ferroptosis-related genes. Besides, treatment with ferroptosis inhibitor N-Acetyl-L-cysteine (NAC) attenuated oxidative damage and improved the construction of cerebrovascular network upon 6PPD exposure. Moreover, using a human vascular endothelial cell line, we further confirmed that 6PPD could trigger abnormal oxidative stress and defective expansion capacity, implying the conserved toxicity cross species. These findings are useful for the elucidation of toxicity underlying 6PPD in cerebrovascular systems of both zebrafish and humans. To investigate the effects of 6PPD on zebrafish development, embryos were treated with 500 μg/L 6PPD from 10 hours post-fertilization (hpf) until 2 days post-fertilization (dpf). For transcriptomic analysis, three biological replicates were prepared for both the 6PPD treatment group and the DMSO control group. For each replicate, the heads of 150 embryos at 2 dpf were dissected, flash-frozen in liquid nitrogen, and sent for transcriptomic sequencing. The raw data analysis was performed to identify differentially expressed genes between the 6PPD-treated and control groups.