|
Fig. 4
Oxidative stress is only induced in grade 3 larvae.
(A) SOD and catalase activities and GSH and MDA (LPO) levels in control and grade 1–3 zebrafish larvae exposed from 7 to 8 days post fertilization (dpf). The data represent the mean ± SEM. The value on each bar indicates the number of pools for each condition, and bars with different letters are significantly different (p < 0.05, one-way ANOVA with Tukey’s multiple comparison test). (B–G) Brightfield (B–D) and fluorescence (E–G) images of the trunks of representative 8 dpf control (B,E), grade 2 (C,F), and grade 3 (D,G) larvae phenotypes. The oxidative fluorescent dye DCFH-DA, which is used for identifying ROS generation in live larvae, showed high ROS generation in grade 3 muscle fibres and spinal cord (G) compared to grade 2 (F) and control (E). Inset at (G) shows brightfield and fluorescence images of the trunk of a grade 3 larva, with the focal plane at the level of the spinal cord. Dorsal and ventral limits of the spinal cord are highlighted by a white dotted line. Abbreviations: sc, spinal cord. (H–J) Grade 3 larvae exhibit a significant reduction in mitochondrial respiration. The basal and maximal respiration (H) and the coupled respiration (I) were strongly reduced in grade 3 larvae with respect to the control larvae (3–11 pools with 20–25 larvae each were analysed for each group; *p < 0.05, ***p < 0.001, Student’s t-test). Moreover, the ratio between coupled respiration and leak was strongly altered (J). (K,L) Prevalence of the grade 3 phenotype can be modified by altering the endogenous levels of GSH. Larvae were pre-incubated with either 50 µM N-acetyl-L-cysteine (NAC) or 0.5 µM diethylmaleate (DEM) for 24 h (from 6 to 7 dpf) followed by co-exposure with 3 µM CPO for 24 h (from 7 to 8 dpf). (K) Increasing the GSH levels with N-acetyl-L-cysteine (NAC) decreased the prevalence of the grade 3 phenotype by 67% [3 groups with 8–19 larvae each were analysed for 3 µM CPO and 3 groups with 13–23 larvae for 3 µM CPO + 50 µM NAC; p < 0.05, Student’s t-test]. (L) Decreasing the GSH levels with diethylmaleate (DEM) increased the prevalence of grade 3 larvae by 82% [6 groups with 22–46 larvae each were analysed for 3 µM CPO and 6 groups with 21–43 larvae for 3 µM CPO + 0.5 µM DEM; p < 0.05, Student’s t-test]. (M,N) Prevalence of the grade 3 phenotype in zebrafish larvae exposed to 3 µM CPO is not significantly reduced by either antioxidant. Larvae were pre-treated with the antioxidants vitamin C and MitoQ for 3 h or 24 h, respectively, followed by co-exposure with 3 µM CPO for an additional 24 h (M); 4 groups with 13–22 larvae each per condition; p > 0.05, one-way ANOVA with Dunnett’s multiple comparison test) or MitoQ (N); 4–8 pools with 19–24 larvae each per condition; p > 0.05, Student’s t-test). Abbreviations: n.s., not significant.