Immunotoxicity and mechanism analysis of zebrafish embryos exposure to benzothiazole and its derivatives

Benzothiazole derivatives (BTHs), including benzothiazole (BTH), 2-hydroxybenzothiazole (OBT), 2-aminobenzothiazole (NTH), and 2-(methylthio)benzothiazole (MTBT), pose significant exposure risks to organisms. Despite their recognized toxicity, the immunotoxic effects of BTHs remain poorly understood. This study systematically evaluated the immunotoxicity of four BTHs (BTH: 50 μM, 100 μM, and 200 μM; OBT: 50 μM, 100 μM, and 200 μM; NTH: 25 μM, 50 μM, and 100 μM; and MTBT: 3.125 μM, 6.25 μM, and 12.5 μM) in zebrafish embryos, including developmental toxicity, innate immune cell responses, oxidative stress levels, and bacterial challenge experiments were conducted to determine the impact of BTHs on pathogen resistance. RNA-seq and qRT-PCR assay were used to determine the mechanisms underlying BTHs-induced immunotoxicity. Results showed that BTH, OBT, NTH, and MTBT exposure caused developmental abnormalities, reduced macrophage and neutrophils numbers, and induced oxidative stress, including superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and reactive oxygen species (ROS). Bacterial challenge assay revealed that BTH, OBT, NTH, and MTBT significantly impaired zebrafish resistance to bacterial infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified key differentially expressed genes (DEGs) in the TLR/NLR-NF-κB signaling pathway following BTH-exposure, which were further validated by qRT-PCR. In summary, BTHs not only exhibit developmental toxicity but also induce immunotoxicity by disrupting the Toll-like Receptors - Nucleotide-binding Oligomerization Domain-like Receptors - Nuclear Factor kappa-B (TLR/NLR-NF-κB) signaling pathway. These findings provide critical insights into the ecological risks of BTHs exposure in aquatic environments.