Toxic effects of trichlorfon on the hepatopancreas of Chinese mitten crab (Eriocheir sinensis): tissue damage, oxidative stress, and Ferroptosis

This study investigated the toxic effects and mechanisms of trichlorfon (TCF) on the hepatopancreas of Chinese mitten crabs (Eriocheir sinensis). The acute toxicity test results showed that the 96 h-LC50 of TCF on E. sinensis was 4.853 mg/L. We conducted an 8-day short-term exposure experiment with TCF concentrations of 0, 0.061, 0.121, and 0.243 mg/L. The results showed that TCF exposure induces oxidative stress and causes tissue damage and lesions to the hepatopancreas, including hepatic tubules exhibiting disruption, swelling, necrosis, and an ill-defined structure. Transcriptomics revealed differentially expressed genes (DEGs) at the different concentrations, KEGG, and GO enrichment analysis showed that the DEGs were mainly involved in the bile secretion pathway and iron ion binding terms. GSEA was used for single-function enrichment analysis of cell growth and death. We found that DEGs at TCF concentrations of 0.121 and 0.243 mg/L were significantly enriched in the Ferroptosis pathway. Moreover, the synaptic vesicle cycle, GABAergic synapse, and serotonergic synapse were significantly enriched in DEGs at 0.243 mg/L, which indicated that TCF may pose a potential threat to the nervous system.. The qPCR detection of Ferroptosis-related genes demonstrated that TCF induces hepatopancreatic Ferroptosis in E. sinensis. We hypothesized that lysosomal autophagy, mainly induced through FTH1 and NCOA4 interactions, released ferric ions, thereby indirectly contributing to Ferroptosis. In addition, WGCNA identified two key modules associated with the phenotype, and further identified the core hub genes were MEX3, RBM46, SLC7A5, and TYR. The results of this study can provide insights into the toxic effects of TCF on aquatic crustaceans.本研究探究了敌百虫（trichlorfon, TCF）对中华绒螯蟹（Eriocheir sinensis）肝胰腺的毒性效应及其作用机制。急性毒性试验结果显示，敌百虫对中华绒螯蟹的96 h-LC50为4.853 mg/L。本研究设置0、0.061、0.121及0.243 mg/L四个敌百虫浓度梯度，开展了为期8天的短期暴露实验。实验结果表明，敌百虫暴露可诱导氧化应激，引发肝胰腺组织损伤与病变，具体表现为肝小管结构紊乱、肿胀、坏死及边界模糊。转录组测序分析显示，不同浓度敌百虫暴露下均存在差异表达基因（differentially expressed genes, DEGs）；京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析及基因本体（Gene Ontology, GO）富集分析结果显示，差异表达基因主要富集于胆汁分泌通路及铁离子结合相关功能条目。采用基因集富集分析（Gene Set Enrichment Analysis, GSEA）对细胞生长与死亡进行单功能富集分析，发现浓度为0.121与0.243 mg/L的敌百虫暴露组的差异表达基因显著富集于铁死亡（Ferroptosis）通路。此外，0.243 mg/L暴露组的差异表达基因还显著富集于突触囊泡循环、γ-氨基丁酸能突触及5-羟色胺能突触通路，提示敌百虫可能对神经系统存在潜在威胁。针对铁死亡相关基因的实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qPCR）检测结果证实，敌百虫可诱导中华绒螯蟹肝胰腺发生铁死亡。本研究推测，主要通过铁蛋白重链1（ferritin heavy chain 1, FTH1）与核受体辅激活蛋白4（nuclear receptor coactivator 4, NCOA4）相互作用诱导的溶酶体自噬过程会释放铁离子，进而间接参与铁死亡的发生。此外，加权基因共表达网络分析（weighted gene co-expression network analysis, WGCNA）筛选得到两个与表型显著相关的关键模块，并进一步鉴定出核心枢纽基因为MEX3、RBM46、SLC7A5及TYR。本研究结果可为阐明敌百虫对水生甲壳类动物的毒性效应提供理论参考。