Early Vascular Developmental Toxicity and Underlying Mechanisms of 1-Bromo-3,6-dichlorocarbazole (1-B-36-CCZ) in Zebrafish Larvae.

Abstract

Polyhalogenated carbazoles (PHCZs) are emerging persistent organic pollutants that have attracted widespread attention due to their environmental occurrence and potential ecological risks. 1-Bromo-3,6-dichlorocarbazole (1-B-36-CCZ), which is a typical homolog of PHCZs produced as a byproduct in the dye industry, has been widely detected in various environmental media. In this study, we employed an integrated approach using an in vivo zebrafish model and network toxicology methods to systematically evaluate the vascular developmental toxicity of 1-B-36-CCZ and elucidate its underlying mechanisms. The experimental results revealed that the 96 h-LC₅₀ of 1-B-36-CCZ in zebrafish larvae was 4.52 mg/L. Sublethal exposures (0.045-45 μg/L) significantly induced an increase in heart rate (p < 0.05) and an enlargement of the pericardial edema area (p < 0.01). Using Tg(flk:eGFP) transgenic zebrafish embryos to assess vascular toxicity at concentrations of 0, 0.045, 0.45, 4.5, and 45 μg/L, we observed that 1-B-36-CCZ exposure significantly reduced the length and anastomosis rate of intersegmental vessels (ISVs) at 30 hpf, and inhibited the development of the common cardinal vein (CCV) at 48 and 72 hpf as well as the subintestinal vessel (SIV) at 72 hpf. Quantitative PCR (qPCR) analysis further revealed that the expression of key angiogenic genes (flk, kdr, and vegfa) was significantly downregulated, thus corroborating the phenotypic observations. Moreover, a "compound-target-pathway" network model predicted that SRC kinase is a key molecular target for 1-B-36-CCZ action. Enrichment analysis of target protein-coding genes and verapamil replication experiments indicated that 1-B-36-CCZ may cause damage to early vascular development in zebrafish larvae by altering intracellular calcium ion content through the activation of the SRC-mediated calcium ion signaling pathway. This study provides new experimental evidence for elucidating the toxic mechanisms of PHCZ-type pollutants and offers a theoretical basis for environmental health risk assessments.