Figure 1

DICNH leads to hatching delay. Zebrafish eggs at 2 hpf were exposed to different concentrations of DINCH and hatching delay was recorded until 124 hpf. Statistical analysis was performed using One-way ANOVA followed by Dunnett's post-test. Error bars represent mean ± SD, n = 90.
Figure 2

DINCH affects the expression of oxidative stress genes. Zebrafish embryos were exposed to 0.01, 0.1, 1 and 10 μM of DINCH for 6 dpf and qRT-PCR analysis was performed for stress related genes including sod1 (A), sod2 (B), sod3 (C), gst (D), cyc1 (E), fkbp4 (F), cat (G), mtf (H), mt2 (I), gadd45a (J) and rad51 (K). Statistical analysis was performed using One-way ANOVA followed by Dunnett's post-test.
Figure 3

Figure 3. DINCH alters lipid metabolism. Zebrafish embryos were exposed to 0.01, 0.1, 1 and 10 μM of DINCH for 6 dpf and ORO staining was performed. Images were taken with 4X objective using a bright field microscope (Olympus BX51). Arrows indicate the area where differences between the control and exposed group were noticeable. n = 10.

Figure 4

DINCH alters fatty acid synthesis, peroxisome proliferator activated receptor, and fatty acid elongation genes. Zebrafish embryos were exposed to 0.01, 0.1, 1 and 10 μM of DINCH for 6 dpf and qRT-PCR analysis was performed lipid metabolism genes including srebp1 (A), srebp2 (B), fasn (C), srebp1 (A), srebp2 (B), fasn (C), elovl1 (D), elovl2 (E), pparg (F), ppara (G) and pparb (H). Statistical analysis was performed using One-way ANOVA followed by Dunnett's post-test.
Figure 5

DINCH alters lipid transport genes. Zebrafish embryos were exposed to 0.01, 0.1, 1 and 10 μM of DINCH for 6 dpf and qRT-PCR analysis was performed for lipid transport and processing including apoeb (A), apoa4 (B), apoa1 (C), star4d (D), ldlr (E), lipc (F) plb (G) and lpl (H). Statistical analysis was performed using One-way ANOVA followed by Dunnett's post-test.
Figure 6

DINCH alters behavior in larvae. Zebrafish embryos were exposed to 0.01, 0.1, 1 and 10 μM of DINCH for 6 dpf and behavior analysis was performed. Distance and acceleration were recorded during light on (A and D), Light off (B and E) and light on (C and F). Kruskal-Wallis test. n = 48.
Figure 7

DINCH affects behavior, cholesterol biosynthesis and homeostasis genes. Zebrafish embryos were exposed to 0.01, 0.1, 1 and 10 μM of DINCH for 6 dpf and qRT-PCR analysis was performed for genes involved in behavior including pcsk9 (A), dmrt3a (B), hmgcs1 (C), mbpa (D), dhcr7 (E) and cfos (F). Statistical analysis was performed using One-way ANOVA followed by Dunnett's post-test.
Figure 8

Overview of the differentially expressed genes in response to DINCH. The schematic diagram shows the regulation and involvement of genes in different signaling mechanisms. Gene involved in lipid synthesis, elongation, hydrolysis and transport were altered in larvae. The stress response and DNA repair genes were also regulated following DINCH exposure. The altered lipid metabolism and transport could also be responsible for the observed cholesterol biosynthesis/homeostasis and brain function alteration in the larvae. The upregulated genes are indicated in red color, the downregulated genes are indicated in green color and the genes that were not regulated are indicated in black color.
Acknowledgments
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