*3.7. Effects of TFZ on Fish Intestinal Cell Line*

As the TFZ concentration increased in the intestinal cell line, the cell activity generally decreased (Figure 7A). The expression of *hsp90* and *grp94* was increased in the 5 mg/L TFZ group relative to the control group (*p* < 0.05, Figure 7B). Additionally, the expression of *grp94* was significantly increased in the 10 mg/L TFZ group compared to the control group (*p* < 0.05, Figure 7B). Compared to the control group, the expression of *hsp90* was markedly increased in the 20 mg/L TFZ group (*p* < 0.01, Figure 7B). Compared to the control group, the expression of *cox2* was obviously increased in the 10 (*p* < 0.01) and 20 mg/L (*p* < 0.05) TFZ groups (Figure 7C). The expression of *srebp1* was significantly increased in the 20 mg/L TFZ group compared to the control group (*p* < 0.05, Figure 7D). The expression of *ppar-γ* was significantly increased in the 5 (*p* < 0.05), 10 (*p* < 0.01), and 20 mg/L (*p* < 0.01) TFZ groups relative to the control group (Figure 7D).

**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.

## **4. Discussion**

From our results, it is clear that TFZ strongly affected the early development of zebrafish in a concentration-dependent manner. After TFZ exposure, the 48 h LC50 in 3 dpf zebrafish embryos and the 24 h LC50 in 6 dpf zebrafish larvae were 4.872 mg/L and 2.580 mg/L, respectively. Previous studies have suggested that the 72 h LC50 of TFZ for rare minnow (*Goboicypris rarus*) embryos was 7.11 (6.69–7.51) mg/L [13], which was to some degree higher than the results in this study. The species tested and exposure time caused these differences in acute toxicity experiments. For *Oncorhynchus mykiss*, the acute 96 h LC50 was 0.57 mg/L in Lewis's study [28], Hermsen's study found that the benchmark concertation of six triazoles ranges from 1.5 mg/L to 25 mg/L in zebrafish embryos [29], which was in accordance with our research.

In addition, we discovered obvious morphological changes induced by TFZ, including pericardial edema, yolk sac swelling, liver size reduction and liver color darkening in 3 dpf zebrafish embryos after exposure for 48 h. Zebrafish embryonic toxicity tests found that pericardial edema and yolk sac retention were extensively noted after triazoles exposure [29–32]. According to Jiang's study, difenoconazole can induce liver degeneration, including the retention of yolk sac in zebrafish [33]. It has been reported that a delay in yolk sac uptake is indicative of dysfunction of the liver during zebrafish larval development following tamoxifen exposure [34]. We found that yolk sac retention and liver degeneration occur in a concentration-dependent manner in zebrafish larvae, which was in accordance

with the results obtained from a previous report [35]. It is noteworthy that the yolk sac is the only nutrient source in zebrafish larvae during embryonic development, and the retention of the yolk sac may affect nutrient absorption and lipid metabolism [36]. We subsequently observed that ALT activity was obviously increased in the 6 dpf zebrafish larvae after TFZ exposure. In conclusion, morphological changes in the liver and a significant increase in ALT activity indicate liver damage.

The antioxidant defense systems were affected by oxidative stress following TFZ exposure. SOD and CAT are potent enzymes for defending against oxygen toxicity because of their inhibition of effects on oxyradical formation [37]. In addition, the MDA content may indirectly represent the extent of lipid peroxidation. According to our study, SOD activity, CAT activity, and MDA content were significantly higher in 6 dpf zebrafish larvae. The increase in SOD activity, CAT activity, and MDA content is likely to toxicant stress and counteract the damage from ROS [38]. However, SOD activity was strongly inhibited in the 3 mg/L TFZ group in 3 dpf zebrafish embryos, indicating that it might destroy the protective system of zebrafish larvae. We observed that the expression of HSPs (including *hsp70*, *grp78*, *hsp90*, and *grp94*) in 3 dpf zebrafish embryos sharply increased in the 2 mg/L TFZ group, indicating a protective effect against protein misfolding [18]. Furthermore, we found that the expression of *hsp90* in the intestinal cell line was significantly increased in the 5 and 20 mg/L TFZ groups, which was in accordance with 3 dpf zebrafish embryos.

Several studies in aquatic organisms have demonstrated the potential for environmental pollutants to disrupt inflammatory gene expression. Inflammatory genes expressed in zebrafish larvae could be increased by glyphosate exposure [39]. Tricyclazole, a pesticide, also altered the transcription of inflammatory factors such as *tnfα* after exposure [24]. Tissue damage caused by environmental stimuli could lead to inflammation [40]. Our study suggests that TFZ exposure significantly increased the expression of *p65-nfκb*, *il-1β*, and *cox2a* which could be infer that potential tissue damage was caused by TFZ. The expression of *cox2* was markedly upregulated in the 10 and 20 mg/L TFZ groups in the intestinal cell line, which was in accordance with zebrafish larvae.

The increased oxidative stress, heat shock response, and inflammatory response are closely related to lipid synthesis in zebrafish [31,41]. In our research, we found that the expression of *srebp1*, *fas*, *acc*, and *ppar-γ* in 3 dpf zebrafish embryos was markedly increased after TFZ exposure, which was in accordance with the expression of *srebp1* and *ppar-γ* in the intestinal cell line. In reverse, TFZ caused liver damage and lipid synthesis through oxidative stress, heat shock response, and inflammation. Further studies should focus on the mechanism that TFZ contributes to lipid synthesis.
