Polyhalogenated carbazoles (PHCZs) are emerging environmental pollutants, yet limited information is available on their embryotoxicity and neurotoxicity. Therefore, the current work was performed to investigate the adverse effects of 3,6-dibromocarbazole (3,6-DBCZ), a typical PHCZs homolog, on the early life stages of zebrafish larvae. It revealed that the 96-hour post-fertilization (hpf) median lethal concentration (LC₅₀) value of 3,6-DBCZ in zebrafish larvae was determined to be 0.7988 mg/L. Besides, 3,6-DBCZ reduced survival rates at concentrations ≥ 1 mg/L and decreased hatching rates at ≥ 0.25 mg/L at 48 hpf. In behavior tests, it inhibited locomotor activities and reduced the frequency of recorded acceleration states in response to optesthesia (a sudden bright light stimulus) at concentrations ≥ 160 μg/L. Meanwhile, 3,6-DBCZ exposure decreased the frequency of recorded acceleration states in the startle response (tapping mode) at concentrations ≥ 6.4 μg/L. Pathologically, with the transgenic zebrafish model (hb9-eGFP), we observed a strikingly decreased axon length and number in motor neurons after 3,6-DBCZ treatment, which may be ascribed to the activation of the AhR signaling pathway, as evidenced by the molecular docking analysis and Microscale thermophoresis (MST) assay suggested that 3,6-DBCZ binding to AhR-ARNT2 compound proteins. Through interaction with AhR-ARNT, a striking reduction of the anti-oxidative stress (sod1/2, nqo1, nrf2) and neurodevelopment-related genes (elavl3, gfap, mbp, syn2a) were observed after 3,6-DBCZ challenge, accompanied by a marked increased inflammatory genes (TNFβ, IL1β, IL6). Collectively, our findings reveal a previously unrecognized adverse effect of 3,6-DBCZ on zebrafish neurodevelopment and locomotor behaviors, potentially mediated through the activation of the AhR pathway. Furthermore, it provides direct evidence for the toxic concentrations of 3,6-DBCZ and the potential target signaling in zebrafish larvae, which may be beneficial for the risk assessment of the aquatic ecosystems.

多卤咔唑（PHCZ）是新兴的环境污染物，但有关其胚胎毒性和神经毒性的信息有限。因此，本研究旨在研究典型PHCZ同源物3,6-二溴咔唑（3,6-DBCZ）对斑马鱼幼虫生命早期阶段的不利影响。结果表明，斑马鱼幼虫受精后96小时(hpf)半数致死浓度(LC ₅₀ )值为0.7988 mg/L。此外，3,6-DBCZ 在浓度 ≥ 1 mg/L 时会降低存活率，在 48 hpf 时浓度≥ 0.25 mg/L 时会降低孵化率。在行为测试中，浓度≥ 160 μg/L 时，它会抑制运动活动并降低记录的响应视觉（突然的强光刺激）的加速状态的频率。同时，浓度≥ 6.4 μg/L 时，3,6-DBCZ 暴露降低了惊吓反应（敲击模式）中记录的加速状态的频率。病理学上，在转基因斑马鱼模型（hb9-eGFP）中，我们观察到3,6-DBCZ处理后运动神经元的轴突长度和数量显着减少，这可能归因于AhR信号通路的激活，这一点可以通过分子对接分析和微尺度热泳（MST）分析表明3,6-DBCZ与AhR-ARNT2复合蛋白结合。 通过与 AhR-ARNT 相互作用，在 3,6-DBCZ 攻击后观察到抗氧化应激（ sod1/2 、 nqo1 、 nrf2 ）和神经发育相关基因（ elavl3 、 gfap 、 mbp 、 syn2a ）显着减少，并伴随炎症基因（ TNFβ 、 IL1β 、 IL6 ）显着增加。总的来说，我们的研究结果揭示了 3,6-DBCZ 对斑马鱼神经发育和运动行为的先前未被认识的不利影响，可能是通过激活 AhR 通路介导的。此外，它为斑马鱼幼虫中 3,6-DBCZ 的毒性浓度和潜在目标信号传导提供了直接证据，这可能有益于水生生态系统的风险评估。