BML-284

Exposure to Oxadiazon-Butachlor causes cardiac toxicity in zebrafish embryos

Yong Huang, Jinze Ma, Yunlong Meng, You Wei, Shuling Xie, Ping Jiang, Ziqin Wang, Xiaobei Chen, Zehui Liu, Keyuan Zhong, Zigang Cao, Xinjun Liao, Juhua Xiao, Huiqiang Lu
a Center for Drug Screening and Research, School of Geography and Environmental Engineering, Gannan Normal University, Ganzhou, 341000, Jiangxi, China
b College of Chemistry and Chemical Engineering, Gannan Normal University, Ganzhou, 341000, Jiangxi, China
c Jiangxi Engineering Laboratory of Zebrafish Modeling and Drug Screening for Human Diseases, Ji’an, Jiangxi, China
d Jiangxi Key Laboratory of Developmental Biology of Organs, Ji’an, 343009, Jiangxi, China
e Department of Ultrasound, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, 330006, Jiangxi, China

A B S T R A C T
Oxadiazon-Butachlor (OB) is a widely used herbicide for controlling most annual weeds in rice fields. However, its potential toxicity in aquatic organisms has not been evaluated so far. We used the zebrafish embryo model to assess the toxicity of OB, and found that it affected early cardiac development and caused extensive cardiac damage. Mechanistically, OB significantly increased oxidative stress in the embryos by inhibiting antioxidant enzymes that resulted in excessive production of reactive oxygen species (ROS), eventually leading to cardiomyocyte apoptosis. In addition, OB also inhibited the WNT signaling pathway and downregulated its target genes includinglef1, axin2 and b-catenin. Reactivation of this pathway by the Wnt activator BML-284 and the antioxidant astaxanthin rescued the embryos form the cardiotoxic effects of OB, indicating that oxidative stress, and inhibition of WNT target genes are the mechanistic basis of OB-induced damage in zebrafish. Our study shows that OB exposure causes car- diotoxicity in zebrafish embryos and may be potentially toxic to other aquatic life and even humans.

1. Introduction
Herbicides are widely used in agriculture to increase crop yield., as well as in floriculture flower farming, and home gardening. However, herbicides and other pesticides are major pollutants, and enter the food chain through the water, plants and animals, and accumulate, over time with significant environmental and health consequences (Nicolopoulou-Stamati et al., 2016; Pouokam et al., 2017).
Oxadiazon-butachlor (OB), an herbicide commonly used for weed management in rice fields (Yu-cai et al., 2010), is absorbed bythe young shoots and secondary roots of the weeds. Oxadiazon [2- teri-butyl-4-(2,4-dichloro-5-isopropylphenyl)-D2-1,3,4 oxadia- zoun-5-one] inhibits protoporphyrin peroxidase (Krijt et al., 1993; Matringe et al., 1989) in the germinating weed seedlings, which retards bud sheath growth and causes rapid tissue decay. Butachlor [2-Chloro-20,60-diethyl-N-(butoxy methyl) acetanilide] (Abdullah et al., 1997) on the other hand inhibits protein synthesis. While, oxadiazon has low acute toxicity (Von, 1994) and prolonged soilretention (Ambrosi et al., 2002; Bari, 2010), butachlor is a potential carcinogen and highly toxic to aquatic life (Ou et al., 2001). How- ever, little is known regarding the toxicity of oxadiazon or buta- chlor, and the underlying mechanisms, in the early life stages of fish. To this end, we assessed the effects of OB on zebrafish, with especially focusing on cardiotoxicity.
The heart is the first organ to develop and function in zebrafish (Bakkers, 2011). Zebrafish (Danio rerio) is an ideal vertebrate model for cardiovascular research (Gong et al., 2019; Huang et al., 2018c; Kopf and Walker, 2009; Roy et al., 2016) since the morphological,physiological, molecular and pathological characteristics of the zebrafish and human heart are similar (Kimmel et al., 1995; Thisse and Zon, 2002). In addition, the zebrafish’s heart consists of a ventricle and an atrium, and the atrioventricular chambers are similar to a 3-week human embryonic heart (Epstein and Epstein, 2005). It is widely used for detecting environmental toxins and evaluating drug toxicity due to ease of genetic manipulation, transparency, small size, low breeding and maintenance costs, and the ability to survive without active circulation in early develop- ment. The early development marker genes of zebrafish’s heart are vmhc (Gong et al., 2019; Huang et al., 2018c; Park et al., 2009; Targoff et al., 2013; Wang et al., 2019b), nppa (Abraham et al., 2009; Deschepper et al., 2001; Grassini et al., 2018; Houweling et al., 2005), myh6 (Huang et al., 2018c; Singleman and Holtzman, 2012), and gata4 (Gong et al., 2019; Han et al., 2019; Huang et al., 2013; Kim et al., 2003; Park et al., 2009; Qian et al., 2017; Takayasu et al., 2008), nkx2.5 (Burcu et al., 2013; Han et al., 2019; Huang et al., 2018c; Huang et al., 2019; Targoff et al., 2013; Wang et al., 2019b), tbx5 (Sanchez-Iranzo et al., 2018; Zhao et al., 2015), tbx2b (Huang et al., 2018a; Huang et al., 2018b; Huang et al., 2018c) are the transcriptional factors. These have been clearly expressed in zebrafish, which is of great help in studying the toxicological mechanism of zebrafish.
Oxidative stress, a condition wherein production of reactiveoxygen species (ROS) overwhelms the cellular antioxidant mech- anisms, is an important factor leading to aging and disease (Choi et al., 2010). It can be triggered by exposure to toxic chemicals, and causes extensive tissue and organ damage (Craig et al., 2007; Hu et al., 2019; Huang et al., 2018a; Olivari et al., 2009). Several endogenous antioxidants can protect the cells against oxidative damage, including the carotenoid pigment astaxanthin that is present in several aquatic organisms (Fassett and Coombes, 2009; Nakajima et al., 2008; Park et al., 2010; Pashkow et al., 2008; Tripathi and Jena, 2010; Ukibe et al., 2009; Wolf et al., 2009). Additionally, the Wnt/b-catenin signaling pathways protects tissues and organs from oxidative stress-induced apoptosis (Almeida et al., 2011; Tao et al., 2013; Wu et al., 2018; Yang et al., 2013; Zancan et al., 2014).
The classical Wnt signaling pathway is crucial for cardiac development of zebrafish (Hoppler et al., 2014; Marvin et al., 2001; Naito et al., 2010; Ozhan and Weidinger, 2015; Pahnke et al., 2015), and promotes the growth and diversification of cardiac precursors to the right ventricle and indoor muscles (Fu et al., 2007), along with repairing damage to the heart tissues (Deb, 2014; Ozhan and Weidinger, 2015).
In this study, we analyzed the cardiotoxic effects of OB on zebrafish embryos in terms of morphological alterations, heart rate, and changes in oxidative stress and apoptosis related markers during heart development. We detected significant toxicity of OB to zebrafish, which might extend to other aquatic organisms and even humans.

2. Materials and methods
2.1. Experimental reagents and chemicals
Oxadiazon (analytical standard, CAS No. 19666-30-9) and butachlor (analytical standard, CAS No. 23184-66-9) were both purchased from Sigma-Aldrich (St. Louis, USA). Oxadiazon- butachlor was prepared by mixing oxadiazon and butachlor in the ratio of 1:6 according to the proportion of pesticide ingredients. MDA, CAT, SOD, ROS and other assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All qPCR reagents were from Takara (Dalian, China). TUNEL apoptosis detection kit (Alexa Fluor 640) was purchased from YeasenBiotechnology Co. Ltd. (Shanghai, China).

2.2. Fish husbandry and embryo collection
Wild-type (WT) AB and transgenic Tg (myl7: GFP) zebrafish lines were purchased from China Zebrafish Resource Centre, and main- tained in flow-through tanks with aerated freshwater at 28 ± 0.5 ◦C under a 14 h: 10 h light/dark cycle according to the Institutional Animal Care and Committee protocols. The fish were fed with freshly hatched brine shrimp as reported by Lu et al. (Lu et al., 2013). o collect embryos, males and females were introduced in cylinders at the ratio of 1:1 or 1:2, and separated by a partition. The next morning, the barrier was removed and the females started to lay eggs. The embryos were collected within half an hour. After sucking out the dead and unfertilized eggs, feces and other debris, the viable embryos were washed several times with egg water, andthen incubated at 28.5 ◦C for 24 h. Melanin production wasinhibited by adding 1-phenyl-2-thiourea (PTU).

2.3. Embryo acute toxicity test
Based on OECD guidelines, well-developed 5 h post-fertilization (hpf) embryos were selected after examining under the Leica ste- reoscopic microscope and dispensed into 6-well plates at the density of 20 embryos per well. The embryos were treated with 0.5,1.0 and 1.5 mg/L OB solution, with the solvent DMSO as the negative control at 28.5 ◦C for varying durations. The reagents were replenished after 24 h, and the embryos were incubated till 96 hpf.
The number of dead embryos were counted at 24, 48, 72and 96 hpf. The heart-beat of the live embryos was observed under the mi- croscope, and the number of beats per 20s was counted. Each concentration was at least 2/3 of the statistical treatment group, and the experiment was repeated three times.

2.4. Fluorescence microscopy
The differentially treated embryos were anesthetized with 0.16% tricaine at 72 hpf, and fixed in 1% low-melting agarose. The indi- vidual zebrafish were positioned with a spatula such that the eyes, body parts and tail aligned in the same horizontal plane. Images were taken with a fluorescence microscope (Leica M205 FA stereo microscope,Germany).

2.5. HE staining
Five zebrafish from each group were collected at 72hpf washed several times with phosphoric acid buffer, and fixed overnight in 4% paraformaldehyde (PFA) Following dehydration through an ethanol gradient, the fixed embryos were embedded in paraffin wax, and cut into sections with the Leica paraffin slicer. The tissue sectionswere collected on glass slides and dried in an oven at 37 ◦C. He-matoxylin and eosin (HE) staining was performed as per standard protocols according to the report (Cheng et al., 2020; Qiu et al., 2019), and the tissues were sealed with neutral resin, coveredwith a glass cover slip, and dried overnight at 37 ◦C. Images weretaken under a microscope (Leica DM2500,Germany).

2.6. Antibody staining and whole-mount TUNEL staining
Twenty embryos were collected from each group at 72 hpf, washed with PBS, and fixed overnight with 4% paraformaldehyde at 4 ◦C. PCNA immunostaining was performed using anti-PCNA anti-body (1:500; Life Technologies) as previously described (Jia et al., 2020). TUNEL staining for apoptotic cells was performed using the TUNEL Apoptosis Detection Kit (Alexa Fluor 640) according tothe manufacturer’s instructions. Briefly, the fixed embryos were permeabilized with protease K, and then incubated with 50 ml TdT reaction mixture at 37 ◦C for 1e2 h in a hybridization chamber. After washing the embryos thrice with PBS containing 0.1% Triton X-100 and 5 mg/mL BSA, the samples were counterstained withDAPI dye for half an hour, washed again, and observed under a laser-scanning confocal microscope (Leica TCS SP8, Germany).

2.7. Oxidative stress analysis
Live embryos were collected at 72hpf, and the protein content was determined using the Bradford assay kit, and the absorbance was measured using a multifunctional micrometer (PerkinElmer Victor nivo, USA). Three replicates per sample (n = 50/sample). For ROS staining, the embryos were incubated in DCFH-DA diluted1:500, at 37 ◦C for 30e60 min in the dark, and observed under afluorescence microscope (Leica M205 FA stereo micro- scope,Germany). The content of malondialdehyde (MDA) and catalase (CAT), the activity of superoxide dismutase (SOD) were measured using suitable kits according to the instructions.

2.8. Real-time fluorescence quantitative PCR (qPCR)
Thirty embryos per group were collected at 72hpf, and washed several times with phosphoric acid buffer. The embryos were ho- mogenized using the TriZol reagent (Invitrogen) to extract the total RNA, which was then reverse transcribed to cDNA using a PrimeScript® RT reagent kit according to the manufacturer’s method (Qiu et al., 2019; Wang et al., 2019a). Real-time PCR was performed in the Applied Biosystems Step-One-plus real-time PCR system (Applied Biosystem, CA, USA) using SYBR Green detection kit (Takara, Dalian, China), with b-actin as the internal control. The primer sequences used in this study were the same as those in (Cheng et al., 2019; Qiu et al., 2019; Wang et al., 2019a; Xiong et al., 2019) and were provided in Supplemental Table 1. Each sample was tested in triplicates.

2.9. Rescue experiments
Twenty well-developed embryos at 5 hpf were seeded per well in a six-well plate, and treated with DMSO 1.0 mg/L OB and 1.0 mg/L OB + 30 nM astaxanthin (Solarbio, UV ≥ 98%) till 72 hpf.
In the second rescue experiment, the embryos were treated with DMSO, different doses of OB (0.5, 1.0 and 1.5 mg/L), and 10 nM BML-284 in combination with each OB dose.

2.10. Statistical analysis
All data were analyzed using the GraphPad Prism8 software, and represented as the mean standard deviation of at least three in- dependent experiments. Two-way ANOVA was used to compare the control and the treatment groups. All values were shown as the mean ± S.E. P values less than 0.05were considered statistically significant.

3. Results
3.1. Morphological observation and acute toxicity
Zebrafish embryos were treated with different concentrations (0.5,1.0,1.5 mg/L)of OB at 72 hpf. As shown in Fig. 1A, OB increased pericardial edema in a dose-dependent manner, resulting in a linearized heart. In addition, high concentrations of OB induced tail curving and yolk sac edema. Furthermore, the mortality rates of the OB-treated zebrafish also increased in a concentration-dependent manner, and was 100% in the embryos treated with 8 mg/L OB at 48, 72 and 96 hpf (Fig. 1B). Lethal concentration 50 (LC50) of OB was9.341 mg/L, 3.747 mg/L, 1.848 mg/L and 0.746 mg/L at 24, 48, 72 and 96 hpf. Finally, the 20s heartbeat frequency decreased linearly in the OB-treated embryos in a dose-dependent manner at 48 and 72 hpf (Fig. 1C). Interestingly, the OB-treated embryos did not hatch, regardless of the concentration (results not shown).

3.2. Cardiac toxicity of OB exposure in zebrafish embryos
OB exposure significantly decreased the number of car- diomyocytes in the developing zebrafish heart in a dose-dependent manner (Fig. 2A). In addition, the myocardial layer thinned, and the atria and ventricles showed a tendency to linearize following OB exposure. As shown in Fig. 2B, the distance between the atrioven- tricular tubes increased in the OB-treated embryos, which likely slowed the slow heart rate and reduced the efficiency of heart pumping. In addition, OB also upregulated myh6, vmhc, nppa andgata4 compared to the control group, while the heart cyclization factor tbx5 was significantly down-regulated (Fig. 2C). The down- stream target genes of the Wnt signaling pathway, such as lef1, b- catenin and axin2, were significantly down-regulated, while the BMP signaling pathway targets such as id1 and id2 were not significantly affected by OB relative to the untreated control (Fig. 2D).

3.3. OB exposure increased oxidative stress in zebrafish embryos
ROS cause significant oxidative damage to i the cellular proteins, nucleic acids and membranes, eventually triggering the apoptosis cascade. The endogenous ROS is usually scavenged by the antioxi- dant enzymes including SOD and CAT. OB exposure transiently elevated CAT activity, while the high dose of 1 mg/L led to a sig- nificant increase (Fig. 3C). In addition, SOD activity showed a slight increase after an initial decline, and decreased thereafter (Fig. 3D), while the content of the lipid oxidation product MDA increased gradually in a dose-dependent manner (Fig. 3B). Intense ROS accumulation was seen in the head, spinal cord and cardiovascular system in the OB-treated embryos (Fig. 3A), indicating that oxida- tive damage is the main cause of fetal heart malformation.

3.4. OB exposure induced cell apoptosis but not influenced the proliferation of cardiomyocytes
To prove the number of cardiomyocytes decreased after expo- sure, we test cell apoptosis and cell proliferation. OB not affectedcardiac cell proliferation (Supplemental Fig. 1). However, OB significantly upregulated proapoptotic genes including bax, cas- pase-3 and p53 at medium and high concentration, and down- regulated the anti-apoptotic bcl2. Thus, the bax/bcl2 ratio was significantly increased in the OB-treated versus control group (Fig. 4B). Consistent with this, the percentage of apoptotic cells significantly increased in the OB-treated embryos in a dose- dependent manner (Fig. 4A).

3.5. Astaxanthin rescued zebrafish embryos from OB-induced toxicity
To further establish that OB-induced oxidation stress was the cause of cardiotoxicity in zebrafish embryos, we treated them additionally with the antioxidant astaxanthin. As shown in Fig. 5A, astaxanthin reversed the morphological damage to the heart in the OB-treated embryos. Interestingly, astaxanthin had no effect on SOD activity (Fig. 5F) but restored CAT activity and decreased MDA levels (Fig. 5D and E). In addition, the expression levels of the cardio-specific vmhc and gata4 (Fig. 5B), and the apoptosis-related bcl2 and bax (Fig. 5C) genes were also restored in the embryos additionally treated with astaxanthin. Taken together, OB-induced cardiotoxicity can be rescued by antioxidant treatment.

3.6. BML-284 recovered cardiac defects caused by OB exposure
Since the WNT signaling pathway was significantly down- regulated after OB exposure, we next determined whether its reactivation could reverse the cardiac malformation caused by OB (Fig. 6A). As shown in Fig. 6B, the Wnt signaling activator BML-284 effectively rescued the zebrafish embryos from OB-induced car- diotoxicity (Fig. 6B). Reactivation of Wnt signaling downregulated ROS levels (Fig. 7A) and restored all indices of oxidative stress (Fig. 7BeD), in addition to the expression levels of genes associated with the Wnt pathway, cardiac development and apoptosis (Fig. 7EeG).

4. Discussion and conclusion
OB is a systemic herbicide widely used in agriculture. However, the toxicological characteristics are still limited, especially their toxicity effects and mechanisms are not clear. To fill the black, we selected zebrafish to study the toxicity and mechanism of OB and found that zebrafish embryos exposed to OB had significant cardiac defects. OB significantly decreased the heart rate and cardiac blood flow, and increased pericardial edema and the mortality rate in a dose-dependent manner.
Zebrafish heart development requires a complex and orderlyprocess, which is regulated by the major genes and transcription factors of heart development (Shu and Chi, 2012; Stainier, 2001). At the molecular level, OB dysregulated the genes associated with cardiac development and we found that the tbx5 gene was signif- icantly down-regulated. The tbx5 gene is involved in the directed migration of a single lateral plate mesoderm cell to the heart pro- ducing region, and required for embryonic cardiac cell cycle pro- gression (Goetz, 2006; Hatcher et al., 2004). Inhibiting the expression of tbx5 blocks the progression of the cell cycle, leading to a reduction in the number of heart cells, defects in cardiac myogenesis, and ultimately to programmed cell death (Goetz, 2006; Lu et al., 2011).
To prove whether there is apoptosis or not after OB exposure, we tested cell apoptosis. The expression of apoptosis-related genes such as proapoptotic genes: bax, caspase-3 and p53 were signifi- cantly up-regulated although the apoptosis inhibiting factors bcl2 were significantly down-regulated, bax/bcl2>1 was significantly up-regulated compared to the control group. Meanwhile, we detected TUNEL analysis confirmed the apoptosis in the car- diomyocytes after OB exposure. Besides, the number of car- diomyocytes decreased found in the HE staining, we assumed that whether the cell proliferation is affected. However, we didn’t see any reduction of OB exposure in cardiac cell proliferation. This suggests that myocardial apoptosis may be caused in part by a reduction in tbx5.
On the other hand, environmental pollutants can trigger oxidative stress by increasing ROS production and/or impairing the antioxidant enzymes and molecules (Andersson-Sjo€land et al., 2015; Cao et al., 2019; Ren et al., 2019; Rossaro and Cortesi, 2013; Stainier, 2001). Oxidative stress occurs upon imbalance of oxidation and antioxidant systems, leading to the accumulation of ROS, which can lead to cell apoptosis (Chowdhury et al., 2009). Besides, Oxidative stress is the pathological basis of numerous diseases, including cancer, immunological disorders, neurodegeneration etc (Almeida et al., 2011; Cao et al., 2020; Hu et al., 2019; Olivari et al., 2009; Qiu et al., 2019). Therefore, we examined the oxidative stress levels of zebrafish larvae exposed to OB. Results showed that OB exposure significantly decreased the activity of SOD and increased the levels of MDA, ROS, whereas CAT levels fluctuated. These results suggest that the increased levels of oxidative stress caused by OB damaged the cardiomyocytes. Astaxanthin, an oxidative stress in- hibitor (Fassett and Coombes, 2009; Nakajima et al., 2008) has been widely reported to be able to: reduce the level of oxidative stress, improve the activity of immune cells to resist foreign substances (Park et al., 2010; Pashkow et al., 2008), reduce DNA damage and play a protective role. Consistent with this, astaxanthin reversed OB-induced cardiotoxicity.
We finally chose Wnt signaling because we detected the target genes of Wnt and BMP signaling and found that Wnt signaling was more significantly downregulated than BMP signaling. Moreover, the BMP signal marker gene (id1) expression have no difference between control and OB group. Studies show that the Wnt signaling pathway and oxidative stress are interrelated, and Wnt signaling can mitigate ROS-induced apoptosis and necrosis (Andersson-Sjo€land et al., 2015; Tao et al., 2013; Wu et al., 2015; Zhang et al., 2013). In addition, the Wnt pathway is also associated with heart regeneration and development (Deb, 2014; Ozhan and Weidinger, 2015). Since the Wnt signaling pathway was signifi- cantly downregulated by OB, we assessed the potential therapeutic effects of Wnt reactivation. The specific Wnt activator BML-284 rescued the zebrafish embryos from OB-induced cardiotoxicity. Also, the tbx5 gene and apoptosis – related genes expression were restored by activation of Wnt signaling.
Taken together, OB exposure causes significant cardiac damagein zebrafish embryos via oxidative stress and apoptosis, and may be potentially toxic to other aquatic life and even humans.

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