三氯杀螨砜对斑马鱼胚胎的生态毒理效应

Ecotoxicological effects of 2,4,5,4′-tetrachlorodiphenyl sulfone on Danio rerio embryos

    摘要
  • 摘要: 目的 探究三氯杀螨砜（TDN）对斑马鱼胚胎的生态毒理效应，为揭示TDN对水生生物的毒性作用机制及有机氯农药的环境生态风险评估提供数据支撑。方法 将斑马鱼胚胎分别暴露于0、6、9、12、15、18 mg/L的TDN溶液中，记录TDN暴露对斑马鱼胚胎存活率、卵黄囊面积、心率和体长的影响。使用试剂盒检测超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性及丙二醛（MDA）含量。实时荧光定量PCR检测斑马鱼胚胎凋亡、炎症和免疫相关基因的相对表达量。采用吖啶橙染色和苏丹黑染色鉴定细胞凋亡水平和嗜中性粒细胞数量。结果 随TDN浓度和暴露时间增加，斑马鱼胚胎存活率逐渐降低，在0~15 mg/L的TDN中暴露72 h，斑马鱼胚胎卵黄囊面积显著（P<0.05，下同）或极显著（P<0.01，下同）增大，心率显著或者极显著降低，体长缩短，15 mg/L TDN处理斑马鱼胚胎出现明显的心包水肿。与0 mg/L TDN处理相比，在6、9、12和15 mg/L的TDN暴露下，斑马鱼胚胎CAT活性均极显著升高，MDA含量均有所升高，9、12与15 mg/L TDN处理SOD活性均极显著升高。与0 mg/L TDN处理相比，6、9、12和15 mg/L TDN处理斑马鱼胚胎促凋亡相关基因Bax和P53相对表达量均有所升高，抗凋亡相关基因Bcl-2相对表达量整体上有所降低，炎症相关基因IL-6、IL-8、CXCL-C1C、TNF-α、Myd88和P65相对表达量均有所升高，免疫相关基因Ifnγ和Mxa和Mxb相对表达量均有所升高。吖啶橙染色和苏丹黑染色结果显示，与0 mg/L TDN处理相比，其他各TDN处理细胞凋亡水平整体上升高，嗜中性粒细胞减少。结论 TDN对斑马鱼胚胎的毒性作用表现在生长发育受损、诱导氧化应激反应、细胞凋亡、炎症反应和抑制免疫功能，其可能通过“氧化应激—凋亡—免疫紊乱”途径发挥作用。含氯杀虫剂的危害机理具有共性，应在环境风险评估中重视其对机体稳态的长期干扰。

     

    Abstract: Objective This study aimed to investigate the ecotoxicological effects of 2,4,5,4′-tetrachlorodiphenyl sulfone （TDN） on zebrafish embryos， and to provide data support for revealing the TDN toxicity mechanism on aquatic organisms and environmental and ecological risks assessment of organochlorine pesticides.Method Zebrafish embryos were exposed to 0， 6， 9， 12， 15， and 18 mg/L TDN solutions to record effects of TDN exposure on embryo survival rate， yolk sac area， heart rate， and body length. Activities of superoxide dismutase （SOD） and catalase （CAT）， along with malondialdehyde （MDA） content， were determined using their respective assay kits. Real-time fluorescence quantitative PCR was used to determine the relative expression of genes related to apoptosis， inflammation， and immunity in zebrafish embryos. Acridine orange and Sudan black staining were used to assess the apoptosis level and neutrophil counts.Result With increasing TDN concentration and exposure time， the survival rate of zebrafish embryos gradually decreased. After 72 h exposure to 0-15 mg/L TDN， the yolk sac area of zebrafish embryos increased significantly （P<0.05， the same below） or highly significantly （P<0.01， the same below）， the heart rate decreased significantly or highly significantly， and the body length decreased. Zebrafish embryos in the 15 mg/L TDN treatment group showed pericardial edemas. Under exposure to 6， 9， 12， and 15 mg/L TDN， the CAT activity of zebrafish embryos exhibited a highly significant increase， MDA content of zebrafish embryos showed some increase for all the embryos， and SOD activities of embryos in 9， 12， and 15 mg/L TDN treatment groups showed a highly significant increase compared with the 0 mg/L TDN treatment group. In the 6， 9， 12， and 15 mg/L TDN treatment groups， compared with the 0 mg/L TDN treatment group， the relative expression of the pro-apoptotic genes Bax and P53 in zebrafish embryos were all increased； the relative expression of the anti-apoptotic gene Bcl-2 was generally decreased； the relative expression of the inflammatory genes IL-6， IL-8， CXCL-C1C， TNF-α， Myd88， and P65 were generally increased； the relative expression of the immune-related genes Ifnγ， Mxa， and Mxb were generally increased. Acridine orange staining and Sudan black staining results showed that compared with the 0 mg/L treatment group， the apoptosis levels in the other groups were generally increased， and the neutrophil count was decreased.Conclusion Toxic effects of TDN on zebrafish embryos manifest as impaired growth and development， induction of oxidative stress， apoptosis， inflammation， and immune suppression through an “oxidative stress-apoptosis-immune disorder” pathway. Chlorinated pesticides share common mechanisms of harm， and their long-term effects on body homeostasis should be noticed in environmental risk assessments.

     

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