The neurotoxicity and Ferroptosis identified in zebrafish embryos. (A) Neuron system fluorescence signals of control group and ESIONPs-exposed group in transgenic zebrafish Tg (eef1a1l1:EGFP). (B) Polarized light intensity of control group and ESIONPs-exposed group of zebrafish muscles. (C) The expression of neuron-developmental markers (pax2a, neurog1, and axin2) at different developmental stages of zebrafish. (D) The expression of muscle markers (acta2, ttn, lpin1) at different developmental stages of zebrafish. (E) ROS generation in zebrafish embryos was detected with fluorescent probe DCFH-DA staining. (F) The prevalence of apoptosis in zebrafish embryos was detected with fluorescent dye AO staining; red arrows indicate the apoptotic cells. (G) The expression of oxidative stress biomarkers (cybb, nox1, rac2) at different developmental stages of zebrafish. (H) The expression of apoptosis biomarkers (jnk1, bcl2a, tp53) at different developmental stages of zebrafish. The fluorescence intensity and polarized light intensity was quantified for individual zebrafish using ImageJ analysis. Scale bar, 200 μm. Value are normalized to Control group, and represent mean ± SE from three independent experiments, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 (Student’s t-test).
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