Journal of Animal Reproduction and Biotechnology 2022; 37(3): 176-182
Published online September 30, 2022
https://doi.org/10.12750/JARB.37.3.176
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Garam An1 , Hahyun Park1 , Taeyeon Hong2 , Gwonhwa Song1,* and Whasun Lim2,*
1Institute of Animal Molecular Biotechnology and Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea
2Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Korea
Correspondence to: Gwonhwa Song
E-mail: ghsong@korea.ac.kr
Whasun Lim
E-mail: wlim@skku.edu
Norflurazon is widely used on agricultural lands and has a high potential to pollute water sources. However, its effects on fish have not been fully elucidated. The purpose of our study was to determine whether norflurazon adversely affects the developmental stage of zebrafish, which are frequently used as a model system to evaluate the environmental impact of pollutants. Norflurazon interfered with the hatching of zebrafish embryos and induced several sublethal deformities including body length reduction, increased yolk sac volume, and enlargement of the pericardial region. We further examined the cardiotoxicity of norflurazon in the flk1:eGFP transgenic zebrafish line. The vascular network, mainly in the brain region, was significantly disrupted in norflurazon-exposed zebrafish. In addition, due to the failure of cardiac looping, norflurazon-exposed zebrafish had an abnormal cardiac structure. These developmental abnormalities were related to the apoptotic process triggered by norflurazon. Overall, the present study demonstrated the non-target toxicity of norflurazon by analyzing the hazardous effects of norflurazon on developing zebrafish.
Keywords: apoptosis, cardiovascular toxicity, developmental toxicity, norflurazon, zebrafish embryo
Herbicide use in agricultural production may potentially contaminate groundwater or surface water and adversely affect non-target species. Therefore, analysis of the ecotoxicological effects of herbicides is important to preserve the aquatic ecosystem. Norflurazon, which is categorized as a pyridazinone herbicide, has been applied to pre-emergent weed management since 1974 (Sathishkumar et al., 2016). Due to its water solubility (28 mg/L), norflurazon use on agricultural lands has the potential to leach to groundwater and surface water (Wilson and Koch, 2013). Wilson et al. (2007) reported that 8,480 mg of norflurazon escaped from the applied site when sprayed at 7.7 kg/ha. Moreover, around 6-8 months were required for norflurazon to be degraded to 50% of its original concentration in aquatic environments.
Considering the possibility of water pollution, some studies have analyzed the toxicity of norflurazon towards aquatic organisms. For example, the planarian
Toxicity assessment using zebrafish embryos has been suggested as a promising method to demonstrate the impact of environmental toxicants (An et al., 2021; Ha et al., 2021). Various studies have confirmed that reactivity to toxicants is highly similar between zebrafish and other fish species (Busquet et al., 2014; Birke and Scholz, 2019). In addition, because of its rapid development and transparency, experiments with zebrafish embryos provide time and cost benefits (Glaberman et al., 2017).
In the present study, the survival and hatching rates of zebrafish embryos were measured following norflurazon treatment. In addition, morphological endpoints in toxicity tests and cardiovascular abnormalities in norflurazon-treated zebrafish were examined. To determine which cellular response may contribute to the reduced viability and deformities of norflurazon-treated zebrafish, we investigated whether norflurazon triggers apoptotic cell death in developing zebrafish. Overall, we conducted a toxicity assessment of norflurazon using a zebrafish model and found that norflurazon may negatively affect the aquatic ecosystem.
Adult wild-type (AB strain) and transgenic (
After 6 h of fertilization, which is the beginning of gastrulation (D’Costa and Shepherd, 2009), normally developed embryos (n=12 for each concentration) were selected and exposed to norflurazon (catalog number: 34364; Sigma-Aldrich, St. Louis, MO, USA). Norflurazon powder was diluted in dimethyl sulfoxide and further diluted to 0, 50, and 100 μM in embryo medium containing phenylthiourea to observe zebrafish embryos without visual interference due to pigmentation. Experimental concentration of norflurazon was determined by referring its LC50 value in other fish species (Munn and Gilliom, 2001). The same volume of dimethyl sulfoxide (0.02%) was diluted in the same medium as a vehicle control. Embryos were transferred to each well of a 24-well plate containing the norflurazon solution. The solution was replaced daily to maintain a specific concentration.
From 24 h to 96 h of norflurazon treatment, the number of live and hatched embryos was recorded. The coagulation of embryos or absence of a heartbeat was used as an indicator of dead embryos. After 96 h of treatment, other sublethal endpoints were evaluated using an optical microscope (DM 2500; Leica, Wetzlar, Germany). In brief, the image of each live larva was captured under the microscope following immobilization with tricaine (catalog number: A5040; Sigma-Aldrich) and mounting with methylcellulose (catalog number: K390; Amresco, Solon, OH, USA). Image analyses were conducted using ImageJ software (NIH, Bethesda, MD, USA).
Transgenic
After 96 h of treatment, norflurazon-treated zebrafish were stained with 5 μg/mL acridine orange (catalog number: A3568; Life Technologies, Carlsbad, CA, USA) for 1 h to visualize apoptotic cells with green fluorescence. Immobilization of zebrafish was carried out in the same method as evaluation of toxicological endpoints, and Axio Imager M1 was used to capture images. Captured images were further analyzed with ImageJ software.
All graphs represent the mean and standard deviation of the experimental data from toxicity tests. Statistical analyses of the data were performed by one-way ANOVA based on the general linear model of SAS software (SAS Institute, Cary, NC, USA). A
To determine whether norflurazon has sublethal effects on developing zebrafish, we treated zebrafish embryos with 0, 50, and 100 μM norflurazon and recorded the number of surviving embryos at 96 h post-fertilization (hpf). The survival rates following norflurazon exposure did not show statistically significant differences (Fig. 1A). However, the proportion of unhatched live zebrafish embryos was markedly increased following norflurazon exposure. At 72 hpf, 86.1 ± 9.6% of zebrafish embryos hatched following 0 μM norflurazon treatment, whereas only 19.4 ± 9.6% and 11.1 ± 4.8% of zebrafish embryos hatched following 50 μM and 100 μM norflurazon treatment, respectively (
Considering that pericardial edema is one of the common characteristics of heart failure (Chen, 2013), we hypothesized that norflurazon might negatively affect the cardiovascular network. To confirm this hypothesis, the
To identify the cellular response responsible for the acute toxicity of norflurazon, we stained norflurazon-treated zebrafish with acridine orange, which intercalates with DNA strands and emits green fluorescence (Smirnova et al., 2021) (Fig. 3A). As several deformities were mainly observed in the posterior region of zebrafish previously, we focused on detecting apoptotic cell death in this region. The fluorescence intensity was 1.2-fold higher in the 50 μM norflurazon group compared with the vehicle group (
In the present study, we evaluated the toxicity of norflurazon towards zebrafish in the developmental stage. The hatching ability of zebrafish embryos exposed to norflurazon was significantly impaired. Moreover, several sublethal deformities and cardiovascular defects were observed in norflurazon-treated zebrafish larvae. The acute toxicity of norflurazon towards zebrafish was accompanied by apoptotic cell death.
Considering the extensive usage of pesticides and the possibility of water contamination, several studies have investigated the hazardous effects of pesticides on aquatic non-target organisms. In this study, a zebrafish model was used to assess the toxicity of the herbicide norflurazon, which is considered as a potential water pollutant owing to its physicochemical properties (Horvat et al., 2005). For example, residual norflurazon was detected up to 3.9 mg/L in surface waters from south Florida (Schuler and Rand, 2008). In addition, 1,684 mg and 8,480 mg of norflurazon was runoff to non-target sites when 4.4 kg/ha and 7.7 kg/ha of norflurazon applicated in flatwoods (Wilson et al., 2007). While these concentrations not accurately reflect exact exposure concentration to fish. Thus, further research revealing exposure amount on fish should be conducted.
We found that norflurazon caused several developmental defects in zebrafish larvae. Norflurazon reduced the hatching rate of embryos. Unsuccessful hatching is frequently observed in response to extrinsic stressors and can be caused by reduced movement due to functional or structural malformation (Hill et al., 2005, Horzmann et al., 2020). This result cannot explain whether norflurazon only delays hatching period or induces lethality by hatching inhibition because hatching ratio was not measured after 96 hpf. However, belatedly hatched larvae usually have lower viability due to developmental deformities (Küçükoğlu et al., 2013, Zoupa and Machera, 2017). Thus, we suggest that norflurazon might also reduce viability of zebrafish by hatching inhibition. In addition, sublethal deformities such as body length reduction and yolk sac enlargement occurred following norflurazon treatment. A shorter length is mainly associated with the irregular development of the skeletal or muscular system (Zoupa and Machera, 2017). A larger yolk sac volume suggests that the normal utilization of nutrients in the yolk is hampered by norflurazon treatment (Sant and Timme-Laragy, 2018). In addition, excessive fluid accumulated in the pericardial area in norflurazon-exposed zebrafish.
Pericardial edema is one of the symptoms used to confirm the cardiovascular toxicity of chemicals (Zoupa and Machera, 2017). We revealed that norflurazon exposure induced not only massive pericardial edema but also an irregular brain vasculature and heart structure in zebrafish. During normal vascularization, migrated precursor cells form primordial hindbrain channels (PHBCs), and central arteries (CtAs) are subsequently sprouted from PHBCs (Gupta et al., 2021). In this study, an irregular cranial vasculature, mainly a reduction in CtAs, was observed following norflurazon treatment. This malformation might result from the altered transcriptional level of genes related to
Apoptosis is a cellular process induced by multiple external stimuli. As demonstrated in previous studies, various pesticides could trigger the apoptotic process through several mechanisms such as oxidative stress and metabolic alterations. For example, deltamethrin was found to impair defense systems against oxidative stress, resulting in reduced viability and malformation with apoptosis (Parlak, 2018). Paraquat exposure was also observed to trigger oxidative stress in a similar manner and induce cell death by modulating the transcriptional levels of genes associated with apoptosis (Wang et al., 2016). Changes in endogenous metabolites induced by imazalil and imazalil-M were found to be involved in apoptosis induction in developing zebrafish (Huang et al., 2022). Therefore, identifying the detailed mechanisms of norflurazon-mediated apoptosis might provide in-depth knowledge for predicting its effects on non-target organisms.
Overall, the present results demonstrated the toxic effects of norflurazon on zebrafish in the developmental stage. Zebrafish exposed to norflurazon in the embryonic stage showed impaired hatchability, pathological phenotypes including a shortened body length, yolk enlargement, and severe edema in the cardiac region, and structural impairment of the cardiovascular system. These developmental defects were mediated by apoptotic cell death. Overall, our study demonstrated the effects of norflurazon on a zebrafish model, suggesting that it could be a threat to the aquatic ecosystem.
None.
Conceptualization, G.S., and W.L.; methodology, G.A., H.P., and T.H.; investigation, G.A., H.P., and T.H.; data curation, G.A., H.P., T.H., G.S., and W.L.; visualization, G.A.; writing-original draft, G.A.; writing-review and editing, G.S., and W.L.; funding acquisition, G.S., and W.L.; supervision, G.S., and W.L.; project administration, G.S., and W.L.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (2020R1A6A3A13075810 and 2019R1A6A1A10073079) and National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2021R1A2C2005841). This study was also supported by the Institute of Animal Molecular Biotechnology, Korea University.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Journal of Animal Reproduction and Biotechnology 2022; 37(3): 176-182
Published online September 30, 2022 https://doi.org/10.12750/JARB.37.3.176
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Garam An1 , Hahyun Park1 , Taeyeon Hong2 , Gwonhwa Song1,* and Whasun Lim2,*
1Institute of Animal Molecular Biotechnology and Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea
2Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Korea
Correspondence to:Gwonhwa Song
E-mail: ghsong@korea.ac.kr
Whasun Lim
E-mail: wlim@skku.edu
Norflurazon is widely used on agricultural lands and has a high potential to pollute water sources. However, its effects on fish have not been fully elucidated. The purpose of our study was to determine whether norflurazon adversely affects the developmental stage of zebrafish, which are frequently used as a model system to evaluate the environmental impact of pollutants. Norflurazon interfered with the hatching of zebrafish embryos and induced several sublethal deformities including body length reduction, increased yolk sac volume, and enlargement of the pericardial region. We further examined the cardiotoxicity of norflurazon in the flk1:eGFP transgenic zebrafish line. The vascular network, mainly in the brain region, was significantly disrupted in norflurazon-exposed zebrafish. In addition, due to the failure of cardiac looping, norflurazon-exposed zebrafish had an abnormal cardiac structure. These developmental abnormalities were related to the apoptotic process triggered by norflurazon. Overall, the present study demonstrated the non-target toxicity of norflurazon by analyzing the hazardous effects of norflurazon on developing zebrafish.
Keywords: apoptosis, cardiovascular toxicity, developmental toxicity, norflurazon, zebrafish embryo
Herbicide use in agricultural production may potentially contaminate groundwater or surface water and adversely affect non-target species. Therefore, analysis of the ecotoxicological effects of herbicides is important to preserve the aquatic ecosystem. Norflurazon, which is categorized as a pyridazinone herbicide, has been applied to pre-emergent weed management since 1974 (Sathishkumar et al., 2016). Due to its water solubility (28 mg/L), norflurazon use on agricultural lands has the potential to leach to groundwater and surface water (Wilson and Koch, 2013). Wilson et al. (2007) reported that 8,480 mg of norflurazon escaped from the applied site when sprayed at 7.7 kg/ha. Moreover, around 6-8 months were required for norflurazon to be degraded to 50% of its original concentration in aquatic environments.
Considering the possibility of water pollution, some studies have analyzed the toxicity of norflurazon towards aquatic organisms. For example, the planarian
Toxicity assessment using zebrafish embryos has been suggested as a promising method to demonstrate the impact of environmental toxicants (An et al., 2021; Ha et al., 2021). Various studies have confirmed that reactivity to toxicants is highly similar between zebrafish and other fish species (Busquet et al., 2014; Birke and Scholz, 2019). In addition, because of its rapid development and transparency, experiments with zebrafish embryos provide time and cost benefits (Glaberman et al., 2017).
In the present study, the survival and hatching rates of zebrafish embryos were measured following norflurazon treatment. In addition, morphological endpoints in toxicity tests and cardiovascular abnormalities in norflurazon-treated zebrafish were examined. To determine which cellular response may contribute to the reduced viability and deformities of norflurazon-treated zebrafish, we investigated whether norflurazon triggers apoptotic cell death in developing zebrafish. Overall, we conducted a toxicity assessment of norflurazon using a zebrafish model and found that norflurazon may negatively affect the aquatic ecosystem.
Adult wild-type (AB strain) and transgenic (
After 6 h of fertilization, which is the beginning of gastrulation (D’Costa and Shepherd, 2009), normally developed embryos (n=12 for each concentration) were selected and exposed to norflurazon (catalog number: 34364; Sigma-Aldrich, St. Louis, MO, USA). Norflurazon powder was diluted in dimethyl sulfoxide and further diluted to 0, 50, and 100 μM in embryo medium containing phenylthiourea to observe zebrafish embryos without visual interference due to pigmentation. Experimental concentration of norflurazon was determined by referring its LC50 value in other fish species (Munn and Gilliom, 2001). The same volume of dimethyl sulfoxide (0.02%) was diluted in the same medium as a vehicle control. Embryos were transferred to each well of a 24-well plate containing the norflurazon solution. The solution was replaced daily to maintain a specific concentration.
From 24 h to 96 h of norflurazon treatment, the number of live and hatched embryos was recorded. The coagulation of embryos or absence of a heartbeat was used as an indicator of dead embryos. After 96 h of treatment, other sublethal endpoints were evaluated using an optical microscope (DM 2500; Leica, Wetzlar, Germany). In brief, the image of each live larva was captured under the microscope following immobilization with tricaine (catalog number: A5040; Sigma-Aldrich) and mounting with methylcellulose (catalog number: K390; Amresco, Solon, OH, USA). Image analyses were conducted using ImageJ software (NIH, Bethesda, MD, USA).
Transgenic
After 96 h of treatment, norflurazon-treated zebrafish were stained with 5 μg/mL acridine orange (catalog number: A3568; Life Technologies, Carlsbad, CA, USA) for 1 h to visualize apoptotic cells with green fluorescence. Immobilization of zebrafish was carried out in the same method as evaluation of toxicological endpoints, and Axio Imager M1 was used to capture images. Captured images were further analyzed with ImageJ software.
All graphs represent the mean and standard deviation of the experimental data from toxicity tests. Statistical analyses of the data were performed by one-way ANOVA based on the general linear model of SAS software (SAS Institute, Cary, NC, USA). A
To determine whether norflurazon has sublethal effects on developing zebrafish, we treated zebrafish embryos with 0, 50, and 100 μM norflurazon and recorded the number of surviving embryos at 96 h post-fertilization (hpf). The survival rates following norflurazon exposure did not show statistically significant differences (Fig. 1A). However, the proportion of unhatched live zebrafish embryos was markedly increased following norflurazon exposure. At 72 hpf, 86.1 ± 9.6% of zebrafish embryos hatched following 0 μM norflurazon treatment, whereas only 19.4 ± 9.6% and 11.1 ± 4.8% of zebrafish embryos hatched following 50 μM and 100 μM norflurazon treatment, respectively (
Considering that pericardial edema is one of the common characteristics of heart failure (Chen, 2013), we hypothesized that norflurazon might negatively affect the cardiovascular network. To confirm this hypothesis, the
To identify the cellular response responsible for the acute toxicity of norflurazon, we stained norflurazon-treated zebrafish with acridine orange, which intercalates with DNA strands and emits green fluorescence (Smirnova et al., 2021) (Fig. 3A). As several deformities were mainly observed in the posterior region of zebrafish previously, we focused on detecting apoptotic cell death in this region. The fluorescence intensity was 1.2-fold higher in the 50 μM norflurazon group compared with the vehicle group (
In the present study, we evaluated the toxicity of norflurazon towards zebrafish in the developmental stage. The hatching ability of zebrafish embryos exposed to norflurazon was significantly impaired. Moreover, several sublethal deformities and cardiovascular defects were observed in norflurazon-treated zebrafish larvae. The acute toxicity of norflurazon towards zebrafish was accompanied by apoptotic cell death.
Considering the extensive usage of pesticides and the possibility of water contamination, several studies have investigated the hazardous effects of pesticides on aquatic non-target organisms. In this study, a zebrafish model was used to assess the toxicity of the herbicide norflurazon, which is considered as a potential water pollutant owing to its physicochemical properties (Horvat et al., 2005). For example, residual norflurazon was detected up to 3.9 mg/L in surface waters from south Florida (Schuler and Rand, 2008). In addition, 1,684 mg and 8,480 mg of norflurazon was runoff to non-target sites when 4.4 kg/ha and 7.7 kg/ha of norflurazon applicated in flatwoods (Wilson et al., 2007). While these concentrations not accurately reflect exact exposure concentration to fish. Thus, further research revealing exposure amount on fish should be conducted.
We found that norflurazon caused several developmental defects in zebrafish larvae. Norflurazon reduced the hatching rate of embryos. Unsuccessful hatching is frequently observed in response to extrinsic stressors and can be caused by reduced movement due to functional or structural malformation (Hill et al., 2005, Horzmann et al., 2020). This result cannot explain whether norflurazon only delays hatching period or induces lethality by hatching inhibition because hatching ratio was not measured after 96 hpf. However, belatedly hatched larvae usually have lower viability due to developmental deformities (Küçükoğlu et al., 2013, Zoupa and Machera, 2017). Thus, we suggest that norflurazon might also reduce viability of zebrafish by hatching inhibition. In addition, sublethal deformities such as body length reduction and yolk sac enlargement occurred following norflurazon treatment. A shorter length is mainly associated with the irregular development of the skeletal or muscular system (Zoupa and Machera, 2017). A larger yolk sac volume suggests that the normal utilization of nutrients in the yolk is hampered by norflurazon treatment (Sant and Timme-Laragy, 2018). In addition, excessive fluid accumulated in the pericardial area in norflurazon-exposed zebrafish.
Pericardial edema is one of the symptoms used to confirm the cardiovascular toxicity of chemicals (Zoupa and Machera, 2017). We revealed that norflurazon exposure induced not only massive pericardial edema but also an irregular brain vasculature and heart structure in zebrafish. During normal vascularization, migrated precursor cells form primordial hindbrain channels (PHBCs), and central arteries (CtAs) are subsequently sprouted from PHBCs (Gupta et al., 2021). In this study, an irregular cranial vasculature, mainly a reduction in CtAs, was observed following norflurazon treatment. This malformation might result from the altered transcriptional level of genes related to
Apoptosis is a cellular process induced by multiple external stimuli. As demonstrated in previous studies, various pesticides could trigger the apoptotic process through several mechanisms such as oxidative stress and metabolic alterations. For example, deltamethrin was found to impair defense systems against oxidative stress, resulting in reduced viability and malformation with apoptosis (Parlak, 2018). Paraquat exposure was also observed to trigger oxidative stress in a similar manner and induce cell death by modulating the transcriptional levels of genes associated with apoptosis (Wang et al., 2016). Changes in endogenous metabolites induced by imazalil and imazalil-M were found to be involved in apoptosis induction in developing zebrafish (Huang et al., 2022). Therefore, identifying the detailed mechanisms of norflurazon-mediated apoptosis might provide in-depth knowledge for predicting its effects on non-target organisms.
Overall, the present results demonstrated the toxic effects of norflurazon on zebrafish in the developmental stage. Zebrafish exposed to norflurazon in the embryonic stage showed impaired hatchability, pathological phenotypes including a shortened body length, yolk enlargement, and severe edema in the cardiac region, and structural impairment of the cardiovascular system. These developmental defects were mediated by apoptotic cell death. Overall, our study demonstrated the effects of norflurazon on a zebrafish model, suggesting that it could be a threat to the aquatic ecosystem.
None.
Conceptualization, G.S., and W.L.; methodology, G.A., H.P., and T.H.; investigation, G.A., H.P., and T.H.; data curation, G.A., H.P., T.H., G.S., and W.L.; visualization, G.A.; writing-original draft, G.A.; writing-review and editing, G.S., and W.L.; funding acquisition, G.S., and W.L.; supervision, G.S., and W.L.; project administration, G.S., and W.L.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (2020R1A6A3A13075810 and 2019R1A6A1A10073079) and National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2021R1A2C2005841). This study was also supported by the Institute of Animal Molecular Biotechnology, Korea University.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
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