Mepanipyrim and cyprodinil induce divergent temporal patterns of AhR-mediated responses in zebrafish (Danio rerio) embryos and larvae


 Mepanipyrim and cyprodinil are widely used to control and/or prevent fungal diseases in fruit culture. They are widely detected in aquatic environment and numerous food commodities including fruit and fruit products. Different from TCDD, mepanipyrim and cyprodinil are more easily degraded and metabolized in the environment. However, the in vivo analysis of their metabolic dynamics is unclear and need to be further confirmed. In this study, zebrafish embryos were constantly exposed to 100 µg/L mepanipyrim or cyprodinil for 7 days. The temporal pattern of CYP1A and AhR2 expression and EROD enzyme activity at different time frames during embryonic and larval development of zebrafish were investigated. Our results showed that mepanipyrim and cyprodinil tend to accumulate in zebrafish during early embryonic developmental stages. Meanwhile, mepanipyrim and cyprodinil exposure could increase the expression level of cyp1a and ahr2 genes and EROD activity by a dynamic pattern in different developmental stages of zebrafish. Besides, their metabolites, which may accumulate in the zebrafish larvae, have strong AhR agonistic activity and showed strong AhR binding ability. Importantly, the risk of exposure to pesticides in embryo stage is huge, and should be paid more attention.


Introduction
Pesticides are proverbially used in agriculture to control insect pests for enhancing the yields and quality . The embryos and larvae of zebra sh, as a valuable models in aquatic ecotoxicology, have gained increasing popularity to assess the effects of various pollutants (Velki et al., 2017;. Besides, zebra sh screenings involving metabolite analysis at the different life stages of zebra sh are also common (Saad et al., 2017;Anselmo et al., 2018). This study was designed to analyze whether the activation of the detoxi cation metabolism through CYP1A could continuously be induced by mepanipyrim and cyprodinil exposure in zebra sh larvae and embryos. For this aim, we have detected the residues of mepanipyrim and cyprodinil in the embryos and larvae of zebra sh. At the same time, the temporal patterns of CYP1A and AhR2 expression and EROD enzyme activity at different time frames during embryonic and larval development of zebra sh were investigated (24, 48, 72, 96, 120, 144, and 168 hours post fertilization (hpf)). In addition, we also predicted the AhR agonistic activity of several possible metabolites of mepanipyrim and cyprodinil by molecular docking method.

Zebra sh maintenance and embryonic exposure
The wild-type TU zebra sh maintenance was based on our previous studies Shen et al., 2021a). The mepanipyrim and cyprodinil were dispersed in DMSO to achieve stock solutions of 1 mg/mL, respectively. TCDD (1 ng/L), mepanipyrim (100 μg/L), and cyprodinil (100 μg/L) exposure solutions were prepared by entering the proper volume of the stock solutions to the zebra sh culture medium. The control group was taken over a coordinative volume of DMSO solvent (0.01%). Eighty embryos (0.5-1.0 hours post-fertilization (hpf)) were cultured in a glass dish containing 40 mL exposure solution (n = 5), which was renewed solutions once daily. The embryos were continuously exposed for 24, 48, 72, 96, 120, 144, and 168 hours to TCDD, mepanipyrim, or cyprodinil.

Residue detection of mepanipyrim and cyprodinil
The actual doses of mepanipyrim and cyprodinil in the exposure solutions were measured according to our previous study . In the residue detection of embryos and larvae, the embryos or larvae were homogenized in 0.2 mL acetonitrile, and then transferred to a 5 mL centrifuge tube with 1 g anhydrous sodium sulfate, 2 ml acetonitrile, and 1 ml acetonitrile-saturated n-hexane. After centrifugation, the acetonitrile layer was extracted and dried with a nitrogen blowing instrument (EYELA MGS-2200, Japan). A solution of 0.1% formic acid-methanol: water (2: 8 v/v) was used for volume determination. The separation was achieved on the chromatographic column (Kinetex 2.6u C18 100A 100*3.0 mm (Phenomenex, USA)), with a gradient elution using mobile phase consisted of ultrapure water (A) and 0.1% formic acid with acetonitrile (B) (S- Table 1 & S-Table 2). The ow rate was 0.25 mL/min. The injection volume was 5 μL. A series of standard concentrations of mepanipyrim and cyprodinil (0.5, 2, 10, 50, 100, and 200 part per billion) were prepared to make the standard curve for quanti cation. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis was performed using Agilent 1290 In nity LC-6490 QQQ (USA).

Real-time quantitative uorescence PCR (RT-qPCR)
Zebra sh embryos and larvae (6 samples/group, 25 embryos or larvae/sample) were mixed for RNA isolation. Total RNA extraction, cDNA synthesis, and RT-qPCR were carried out according to our previous studies (

Measurement of the EROD activity
Zebra sh embryos and larvae (6 samples/group, 25 embryos or larvae/sample) were collected for EROD activity detection. A total volume of 75 μL EROD buffer (3 μL for per embryo or larva) was added to 1.5 mL reaction tube. Next, the samples were dispersed using a dispersing device (TIANGEN Biotech CO., LTD, Beijing) until completely dissociated. The homogenized samples were centrifuged at 3000 rotation per minute (rpm) and 4 ℃ for 15 min. Total protein concentrations of the homogenized samples were analyzed using BCA Protein Assay Kit (EpiZyme, Shanghai, China). A resoru n (Sigma-Aldrich, Germany) standard was used for drawing the sample-speci c standard curves. The supernatant of the homogenized samples were transferred to a white opaque 96-well plate to detect the EROD activity. In detail, 0.6 μM 7-ethoxyresoru n (Sigma-Aldrich, Germany) was added to each well, and was incubated in darkness at room temperature for 10 min. Thereafter, 3.35 mM NAPDH (Sigma-Aldrich, Germany) was added to each well, and was incubated in darkness at room temperature for 15 min. Lastly, 1.35 mM icecold acetonitrile-uorescamine (Sigma-Aldrich, Germany) solution was added to stop the reaction. After 15 min, the uorescence was measured by a POLARstar Omega automatic analysis system (BMG Labtech, Ortenberg, Germany) at 544 nm excitation and 590 nm emission. The EROD activity was calculated as pmol resoru n/min/mg protein.

Molecular docking
To examine the potential AhR activity of pesticides or their metabolites, AutoDock suite of programs was used to implement the molecular docking process. The ligand-binding domain sequence of AhR protein was obtained from UniProt (Entry ID: P35869). The detailed molecular docking process is based on our previous study (Tang et

The residues of mepanipyrim and cyprodinil in zebra sh embryos and larvae
The actual measured concentrations of mepanipyrim and cyprodinil in the exposure solutions were 65.60 ± 11.71 and 62.75 ± 4.94 µg/L, respectively (Table 1). In order to better understand the metabolism of mepanipyrim and cyprodinil in the developmental process of zebra sh embryos and larvae, we detected the residues of mepanipyrim and cyprodinil in different developmental stages of zebra sh. The measured concentrations of mepanipyrim in 24 and 48 hpf embryos of zebra sh were 5.16 ± 0.16 and 5.27 ± 0.88 µg/g, respectively (Fig. 1a, Table 2). However, with the development of embryos, mepanipyrim was gradually degraded and metabolized in larvae. The measured concentrations of mepanipyrim in 72, 96, 120, 144, and 168 hpf larvae of zebra sh were 1.83 ± 0.20, 0.98 ± 0.30, 0.15 ± 0.01, 0.06 ± 0.02, and 0.08 ± 0.01 µg/g, respectively (Fig. 1a, Table 2). Compared with mepanipyrim, cyprodinil was easier to accumulate in zebra sh larvae. The measured concentrations of cyprodinil in 24, 48, and 72 hpf embryos or larvae of zebra sh were 18.71 ± 1.94, 19.56 ± 2.32, and 18.41 ± 0.83 µg/g, respectively (Fig. 1b, Table  2). However, cyprodinil was also gradually degraded and metabolized in larvae after 72 hours. The measured concentrations of cyprodinil in 96, 120, 144, and 168 hpf larvae of zebra sh were 2.40 ± 0.13, 0.15 ± 0.02, 0.36 ± 0.06, and 0.53 ± 0.07 µg/g, respectively (Fig. 1b, Table 2).  3.2. The mRNA expression of cyp1a and ahr2 genes in mepanipyrim or cyprodinil exposed embryos and larvae We next examined the temporal pattern of cyp1a and ahr2 mRNA expression during zebra sh early developmental process. The mRNA expression patterns of cyp1a gene after exposure to mepanipyrim or cyprodinil show differences compared with TCDD exposure. Expression of cyp1a gene in embryos and larvae was dynamically induced by mepanipyrim and cyprodinil (Fig. 2a). However, the expression of cyp1a in embryos and larvae was continuously induced by TCDD. The number of cyp1a transcripts was constantly increased by TCDD exposure and reached about 2000-fold change in 7 dpf larvae compared with 24 hpf solvent control embryos (Fig. 2a). At the same time, the mRNA expression of ahr2 gene was also showed a dynamic induction by mepanipyrim and cyprodinil exposure (Fig. 2b). Nevertheless, the mRNA expression of ahr2 gene was showed a continuous induction by TCDD exposure (Fig. 2b).
3.3. The EROD activities of mepanipyrim or cyprodinil exposed embryos and larvae . To further con rm our results, we detected the EROD activity of mepanipyrim and cyprodinil exposed embryos and larvae. As shown in Fig. 3, the EROD activities of embryos and larvae were dynamically altered by mepanipyrim and cyprodinil exposure, while TCDD exposure lead strong increase in EROD activities.

The AhR agonistic activities of mepanipyrim and cyprodinil metabolites
To detect the AhR agonistic activity of potential metabolites of mepanipyrim and cyprodinil, molecular docking method was used to explore the binding energies between their metabolites and AhR. The smaller binding energies indicating better interaction activity. As shown in Table 3, the binding energy of strong AhR agonist TCDD interacting with AhR was − 8.5 kcal/mol, and the binding energy of mepanipyrim and cyprodinil interacting with AhR were − 9.3 and − 9.5 kcal/mol, respectively. In addition, the binding energy of two potential metabolites (No. 5

Discussion
Developing Our results showed that mepanipyrim and cyprodinil tended to accumulate in zebra sh during early developmental stages. With the development of zebra sh, 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).  2018) found that TCDD exposure could cause prominent elevation of EROD activity in larval zebra sh. In the present study, our results showed that 1 ng/L TCDD exposure could dramatically increase the EROD activity in different development stages of zebra sh. 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 zebra sh. It also implied that the potential metabolites of mepanipyrim and cyprodinil could increase the EROD activity in zebra sh larvae.
Unlike persistent organic pollutants, pesticides are easier to be degraded and metabolized (Velki et al., 2017). But their metabolites may be di cult 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 zebra sh 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 zebra sh 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 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 zebra sh 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 veri cation. 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.

Conclusion
This study was designed to analyze whether the activation of the detoxi cation metabolism through CYP1A could be continuously induced by mepanipyrim and cyprodinil exposure in zebra sh larvae and embryos. Our results showed that mepanipyrim and cyprodinil tended to accumulate in zebra sh during early embryonic developmental stages. With the development of embryos, mepanipyrim and cyprodinil were gradually degraded and metabolized in larvae. Meanwhile, mepanipyrim and cyprodinil exposure could increase the expression of cyp1a and ahr2 genes and EROD activity by a dynamic pattern in different developmental stages of zebra sh. Besides, their metabolites, which may accumulate in the zebra sh larvae, have strong AhR agonistic activity and showed strong AhR binding ability.

Con icts of interest statement
The authors declare that they have no con ict of interest. Availability of data and materials The obtained and analyzed data of this study are available from the corresponding author on reasonable request.  Tables   Due to technical limitations, Table 3 is only available as a download in the Supplemental Files section. Figure 1 Measured concentrations of mepanipyrim (a) and cyprodinil (b) (ng/g ± SD) in zebra sh larvae or embryos (n = 3). Zebra sh embryonic exposure to 100 μg/L mepanipyrim or cyprodinil for 1, 2, 3, 4, 5, 6, and 7 days (D), the residues of mepanipyrim or cyprodinil in different developmental stages of zebra sh were detected. N.D. represents no detection.

Figure 2
The mRNA expression of cyp1a and ahr2 genes in zebra sh embryos and larvae. The expression of cyp1a (a) and ahr2 (b) exhibited dynamic change by 100 μg/L mepanipyrim or cyprodinil exposure, and strongly inducted by 1 ng/L TCDD exposure. The data (mean ± standard errors, n = 6) were analysed using the Duncan's post hoc test after one-way analysis of variance (ANOVA). ***P < 0.001; *0.01 < P < 0.05; n.s. indicates no signi cant difference. D: days.