Fig 1.
Sensitivity of zebrafish to cyanide depends on the developmental stage.
The LD50 for 3h exposure to KCN in zebrafish is shown as a function of the developmental stage in days post fertilization (dpf). The best-fit sigmoidal curve calculated by non-linear regression analysis is plotted in the figure. LD50 values were calculated by non-linear regression fitting of dose-response curves showing survival after cyanide exposure in zebrafish embryos and larvae at different stages (S1 Dataset).
Fig 2.
The transcriptional response to cyanide exposure is very limited in zebrafish embryos and larvae.
Data from a microarray experiment comparing 1 dpf embryos and 7 dpf larvae exposed to KCN at the indicated doses was used to generate a heatmap showing expression changes of 10,146 genes as compared to the respective vehicle treatment. On the right an enlargement is shown of the clustered regions that show differential expression at the top and the bottom of the range.
Table 1.
Gene expression changes after exposure to cyanide at different ages.
Fig 3.
Comparison of the metabolomic responses of 1 dpf and 7 dpf zebrafish embryos to cyanide exposure.
Results from the metabolomic analysis of 1 dpf and 7 dpf zebrafish embryos exposed to 500 μM or 20 μM KCN, respectively, or vehicle for 2 h. Pathway enrichment analysis results are plotted according to p-values and pathway impact scores. Pathways whose regulation in the response to cyanide is significantly different between different ages after correction for the false discovery rate are indicated in the figure; pathways related to energy metabolism are marked in boldface type.
Fig 4.
Comparison of the metabolomic signature of 1 dpf and 7 dpf zebrafish.
A) Representation of the pathway enrichment analysis of the metabolite concentration levels at baseline in 1 dpf embryos vs 7 dpf larvae. Pathways are plotted according to p-values and pathway impact scores. The top significant pathways are indicated in the figure; pathways related to energy metabolism are in boldface type. B) Measurements of levels of TCA cycle intermediates in 1 and 7 dpf zebrafish. ***: P<0.001 vs 7 dpf larvae by Sidak’s multiple comparison test after two-way ANOVA.
Fig 5.
Hit compounds providing protection against cyanide toxicity.
Survival of 7 dpf zebrafish larvae exposed to a lethal dose of 20 μM cyanide for 24h, in combination with the 8 compounds that were found to be protective in our focused drug screen. EC50 values were calculated by linear regression fitting of sigmoidal dose-response curves to the data (S4 Dataset).
Fig 6.
Pyruvate dehydrogenase kinase regulates sensitivity to cyanide.
Survival curves of 4 dpf zebrafish control larvae and larvae injected with a synthetic morpholino (MO) targeting PDK1 or PDK2a after challenge with 100 μM KCN. N = 7–11 larvae per group. ***: P<0.001 by Log-rank (Mantel-Cox) test.
Fig 7.
Glyoxylate reverses changes in cellular metabolism induced by cyanide.
Selected metabolite levels were measured with and without KCN administration in pooled 7 dpf zebrafish larvae treated with sodium glyoxylate or vehicle. Data from replicate measurements are normalized to the level measured in 7 dpf control larvae. Increased levels of glucose and decreased levels of lactate, gluconic acid, and glycerol-3-phosphate indicate that glyoxylate treatment reduced KCN-induced glucose oxidation. Reduction of KCN-induced 3-hydroxybutyrate levels reflects a decreased level of ketosis during glyoxylate treatment. Glyoxylate treatment also leads to a mild improvement in ATP levels after KCN administration.