As an efficient broad-spectrum insecticide, flubendiamide is widely used in agricultural production. Recently, the toxicological effects of flubendiamide on non-target organisms have received more and more attention (Nareshkumar et al. 2017, Ranjan et al. 2018). Some studies have reported the adverse effects of flubendiamide on non-target organisms such as earthworms (Eisenia fetida), Drosophila melanogaster and Daphnia magna (Cui et al. 2017, Liu et al. 2017, Sarkar et al. 2017). Unfortunately, the bioaccumulation and toxic effects of flubendiamide on zebrafish have not been reported. Therefore, the bioaccumulation behavior of flubendiamide on zebrafish was analyzed by using LC-MS/MS. In addition, the toxic effects involving growth phenotypes, oxidative stress and cell apoptosis of flubendiamide on zebrafish were explored in this study.
First of all, the results of the bioaccumulation experiment showed that the concentrations of flubendiamide could reach bioaccumulation equilibriums in zebrafish after 14 days of flubendiamide exposure. In addition, the BCFs of flubendiamide in zebrafish were 1.125–2.011. These results indicated that zebrafish had a low bioaccumulation capacity for flubendiamide according to the classification standards of bioaccumulation capacities (Jia et al., 2017). Meanwhile, there were no significantly change in body weight, body length and K-factors with flubendiamide exposure. However, the HIS was increased significantly after 14 days of 1.0 mg/L flubendiamide exposure. This implied that flubendiamide exposure may affect liver functions of zebrafish and short-term exposure to flubendiamide could not adversely affect the growth of zebrafish. The liver tissue is the largest gland in vertebrates, and it plays an important role in the metabolism of carbohydrates, fats, proteins, vitamins and hormones (Qiu et al., 2019). Furthermore, the effects of flubendiamide on liver tissue of zebrafish was studied by histopathological analysis. After 14 days of 0.1, 0.5 and 1 mg/L flubendiamide exposure, a series of changes occurred in liver tissue structure of zebrafish including lymphocytic infiltration, hepatocellular vacuolization and cell necrosis. These results further confirmed that flubendiamide can cause liver damage in zebrafish.
A large number of studies have shown that the damage of liver tissue is often accompanied by the occurrence of oxidative stress (Meng et al. 2021, Meng et al. 2019, Wang et al. 2015). Next, we analyzed the impacts of flubendiamide on the activities of SOD, CAT and GPx and the contents of MDA and GSH in liver of zebrafish. The results showed that activity of CAT in liver of zebrafish had changed significantly after flubendiamide exposure. As an important antioxidant enzyme, CAT can efficiently catalyze H₂O₂ into water and oxygen, which has the function of scavenging free radicals and protecting cells from damage in organisms (Zhang et al. 2018).This implied that flubendiamide could destroy the antioxidant enzyme system of zebrafish. In addition, exposure to flubendiamide caused a significant decrease in the content of GSH in the liver of zebrafish. GSH is an important regulatory metabolite in cells. The decrease of content of GSH is a potential early activation signal of apoptosis, and the subsequent generation of oxygen free radicals could promote cell apoptosis (Meng et al. 2019). Importantly, as a product of lipid peroxidation, the content of MDA is usually used to reflect the degree of oxidative damage (Gupta et al. 2009).In our study, the content of MDA in liver of zebrafish had increased significantly with flubendiamide exposure. It is indicated that the oxidative stress in liver of zebrafish induced by flubendiamide. In addition, the increase in the content of MDA may severely damage cell membranes (Teng et al. 2019). Therefore, we observed structural damage to the liver tissue of zebrafish in the flubendiamide treatment groups.
The appearance of oxidative stress and lipid peroxidation may lead to apoptosis has been confirmed (Teng et al. 2019). It was necessary to further explore the effects of flubendiamide on the cell apoptosis in liver tissue of zebrafish. Therefore, the mRNA expression of a series of cell apoptosis related-genes were determined by qPCR analysis. In a series of genes that promote apoptosis, the increased mRNA expression of p53 and puma can induce apoptosis (Wang et al. 2004, Yu &Zhang 2008). In addition, caspase-3 and caspase-9 are the key executive genes in the process of apoptosis (Soengas et al. 1999). However, apaf-1 can activate the mRNA expression of caspase-3 and caspase-9 (Zimmermann et al. 2001). In particular, the mRNA expression levels of p53, puma, caspase-3, caspase-9 and apaf-1 in liver of zebrafish were significantly increased after flubendiamide exposure. Moreover, as the target gene of p53, bax can promote the release of cytochrome C from mitochondria. In addition, bcl-2 plays an important role in inhibiting cell apoptosis. The ratio of expression levels of bcl-2/bax expression has a decisive influence on the release process of cytochrome C in mitochondria (Zimmermann et al. 2001). Therefore, the decrease of the ratio of expression levels of bcl-2/bax can be used as a sign of the occurrence of apoptosis process (Whiteman et al. 2007). Consistently, the ratios of expression levels of bcl-2/bax in liver of zebrafish were significantly decreased in the flubendiamide treatment groups. This resulted suggested that exposure to flubendiamide could induce apoptosis in liver of zebrafish. In conclusion, our results from this study showed that flubendiamide may cause liver damage of zebrafish by inducing oxidative stress and cell apoptosis.