Developing fish are one of the most sensitive organisms to TCDD and other DLCs (Teraoka et al., 2003). The CYPs play a crucial role in the metabolism and degradation of hazardous compounds (Guengerich, 2017; Loerracher et al., 2020). Among the CYPs, cyp1a is the best characterized and is the strongest response in TCDD bio-activation in fish (Chuang et al., 2004; Jonsson et al., 2009; Chen & Chan, 2018). As a persistent organic pollutant, TCDD is very difficult to be degraded and metabolized in organisms (Sorg et al., 2009; Sorg, 2014). Due to its persistent existence in organisms, it can constantly induce the expression of AhR targeting gene cyp1a (Roy et al., 2011; Hervé et al., 2010; Bock, 2018; Roy et al., 2018). CYP1A protein is first detected at 36 hpf zebrafish larvae after exposure to TCDD, and constitutive expression of CYP1A is demonstrated in the 120 hpf larval zebrafish (Andreasen et al., 2002; Otte et al., 2010). Many studies have shown that low concentration of TCDD could strongly induce the expression of cyp1a in many tissues of zebrafish, especially in the liver and gut (Kim et al., 2013; Shen et al., 2018; Xie et al., 2018). Similarly, we found that 1 ng/L TCDD exposure could dramatically induce the expression of cyp1a by a constant increasing pattern in zebrafish larvae. These results further indicate the persistence of TCDD in organisms.
CYP1A is commonly associated with the biotransformation of environmental contaminants, especially the DLCs (Cardoso et al., 2019; Licata et al., 2019). Many DLCs including pesticides belong to the substrates of CYP1A enzyme biotransformation, which are usually converted to more hydrophilic metabolites for excretion (Roy et al., 2019). Besides, CYP1A induction can also produce toxic effects by promoting the production of active metabolites (Voelker et al., 2008). We found that mepanipyrim and cyprodinil exposure could induce the expression of cyp1a by a dynamic pattern in different developmental stages of zebrafish. Meanwhile, the potential metabolites of mepanipyrim and cyprodinil also have AhR agonistic activity, indicating the induction of cyp1a expression could also be mediated by their metabolites.
As a target gene of AhR, the change of cyp1a expression is closely related to the successful entry of AhR into nucleus and the formation of AhR/ARNT dimer acting on dioxin-responsive elements (Denison et al., 2017; Vogel et al., 2020). AhR located in the cytoplasm remains inactive before binding ligands (Reynaud & Deschaux, 2006; AnvariFar et al., 2018). The storage of AhR in the cytoplasm always maintains a dynamic balance (Bell & Poland, 2000; Mejía-García, et al., 2015). It can play a nuclear receptor role only after ligand binding or activation by phosphorylation (Guyot et al., 2013). Many studies have reported that DLCs can strongly induce the expression of ahr2 (Guo et al., 2017; Zhou et al., 2020). The increased mRNA expression of ahr2 have become an important toxic indicator of DLCs (Cheng et al., 2020). In the present study, we found that the mRNA expression of ahr2 also showed a dynamic change by mepanipyrim or cyprodinil exposure, and was consistent with the expression of cyp1a in zebrafish. The continuous activation of AhR signaling pathway is a crucial condition to ensure that mepanipyrim and cyprodinil can be effectively degraded and metabolized (Hale et al., 2017).
Over-expression of CYP1A is involved in production of reactive oxygen species (ROS) (Zangar et al., 2004). Previous study found that TCDD could induce ROS production by up-regulating the expression of CYP1A, which could further cause damage to DNA (Kopf & Walker, 2010; Kalaiselvan et al., 2016; Liu et al., 2019). Besides, pesticides exposure can result in the transformation of toxic substances into ROS, which can ultimately cause cell injury (Singh et al., 2018; Shen et al., 2021b). In the present study, we found that the mRNA expression of cyp1a in zebrafish larvae was significantly increased by mepanipyrim or cyprodinil exposure, indicating that mepanipyrim and cyprodinil may increase the level of ROS. Under normal physiological conditions, the production of ROS can promote the growth and development of cells or organisms. However, excessive production of ROS can cause great damage to cell components, such as biomembrane and cell membrane, which will seriously affect the “dynamic balance” of cells or organisms (Suzuki et al., 2012; Bhagat et al., 2020).
Because of the short half-life, the pesticides exhibit a short term toxic effects after application and/or exposure (Korkmaz et al., 2018). During pregnancy, pesticides have the potential to pass through the placental barrier and disturb the fetal development (Dewailly et al., 2014). In addition, pesticides are likely to accumulate in the organisms in early embryonic developmental stages (Gavelle et al., 2016; Kuang et al., 2020). Our results showed that mepanipyrim and cyprodinil tended to accumulate in zebrafish during early developmental stages. With the development of zebrafish, mepanipyrim and cyprodinil began to be degraded and metabolized. These results implied that organisms were sensitive to pesticides in early life, and pesticides were more likely to accumulate in early life stages of organisms. At the same time, pesticides exposure in early life may affect the normal development of organisms. Therefore, the risk of exposure to pesticides in embryo stage is huge, and should be paid more attention. Furthermore, pregnant women, in particular, should avoid to be exposed to pesticides, such as the diets with pesticide residues during pregnancy (Dewailly et al., 2014).
The induction of EROD activity is often used to assess the AhR agonistic activity of dioxins and DLCs in organisms (Eichbaum et al., 2014; Kais et al., 2017; Ji et al., 2021). TCDD can dramatically increase the EROD activity in the liver, kidney, and gills of seabream, Sparus aurata (Ortiz-Delgado et al., 2008). Basal EROD enzymatic activity is detected in 6 hpf zebrafish embryos, after then, it has constantly increased by a slow pattern (Meyer-Alert et al., 2018; Meyer-Alert et al., 2019). Previously, Xu et al. (2018) found that TCDD exposure could cause prominent elevation of EROD activity in larval zebrafish. In the present study, our results showed that 1 ng/L TCDD exposure could dramatically increase the EROD activity in different development stages of zebrafish. It was consistent with the increase of cyp1a mRNA expression induced by TCDD. At the same time, we found that mepanipyrim and cyprodinil exposure could increase the EROD activity with a dynamic pattern in different developmental stages of zebrafish. It also implied that the potential metabolites of mepanipyrim and cyprodinil could increase the EROD activity in zebrafish larvae.
Unlike persistent organic pollutants, pesticides are easier to be degraded and metabolized (Velki et al., 2017). But their metabolites may be difficult to be degraded and could accumulate in the organism for a long time. Our study found that embryonic exposure to diuron for 7 days, diuron could quickly be degraded into its two major metabolites N-demethyldiuron (DCPMU) and N-didemethyldiuron (DCPM) in zebrafish larvae. Meanwhile, DCPMU and DCPU could gradually accumulate in the larvae with the extension of exposure time (unpublished data). In the present study, we found that mepanipyrim and cyprodinil could also be quickly degraded, and their residues in larvae decreased with the exposure time. Therefore, we speculated that their potential metabolites may accumulate in the zebrafish larvae. Previously, it has been reported that mepanipyrim and cyprodinil exhibit a very strong AhR agonistic activity and can dramatically induce the expression of AhR-regulated genes (Tang et al., 2020; Zhu et al., 2021). Presently, we found that mepanipyrim or cyprodinil had strong binding with AhR, even stronger than TCDD. Besides, their metabolites also had strong AhR agonistic activity and showed strong AhR binding ability. The residues of mepanipyrim or cyprodinil in 5 dpf zebrafish larvae were very low, but the cyp1a expression and EROD activity did not return to normal level, indicating that their metabolites could accumulate in larvae. This is very consistent with our hypothesis. Unfortunately, there are no commercial metabolites available in the market, so it is impossible to carry out substantive verification. To sum up, a direct temporal relation between CYP1A or EROD induction and mepanipyrim or cyprodinil exposure is still vaguely indicated by the present results and further research is required.