Triflumizole Induces Developmental Toxicity, Liver Damage, Oxidative Stress, Heat Shock Response, Inflammation, and Lipid Synthesis in Zebrafish

Abstract

Triflumizole (TFZ) toxicity must be investigated in the aquatic environment to understand the potential risks to aquatic species. Accordingly, the adverse effects of TFZ exposure in zebrafish were investigated. Results demonstrate that, after TFZ exposure, the lethal concentration 50 (LC₅₀) in 3 d post-fertilization (dpf) embryos and 6 dpf larvae were 4.872 and 2.580 mg/L, respectively. The development (including pericardium edema, yolk sac retention, and liver degeneration) was apparently affected in 3 dpf embryos. Furthermore, the alanine aminotransferase (ALT) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, and malondialdehyde (MDA) content in 6 dpf larvae were significantly increased. Additionally, the expression of heat shock response genes (including hsp70, grp78, hsp90, and grp94), inflammatory genes (including p65-nfκb, il-1β, and cox2a), and lipid synthetic genes (including srebp1, fas, acc, and ppar-γ) in 3 dpf embryos was significantly increased, which was also partially observed in the intestinal cell line form Pampus argenteus. Taken together, TFZ could affect the development of zebrafish, accompanied by disturbances of oxidative stress, heat shock response, inflammation, and lipid synthesis. Our findings provide an original insight into the potential risks of TFZ to the aquatic ecosystem.

Keywords: heat shock response; inflammation; lipid synthesis; oxidative stress; triflumizole; zebrafish.

Figure 1

Exposure patterns and the survival percentage of zebrafish after triflumizole exposure. (A) Three dpf embryos were exposed to 1, 2, and 3 mg/L triflumizole for 48 h, (B) Six dpf larvae were exposed to 0.5, 1, and 1.5 mg/L triflumizole for 24 h. (C) The percentage of survival of 3 dpf embryos after triflumizole exposure for 48 h. (D) The percentage of survival of 6 dpf larvae after triflumizole exposure for 24 h. dpf: d post-fertilization; LC₅₀: lethal concentration 50.

Figure 2

Effects of development after triflumizole exposure in zebrafish. (A) 3 dpf zebrafish embryos with morphological changes after triflumizole exposure for 48 h. (B) 6 dpf zebrafish larvae with liver degeneration after triflumizole exposure for 24 h. Blue arrows and red arrows indicate pericardial edema and liver degeneration, respectively. H: heart, the green dotted line; L: liver, the red dotted line; I: intestine, the blue dotted line; Y: yolk sac, the yellow dotted line. Scale bars = 500 μm; Scale bars = 100 μm.

Figure 3

Effects of triflumizole on enzyme activities in zebrafish. For 3 dpf zebrafish embryos, SOD activity (A), CAT activity (B), MDA content (C), and ALT activity (D) after triflumizole exposure for 48 h. For 6 dpf zebrafish larvae, SOD activity (E), CAT activity (F), MDA content (G), and ALT activity (H) after triflumizole exposure for 24 h. Data are expressed as the mean of three replicates ± standard error (SEM). Asterisks denote significant differences between the control group and TFZ groups (determined by Dunnett post hoc comparison, * p < 0.05, ** p < 0.01). SOD: superoxide dismutase; CAT: catalase; MDA: malondialdehyde; ALT: alanine transaminase.

Figure 4

The expression of heat shock response genes (including hsp70, grp78, hsp90, and grp94) in zebrafish. (A) For 3 dpf zebrafish embryos, the expression of hsp70, grp78, hsp90, and grp94 after triflumizole exposure for 48 h. (B) For 6 dpf zebrafish larvae, the expression of hsp70, grp78, hsp90, and grp94 after triflumizole exposure for 48 h. Data are expressed as the mean of three replicates ± standard error (SEM). Asterisks denote significant differences between the control group and TFZ groups (determined by Dunnett post hoc comparison, * p < 0.05, ** p < 0.01). hsp70: heat shock protein 70; grp78: glucose-regulated protein 78; hsp90: heat shock protein 90; grp94: glucose-regulated protein 94.

Figure 5

The expression of inflammatory genes (including tnfα, il-1β, p65-nfκb, cox2a, and cox2b) in zebrafish. (A) For 3 dpf zebrafish embryos, the expression of tnfα, il-1β, p65-nfκb, cox2a, and cox2b after triflumizole exposure for 48 h. (B) For 6 dpf zebrafish larvae, the expression of tnfα, il-1β, p65-nfκb, cox2a, and cox2b after triflumizole exposure for 24 h. Data are expressed as the mean of three replicates ± standard error (SEM). Asterisks denote significant differences between the control group and TFZ groups (determined by Dunnett post hoc comparison, ** p < 0.01). tnfα: tumor necrosis factor α; il-1β: interleukin 1, beta; p65-nfκb: p65-nuclear transcription factor κB; cox2a: cyclooxygenase type 2 a; cox2b: cyclooxygenase type 2 b.

Figure 6

The expression of lipid synthesis genes (including srebp1, fas, acc, and ppar-γ) in zebrafish. (A) For 3 dpf zebrafish embryos, the expression of srebp1, fas, acc, and ppar-γ after triflumizole exposure for 48 h. (B) For 6 dpf zebrafish larvae, the expression of srebp1, fas, acc, and ppar-γ after triflumizole exposure for 48 h. Data are expressed as the mean of three replicates ± standard error (SEM). Asterisks denote significant differences between the control group and TFZ groups (determined by Dunnett post hoc comparison, * p < 0.05, ** p < 0.01). srebp1: sterol regulatory element binding transcription protein 1; fas: fas cell surface death receptor; acc: acetyl-coa carboxylase; ppar-γ: peroxisome proliferator-activated receptor gamma.

Figure 7

Effects of the fish intestinal cell line after triflumizole exposure for 24 h. (A) The cell viability. (B) The expression of hsp70, grp78, hsp90, and grp94 after triflumizole exposure. (C) The expression of p65-nfκb, p105-nfκb, and cox2 after triflumizole exposure. (D) The expression of srebp1, lpl, ppar-γ and ppar-α after triflumizole exposure. Data are expressed as the mean of three replicates ± standard error (SEM). Values without a common superscript letter differ (p < 0.05, Tukey’s test). Asterisks denote significant differences between the control group and TFZ groups (determined by Dunnett post hoc comparison, * p < 0.05, ** p < 0.01). Data are expressed as the mean of three replicates ± standard error (SEM). p65-nfκb: p65-nuclear transcription factor κB; p105-nfκb: p105-nuclear transcription factor κB; cox2: cyclooxygenase 2; lpl: lipoprotein lipase; ppar-α: peroxisome proliferator-activated receptor alpha.