Exposure To Pyrazosulfuron-ethyl Induces Immunotoxicity And Behavioral Abnormalities In Zebrafish Embryos

Objective:Inrecent years,with thecontinuous development of agriculture,pesticideshave been widely produced,applied and improperly handled in large quantities.Chemical pesticides can be detected in variousenvironmental mediaand soil environments as well as aquatic ecosystems.However,we have limited information about the potential toxic effects of pesticides on aquatic biological safety and aquatic life.Pyrazosulfuron-ethyl is one of thewidely used herbicides in agricultureand can cause severe toxicity.In orderto clarify the toxic effects of this pesticide,zebrafish embryos and juveniles were tested withpyrazosulfuron-ethyl and zebrafish as an experimental animal model.To attempt toconstruct links between toxicity through individual characterization from molecularlevel and toxic effects toprovide data support andtheoretical basis for the potential risks of pesticide exposuretoaquatic organisms and aquatic ecological environments.Methods:The Tg(lyz: DsRed)zebrafish transgenic fish line at 10 hpf(hour post fertilization)after fertilization was randomly assigned to a 6-well plate with 20 plates per well in three replicates.We exposed zebrafish embryos to 1 mg/L,5 mg/L,and 9 mg/L pyrazosulfuron-ethyl to 72 hpf,changed fresh solution once a day,observed juvenile zebrafish development under a Leica microscope,and counted their heart rate,hatching rate,malformation rate,and mortality.Meanwhile,we measured the body length through the ZEN software of the Zeiss microscope.Fifteen fish were measured for each group,and the experiments were performed in triplicate.In addition,we performed neutral red and Sudan black staining to observe the development of immune cells.Using fish lines from the wild-type AB strain,juveniles were collected after drug treatment to 72 hpf for ROS staining,and total proteins of each experimental group were extracted to determine the enzyme activity levels of superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA).The expression levels of immune development-related genes such as CXCL-C1C、IL-8、IFN-γ、MYD88、TLR-4、IL-1β were determined by quantitative real-time PCR.High-concentration groups were rescued using astaxanthin and DAPT,the Notch signaling pathway inhibitor,to observe thechanges in theimmune development of juvenile zebrafish before and after drug treatment.Data were statistically analyzed using SPSS 18.0softwareand statistical maps were madewith Graph Pad Prism 8.Results:1.Compared with the control group,the zebrafish had decreased autonomous movement,shorter body length,abnormal heart development,and decreased incubation rate,especially between the 9mg/L pyrazosulfuron-ethyl group and the control group(P <0.001).At the same time,the exposure results of different concentrations of pyrazosulfuron-ethyl showed dose-dependent expression,and the heart rate of juvenile zebrafish in the drug-treated group gradually decreased and the treatment time increased.2.The results of Sudan black staining and neutral red staining showed that the number of neutrophils and macrophages in the drug-treated group was significantly different from the control group.The real-time PCR showed immune development related genes,TLR-4,My D 88 and IL-1βexpression.3.Oxidative stress test results after pyrazosulfuron-ethyl treatment showed fluorescence enhancement of ROS staining with increasing drug concentration,showing a significant gap from the control group.The index of oxidative stress-related enzyme activity was also abnormal: superoxide dismutase(SOD)activity decreased,while malondialdehyde(MDA)and catalase(CAT)increased,indicating oxidative stress in pyrazosulfuron-ethyl induced zebrafish immune toxicity and oxidative stress.After rescue treatment,the antioxidant astaxanthin(ATX)had significant efficacy,and the ROS test results of the ATX rescue group showed that the oxidative stress level was reduced,which provided strong evidence for pyrazosulfuron-induced immune development toxicity in zebrafish.4.After detection,the expression level of genes associated with Notch signaling decreased significantly,while the Notch signaling pathway inhibitor DAPT treatment,the number of immune cells in the group was increased significantly,indicating that Notch signaling was involved in the toxic immune development response caused by pyon,while the ROS staining also showed a strong decrease of green fluorescence.Therefore,we concluded that pysulfuron-induced defects in zebrafish heart development might be achieved by upregulated ROS and Notch signaling pathways.Pyrazosulfuron-ethyl induced the upregulation of Notch signaling and decreased the expression of CXCL-C1 C,IL-8,IFN-γ,MYD 88,TLR-4 and IL-1β,suggesting that pyrazosulfuron-ethyl may reduce theexpression of theseinflammation-related genes by upregulating Notch signaling.Conclusion:1.Pyransulfuron-ethyl exposure induces defects inimmune development inzebrafish embryos.2.Pyransulfuron-ethyl affects motorbehavioral abnormalities inzebrafish embryos.3.Pyransulfuron-ethyl exposure destroys the immune system through oxidative stress.4.Pyransulfuron-ethyl affects immune developmental toxicity in zebrafish embryos through upregulation of Notch signaling.