(−)‐Epicatechin gallate ameliorates cyprodinil‐induced cardiac developmental defects through inhibiting aryl hydrocarbon receptor in zebrafish

Cyprodinil is a widely used fungicide with broad‐spectrum activity, but it has been associated with cardiac abnormalities. (−)‐Epicatechin gallate (ECG), a natural polyphenolic compound, has been shown to possess protective properties in cardiac development.


| INTRODUCTION
Congenital heart disease (CHD) is the most prevalent type of anatomical malformation present at birth, affecting 0.8%-1% of live-born infants, and its global prevalence is on the rise (Liu et al., 2019).Despite its high frequency, the underlying causes of CHD remain largely unknown (Huhta & Linask, 2013).While genetic factors contribute to approximately 15% of CHD cases, there is increasing recognition of the environmental factors that may play a significant role, with up to 30% of cases linked to environmental influences, including high temperatures, radiation, noise, medications, as well as biological factors such as viruses, bacteria, and parasites (Liang et al., 2017;Liu et al., 2013).One of the environmental pollutants associated with the factors mentioned above is cyprodinil, a pyrimidine amine pesticide widely used in the cultivation of vegetables, fruits, and rice.Its widespread use is driven by its effectiveness in combating gray mold in agricultural settings (Chen et al., 2016).Notably, cyprodinil has been detected in aquatic environments at concentrations ranging from 0.73 to 2.89 μg/L, indicating its potential for environmental exposure (Tang et al., 2020).Increasing evidence suggests that cyprodinil, acting as an agonist for the aryl hydrocarbon receptor (AhR), exerts detrimental effects on cardiac development, raising concerns about potential cardiac developmental injuries associated with cyprodinil exposure (Fang et al., 2013).This highlights the significance of understanding the impact of cyprodinil on cardiac development.
However, the current approach to treating pesticideinduced cardiac damage has certain limitations.For instance, the management of cardiotoxicity resulting from organophosphorus and carbamate pesticide poisoning primarily involves maintaining vital signs, including mechanical ventilation, decontamination procedures to prevent further uptake, and the administration of antinicotinic and muscarinic agents.However, there is a notable absence of a specific treatment protocol for toxicity caused by organochlorine pesticides, glyphosate herbicides, or bipyridyl herbicides.Consequently, the management of poisoning caused by these substances is largely restricted to decontamination measures, aggressive symptomatic care, and supportive treatment, as no known antidote has been identified (Anakwue, 2019).Therefore, the search for a natural product that can effectively mitigate pesticide-induced cardiotoxicity is of immense significance and warrants further research.
(À)-Epicatechin-3-gallate (ECG), a flavonoid organic compound found in green tea (Camellia sinensis), has attracted significant attention for its potential in addressing various health issues.Flavonoids, as a common and diverse group of polyphenols, possess strong antioxidant properties due to the presence of numerous hydroxyl groups in their molecules (Cardoso et al., 2020;Steinmann et al., 2013).Unfermented green tea is the richest source of catechins, although they are also naturally occurring in black tea, coffee, berries, grapes, and wine.Research has uncovered diverse biological effects of ECG, including antioxidant, anti-inflammatory, antiviral, anti-bacterial, anti-aging, and hypotensive properties (Ahmad & Makhtar, 1999;Beltz et al., 2006;Jigisha et al., 2012;Leung et al., 2001;Zhang et al., 2018).ECG has also shown promise in cancer prevention, with potential benefits for lung, esophagus, stomach, intestinal, pancreatic, breast, prostate, and bladder cancers (Bernatoniene & Kopustinskiene, 2018;Gupta et al., 2014;Masek et al., 2017;Singh et al., 2011).Notably, ECG has been extensively researched for its protective effects on the heart.It has demonstrated the ability to protect against ischemia/reperfusion (I/R) injury by modulating Na/K-ATPase/Src receptor function (Qi et al., 2019).Furthermore, ECG has also been reported to protect against heart toxicities caused by doxorubicin and nitric oxide, as well as in the context of coronary heart disease and post-myocardial infarction (Cai et al., 2015;Mukhtar & Ahmad, 2000;Paquay et al., 2000;Ravindran et al., 2022).While the cardioprotective effects of ECG have been acknowledged, a comprehensive understanding of the underlying mechanisms is yet to be fully elucidated.
Zebrafish (Danio rerio) is a non-mammalian vertebrate model that has gained prominence in developmental genetics, functional genomics, and cardiac morphology-function studies (Coppola et al., 2023;Genge et al., 2016).This is attributed to the transparency of their larval stages, which allows for the easy observation of critical developmental milestones and direct examination of the heart (Chang et al., 2023).One key advantage of using zebrafish as a research model is the similarity between their genome and that of humans (Bakkers, 2011).Additionally, zebrafish have a high reproductive capacity, making them cost-effective to maintain.Importantly, zebrafish share essential similarities with humans in terms of cardiac physiology, including fundamental contractile dynamics.As a result, zebrafish have emerged as a robust model for investigating channelopathies and cardiomyopathies, offering valuable insights into cardiac function (Bakkers, 2011).
In this study, we aimed to investigate the potential protective role of ECG against the detrimental effects of cyprodinil on zebrafish development and to elucidate the underlying mechanistic pathways involved.Zebrafish embryos were exposed to cyprodinil with and without ECG, and our results revealed a significant mitigation of cyprodinil-induced developmental dysfunction in zebrafish embryos in the presence of ECG.Notably, ECG exerted a strong influence on various developmental parameters, including embryonic movement, hatching rate, the percentage of larvae with pericardial anomaly, and tachycardia, acting as a potent protective agent against the adverse effects of cyprodinil.Furthermore, our study explored the molecular mechanisms underlying these protective effects.We found that ECG effectively counteracted cyprodinil-induced alterations in mRNA expression, particularly those involved in cardiac development and calcium channels.Intriguingly, our research also uncovered the potential inhibitory effect of ECG on AhR signaling pathways, which were upregulated by cyprodinil.This work not only demonstrated the protective potential of ECG against cyprodinil but also provided insights into a novel approach for enhancing the regulation and safe use of this chemical compound.By harnessing the synergistic benefits of ECG, a natural small molecule, we propose an innovative strategy with promising implications for promoting the safe use of cyprodinil.

| Chemicals and reagents
Cyprodinil was obtained from Dr. Ehrenstorfer (Augsburg, Germany).ECG and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA).All other chemicals and reagents used in this study were purchased from reputable manufacturers.

| Zebrafish maintenance and embryo collection
Adult zebrafish (Danio rerio) of the TU strain were bred and maintained in our laboratory according to standard protocols.Zebrafish were housed in a recirculating aquatic system at a constant temperature of 28 C with a 14-h light/10-h dark cycle.The fish were fed twice daily with live brine shrimp.To obtain zebrafish embryos, adult zebrafish were transferred to breeding tanks with a ratio of one male to two females.The tanks were equipped with plastic mesh dividers to prevent cannibalism and to facilitate controlled breeding.The breeding tanks were checked every morning, and if spawning was observed, the eggs were collected immediately.To collect the embryos, a glass Petri dish filled with embryo medium was placed under the breeding tank and gently tapped to release the eggs.The embryos were then carefully transferred to a fresh Petri dish containing embryo medium using a wide-bore glass pipette.Embryo medium was prepared by dissolving Instant Ocean sea salt (Aquarium Systems) in distilled water.The salt concentration was adjusted to 60 mg/L.The pH of the medium was maintained at 7.2-7.4using sodium bicarbonate.The medium was filtered through a 0.22 μm filter and stored at 4 C until use.

| Zebrafish embryo exposure
A stock solution of cyprodinil was prepared by dissolving 10 mg of cyprodinil powder in 20 mL of DMSO.The solution was vortexed thoroughly to ensure complete dissolution.The cyprodinil stock solution had a final concentration of 500 μg/mL.A stock solution of ECG was prepared by dissolving 44.2 mg of ECG powder in 100 mL of distilled water.The solution was sonicated for 10 min to facilitate dissolution.The ECG stock solution had a final concentration of 442 μg/mL (1 mM).Zebrafish embryos collected within 1 h post fertilization (hpf) were randomly divided into experimental groups with 6 replicates per group.Each group was exposed to cyprodinil and/or ECG, while the solvent control group was treated with an equal volume of DMSO (0.01%) diluted in embryo medium.The embryos were placed in glass Petri dishes containing 10 mL of the respective exposure solution, ensuring that the embryos were fully submerged.After the 72-h exposure period, the zebrafish embryos were carefully collected.The embryos were observed and imaged using a stereomicroscope (Nikon, Tokyo, Japan) at predetermined time points to assess heart development and any associated defects.Images were captured using a digital camera attached to the microscope.Subsequent analysis and quantification of heart defects were performed using the ImageJ software.

| Assessment of cardiac malformations and function
Zebrafish larval cardiac morphology, particularly the percentage of larvae with pericardial anomaly, was assessed at 72 h post-exposure.The pericardium is a transparent sac surrounding the heart, and its abnormality indicates the presence of cardiac developmental defects.The percentage of larvae with pericardial anomaly was qualitatively evaluated by observing the larvae under a stereomicroscope and comparing them to control larvae.Zebrafish larval cardiac function was assessed using several parameters, including heart rate (HR), enddiastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and cardiac output (CO).These parameters provide insights into the overall efficiency and performance of the zebrafish larval heart.The percentage of larvae with pericardial anomaly was calculated by the number of pericardial edema zebrafish/ the number of survival zebrafish * 100.HR was determined by counting the number of consecutive cardiac cycles within a defined time period, usually 30 s. EDV represents the maximum end-diastolic volume, which we measure when the zebrafish heart is at maximal diastole.ESV, on the other hand, denotes the minimum end-systolic volume, which we measure when the zebrafish heart contracts to its minimal volume.SV was calculated as the difference between the EDV and the ESV.SV represents the volume of blood ejected by the heart with each contraction.CO was calculated as the product of SV and HR, CO represents the volume of blood pumped by the heart per unit of time and provides a comprehensive measure of cardiac performance.

| Real-time quantitative PCR (qPCR)
We employed TRIzol reagent (TaKaRa, Tokyo, Japan) for the extraction of total RNA from the 72 hpf whole embryos following the manufacturer's protocol.This method effectively minimizes contamination from genomic DNA, thereby yielding high-purity RNA.We extracted RNA from 20 embryos per well, with 4 replicates per group.The concentration and purity of the extracted RNA were determined using a spectrophotometer.Subsequently, cDNA synthesis was performed using a reverse transcription kit, where 1 μg of total RNA was used as the template (TransGen, Beijing, China).The reverse transcription reaction was carried out at a specific temperature and time according to the kit's protocol.Primer sequences for the cardiac development-related genes and the reference gene ( gapdh) were designed based on the zebrafish genome using Primer Premier 5.0 (Primer company, Canada).To minimize amplification of genomic DNA to the greatest extent possible, we designed primers specific to qPCR that span exon-exon junctions.The primer sequences for the tested genes and the reference gene gapdh are listed in Table S1.Real-time quantitative PCR was performed using a Mx3000P Real-Time PCR system (Stratagene, La Jolla, CA, USA) and a SYBR Green-based master mix.Each reaction contained 10 μL of master mix, 1 μL of cDNA template, and 0.5 μL of forward and reverse primers (10 μM each), with nuclease-free water added to reach a final volume of 20 μL.The thermal cycling conditions consisted of an initial denaturation step followed by a specific number of amplification cycles, each consisting of denaturation, annealing, and extension steps.The fluorescence signal was measured at each amplification cycle to determine the amplification efficiency and Ct values.The relative quantification of gene expression was determined using the comparative Ct method (2 ÀΔΔCt method).The expression level of each target gene was normalized to the expression level of the reference gene gapdh.Furthermore, normalization to the DMSO control group was performed for each gene.The fold change in gene expression among experimental groups and the control group was calculated.

| Data processing
Statistical analysis was performed using the GraphPad Prism software.Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test.A p-value less than 0.05 was considered statistically significant.All data were presented as mean ± standard error (SEM).
The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies (Tveden-Nyborg et al., 2023).

| ECG alleviated cyprodinil-induced developmental toxicity in zebrafish larvae
In this study, we utilized cyprodinil as a positive drug to induce heart injury in zebrafish and investigated the potential protective effect of ECG on cardiac function.To determine the appropriate concentration of cyprodinil, we referred to a previous study conducted by our laboratory (Tang et al., 2020).The results showed that exposure to cyprodinil at concentrations of 250 and 500 μg/L significantly decreased the survival rate of zebrafish embryos (Figure 1a).The larval survival rate (%) decreased from 90.56 ± 2.34 in the control group to 83. 83 ± 2.11, 80.00 ± 2.43, 60.56 ± 3.15, and 31.11 ± 2.22 after exposure to 10, 100, 250, and 500 μg/L cyprodinil, respectively.Additionally, ECG at all tested concentrations (1, 10, 100 μM) exhibited a detectable effect on embryo survival rate (Figure 1b).The larval survival rate (%) decreased from 89.44 ± 2.91 in the control group to 31.11 ± 3.30 after treatment with 500 μg/L cyprodinil, but recovered to 70.00 ± 2.98, 72.78 ± 3.89, and 74.44 ± 3.82 after exposure to 1, 10, and 100 μM ECG, respectively.Based on these results, we selected the concentration of 500 μg/L cyprodinil and 1 μM ECG (442 μg/L) for subsequent experiments.
To evaluate the effects of cyprodinil and ECG on zebrafish embryo development, we examined the movement and hatching rate of exposed embryos.As shown in Figure 1c, cyprodinil decreased the movement of embryos (times per minute) from 4.18 ± 0.92 in the control group to 2.44 ± 0.38, while treatment with ECG rescued this effect, resulting in a movement rate of 3.98 ± 0.79 after exposure to cyprodinil plus ECG.The hatching rate was not significantly different among the groups at 48 hpf, but it was significantly decreased in the cyprodinil group at 60 and 72 hpf.However, the addition of ECG mitigated this decrease in hatching rate over time.Given the widespread use of cyprodinil and its potential harmful effects on aquatic organisms (Go et al., 2015;Kim et al., 2018), our findings suggest that cyprodinil inhibition suppresses embryonic movements and hatching in zebrafish, and ECG treatment would rescue this effect.

| ECG ameliorated cyprodinilinduced cardiac development defects in zebrafish larvae
Previous studies have shown that cyprodinil can induce heart injury in zebrafish by activating the aryl hydrocarbon receptor (AhR) (Shen et al., 2023).In this study, we investigated the cardiac developmental toxicity of zebrafish embryos and observed that ECG could rescue The percentage of larvae with pericardial anomaly caused by cyprodinil (Figure 2a).The percentage of larvae with pericardial anomaly in the control group was 1.19 ± 0.75%, while in the ECG group it was 2.30 ± 1.13%.However, in the cyprodinil group, The percentage of larvae with pericardial anomaly increased significantly to 26.22 ± 5.95%, but this was reduced to 5.62 ± 2.24% in the cyprodinil plus ECG group (Figure 2b).Furthermore, we assessed the HR of zebrafish embryos and found that it was 147.4 ± 1.79 and 145.3 ± 1.70 beats per minute in the control and ECG groups, respectively.However, exposure to cyprodinil significantly increased the HR to 196.1 ± 2.55 bpm, which was then reduced to 149.5 ± 1.59 bpm in the cyprodinil plus ECG group (Figure 2c).These results indicate that cyprodinil disrupts the cardiac rhythm of zebrafish, but this effect can be mitigated by ECG.We further examined indicators of cardiac systole and diastole, including EDV, ESV, SV, and CO (Figure 2d-g).There were no meaningful alterations in either EDV or ESV among the different groups.However, SV showed a decreasing trend after cyprodinil treatment, and this decrease was significantly rescued in the cyprodinil plus ECG group (Figure 2f).On the other hand, CO did not show significant changes among all the groups (Figure 2g).In conclusion, our findings indicate that cyprodinil induces cardiac dysplasia in zebrafish embryos, and ECG partially alleviates these variations.Overall, the cardiac function of larvae was adversely affected by cyprodinil, but co-treatment with ECG provided protection against alterations in heart function.

| ECG recovered the low transcript of genes related to heart development caused by cyprodinil
In our pursuit to unravel the molecular mechanisms underlying the cardiac malformations induced by cyprodinil, we conducted a series of investigation to shed light on the potential of ECG in ameliorating these effects.We focused on examining the transcriptional patterns of key genes associated with cardiac development in zebrafish larvae at 72 hpf, both in the absence and presence of ECG intervention.Among the critical components of cardiac sarcomeres, cardiac myosin plays a crucial role as a structural entity (Chen et al., 2008).During early stages of development in zebrafish (before 72 hpf), atrial myosin heavy chain (amhc) is specifically expressed in atrial myocardiocytes, while ventricular myosin heavy chain (vmhc) is specifically expressed in ventricular myocardiocytes (Li et al., 2017;Yelon et al., 1999).As shown in Figure 3, the expression level of amhc was significantly decreased to 0.48-fold in the cyprodinil group, but increased to 1.02-fold in the cyprodinil plus ECG group.Similarly, the transcript level of vmhc exhibited a downward trend in the cyprodinil-treated groups, reaching 0.34-fold, but increased to 1.19-fold in the cyprodinil plus ECG group.These findings suggest that ECG can rescue the cardiac atrium and ventricle developmental abnormalities induced by cyprodinil in zebrafish.
Gata4 and tbx5 are essential cardiac transcription factors (Bruneau et al., 1999;Kuo et al., 1997).Previous studies have demonstrated that tbx5 and gata4 cooperatively interact on DNA to regulate heart development (Gonzalez-Teran et al., 2022).In our study, the mRNA levels of gata4 were 0.95-fold, 0.51-fold, and 0.87-fold change in the ECG, cyprodinil, and cyprodinil plus ECG groups, respectively.Similarly, the tbx5 transcript levels were 1.08-fold, 0.55-fold, and 1.09-fold change.Considering the importance of gata4 and tbx5 in cardiac development, the lower transcript levels of these genes may contribute to tachycardia and a decrease in stroke volume (SV).Gata4, tbx5, and nkx2.5, which plays a pivotal role in cardiomyocyte differentiation, collectively orchestrate the complex events that govern cardiac development (de Sena-Tom as et al., 2022).Gata4 also interacts with nkx2.5, participating in maintenance of cardiac function and structure (Zhou et al., 2022).We found that the expression level of nkx2.5 was significantly decreased to 0.55-fold in the cyprodinil group, but increased to 1.10-fold in the cyprodinil plus ECG group.This suggests that nkx2.5 plays a crucial role as a cofactor, working in conjunction with tbx5 and gata4, to intricately modulate the transcriptional machinery responsible for downstream gene regulation.These findings make efforts for the development of new therapeutic approaches aimed at targeting these key factors and improving cardiac health.

| ECG alleviated the low expression of cardiac calcium channels-related genes caused by cyprodinil
Intrigued by the intricate interplay of molecular mechanisms in cardiac development, we sought to explore the effects of cyprodinil and ECG on cardiac calcium channels-related genes in zebrafish.Previous studies have highlighted the crucial roles of tbx5 and gata4 in orchestrating functional gene induction, including the renowned ryr2a (Zhou et al., 2012).At the core of cardiac excitation-contraction coupling lies the type 2 ryanodine receptor (Ryr2), a pivotal Ca 2+ release channel essential for proper cardiac function (Bers, 2002).Calcium ions play a fundamental role in the diastolic and systolic processes of the heart, ensuring normal cardiac morphology and function (Guo et al., 2023).Through meticulous scrutiny of expression levels, we uncovered intriguing findings (Figure 4) suggesting that cyprodinil and/or ECG exert their influence on cardiac development through mechanisms distinct from ryr2.Within this intricate web of molecular interactions, another key player emerged: ncx1, which encodes the exchanger 1 (Ncx1) protein.
Ncx1 assumes a pivotal role in the early stages of cardiac growth, serving as a beacon of hope and a marker of nascent cardiac development (Menick et al., 2007).Our findings revealed that the expression of ncx1h was significantly downregulated to 0.39-fold in the cyprodinil group, but increased to 0.80-fold in the cyprodinil plus ECG group.Similarly, the transcript level of cacna1aa, a gene involved in L-type voltage-dependent calcium channels, was lower in the cyprodinil-treated group (0.62-fold), but increased to 1.02-fold in the cyprodinil plus ECG group.Cadherin2, encoded by cdh2, is a crucial cell-adhesion molecule involved in zebrafish cardiovascular development.The previous studies showed that N-cadherin (Cdh2) as a mechanism underlying the formation of cell-cell junctions between trabecular and compact layer cardiomyocytes (Cherian et al., 2016).During development, the heart undergoes a series of morphological changes to maximize its function, with trabeculation being one of the key processes.Trabeculae initially appear as myocardial ridges in the outer curvature of the ventricle and are crucial for cardiac function (Sedmera et al., 2003).Disruption of cadherin2 function can impair myocardiocyte differentiation and physiological cardiovascular performance (Bagatto et al., 2006).In our study, the transcript level of cdh2 showed an increase in the cyprodinil-treated group (2.15-fold), but decreased to 0.89-fold in the cyprodinil plus ECG group.These results suggest that cyprodinil may lead to a decrease in the influx of calcium ions from extracellular mediators into the cytoplasmic matrix, while ECG treatment can mitigate this effect (Lin et al., 2023).The restoration of gene expression levels in the cyprodinil plus ECG group suggests that ECG treatment can alleviate the adverse effects of cyprodinil on calcium channels-related genes, potentially restoring calcium homeostasis and maintaining normal cardiac function.
ATP can activate calcium ion-dependent signals that are responsible for heart failure (Lopaschuk et al., 2021).Previous studies have reported that ECG can inhibit the activity of ATP-sensitive potassium (K ATP ) channels at micromolar concentrations (Jin et al., 2007).To further explore the effects of cyprodinil and ECG on ATP-related genes, we examined the transcript levels of atp2a2a.Our study revealed that the expression of atp2a2a was 0.91-fold in the ECG group, 0.55-fold in the cyprodinil group, and 0.98-fold in the cyprodinil plus ECG group.These findings indicate that cyprodinil treatment resulted in a decrease in the expression of atp2a2a, while ECG treatment partially restored its expression.The dysregulation of atp2a2a suggests potential disruptions in ATPdependent calcium signaling, which could contribute to cardiac dysfunction.The restoration of gene expression levels by ECG treatment suggests its potential in mitigating the adverse effects of cyprodinil on ATP-dependent calcium signaling and cardiac function.

| ECG repaired cyprodinil-induced over-expression of AhR and its downstream genes
In addition to its protective effects against cyprodinil, our study investigated whether ECG could act as an antagonist to the AhR pathway, considering its potential role in mitigating the toxic effects induced by cyprodinil.In zebrafish, three genes of the ahr family are expressed: ahr1a1, ahr1b1, and ahr2, with ahr2 being considered the primary receptor (Mi et al., 2023).Our findings revealed a significant upregulation of ahr2 mRNA expression by 3.23-fold after cyprodinil treatment.However, when cyprodinil was administered in combination with ECG, the upregulation of ahr2 expression was reduced to 0.95-fold (Figure 5).These results suggest that ECG can repair the cyprodinil-induced over-expression of AhR and its downstream genes, potentially modulating the AhR pathway and mitigating the adverse effects of cyprodinil.
In the battle against environmental contaminants, cells employ metabolic enzymes as their secret weapon in a two-phase detoxification process known as phase I and phase II (Badal & Delgoda, 2014).Leading the charge in phase I is the mighty Cytochrome P450 (CYP) family, an enzyme involved in metabolism that determines the rate at which pollutants are metabolized (Burkina et al., 2018).Guiding the regulation of the esteemed CYP1 family's metabolic enzyme expression is the renowned AhR, which orchestrates cellular detoxification (Kyoreva et al., 2021).Exogenous ligands of AhR, such as dioxins, unleash reactive oxygen species (ROS) during catabolism, setting off a cascade of detrimental effects (Sorg, 2014).Amidst this chaos, a glimmer of hope emerges.cyp1a1 plays the role of AhR target gene.As cyp1a1 expression increases, the toxicity induced by environmental contaminants may be tamed, offering a shield against their ravages.In our study, we sought to unravel the intricate dance between cyprodinil, ECG, and the enigmatic cyp1a1.We discovered that cyprodinil alone significantly upregulated the expression of cyp1a1 mRNA by an impressive 2.48-fold, a figure that diminished to 1.08-fold when cyprodinil was administered alongside ECG.However, a limitation of this study is the lack of protein-level validation due to the absence of zebrafish antibodies, which also offers a new direction for our future investigation into the rescue of cyprodinil-induced zebrafish cardiac developmental toxicity through AhR activation by ECG.These results suggest that ECG is likely to rescue the zebrafish cardiac developmental abnormalities induced by cyprodinil by inhibiting the AhR receptor signaling pathway.

| CONCLUSIONS
In conclusion, our study provides further evidence supporting the activation of the AhR by cyprodinil and confirms its detrimental effects on zebrafish development.These effects include impairments in embryonic movement, hatching rate, percentage of larvae with pericardial anomaly, and tachycardia.Additionally, we observed significant alterations in genes associated with cardiac development, such as amhc, vmhc, tbx5, and gata4.Furthermore, changes were observed in genes related to cardiac calcium channels, including ncx1h, atp2a2a, and cdh2.The variations observed in ahr2 and cyp1a1 suggest a potential mechanism underlying the adverse effects of cyprodinil on cardiac development and function.Importantly, our study demonstrates that the administration of ECG was able to rescue all the observed changes by suppressing the activity of cyprodinil through AhR.This finding suggests a novel approach for regulating and safely utilizing cyprodinil in combination with ECG, which could have positive implications for organisms affected by pesticide exposure.
To conclude, our findings highlight the importance of understanding the molecular mechanisms underlying the developmental toxicity of cyprodinil and provide insights into potential strategies for mitigating its adverse effects.Further studies are warranted to explore the deeper mechanisms of ECG as a protective agent against cyprodinil-induced cardiac abnormalities and to investigate its efficacy in other organisms exposed to similar pesticides.

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I G U R E 1 Influence of zebrafish embryo development after treatment with cyprodinil and ECG.(a) Survival rate of zebrafish embryos at 72 hpf treated with different concentration of cyprodinil.(b) Survival rate of zebrafish embryos at 72 hpf treated with ECG at different concentration of 1, 10, 100 μM, respectively.The dose of cyprodinil was 500 μg/L.(c) The embryonic movement at 24 hpf.(d) The hatching rate at 48, 60, and 72 hpf.The data are presented as mean ± SEM (n = 6).Distinct letters above the bar suggest statistical significance.

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I G U R E 2 Cardiac structure-function in zebrafish after treatment with cyprodinil and ECG after 72 h.(a) Represent images of zebrafish embryos at 72 hpf.Arrows: Pericardial normal morphology or anomaly.Scale bar = 500 μm.(b) The percentage of larvae with pericardial anomaly.(c) The heart rate.(d) EDV.(e) ESV.(f) SV.(g) CO.The data are presented as mean ± SEM (n = 6).Distinct letters on the bar indicate statistical significance.

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I G U R E 3 The mRNA expression of cardiac development-related genes in zebrafish exposed to cyprodinil and ECG.The data are presented as mean ± SEM (n = 4).Distinct letters on the bar indicate statistical significance.

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I G U R E 4 The mRNA expression of cardiac calcium channels-related genes in zebrafish exposed to cyprodinil and ECG.The data are presented as mean ± SEM (n = 4).Distinct letters on the bar indicate statistical significance.F I G U R E 5 Alteration of AhR pathway of zebrafish administration with cyprodinil and ECG after 72 h.The transcript of ahr2 and cyp1a1 of zebrafish exposed to cyprodinil and ECG.The data are presented as mean ± SEM (n = 4).Distinct letters on the bar indicate statistical significance.