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

## TL;DR

This study shows how a chemical called 1-B-36-CCZ harms blood vessel development in zebrafish larvae and suggests a possible mechanism involving calcium signaling.

## Contribution

The study identifies SRC kinase and calcium signaling as a novel mechanism for vascular toxicity caused by 1-B-36-CCZ in zebrafish.

## Key findings

- 1-B-36-CCZ exposure reduces intersegmental vessel length and anastomosis rate in zebrafish embryos.
- SRC kinase and calcium signaling are implicated in the vascular toxicity of 1-B-36-CCZ.
- Verapamil, a calcium channel blocker, mitigates the developmental and vascular damage caused by 1-B-36-CCZ.

## Abstract

1-Bromo-3,6-dichlorocarbazole (1-B-36-CCZ) is a typical homolog of polyhalogenated carbazoles (PHCZs) that is widely present in various environmental media. In this study, we used a zebrafish model to evaluate the developmental and vascular toxicity of 1-B-36-CCZ. By integrating network toxicology methods, we found that the potential mechanism of 1-B-36-CCZ toxicity may involve the activation of the calcium signaling pathway via SRC kinase, thus leading to vascular toxicity. This potential mechanism was further validated by the efficacy of verapamil, a calcium channel blocker, in ameliorating the developmental abnormalities and vascular damage induced by 1-B-36-CCZ. These results provide new experimental data for elucidating the toxic mechanisms of PHCZ-type pollutants and offer a theoretical basis for environmental health risk assessments.

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.