Elsevier

Environmental Pollution

Volume 253, October 2019, Pages 722-730
Environmental Pollution

Mitochondrial dysfunction, apoptosis and transcriptomic alterations induced by four strobilurins in zebrafish (Danio rerio) early life stages

https://doi.org/10.1016/j.envpol.2019.07.081Get rights and content

Highlights

  • Larval zebrafish was most susceptible to pyraclostrobin in four strobilurins tested.

  • RNA-seq revealed strobilurins regulated cell apoptosis and cancer related pathways.

  • Strobilurin fungicides caused mitochondrial dysfunction and oxidative stress.

  • Strobilurins induced cell apoptosis through the mitochondrial pathway.

Abstract

Though the toxicity of strobilurins on non-target aquatic organisms has been characterized, the associated toxic mechanisms have not been fully explored. The present study showed that the larval stage was the most sensitive developmental stage in zebrafish, and pyraclostrobin (PY) had the highest acute toxicity to embryos, larvae, juvenile and adult with 96 h-LC50 at 0.048 mg/L, 0.029 mg/L, 0.039 mg/L, 0.031 mg/L respectively, when compared with the toxicity of trifloxystrobin (TR), kresoxim-methyl (KM) and azoxystrobin (AZ) at corresponding developmental stage. Then we investigated the transcriptomics and developmental toxicity of TR, KM, AZ and PY on zebrafish embryos after 72 h exposure. RNA-seq revealed that the pathways related to cell apoptosis and cancer, and cellular components organelle membrane and mitochondrion, were markedly affected after TR, KM, AZ and PY exposure during zebrafish early life stages. The results were further confirmed by the induction of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) activities, the elevation of H2O2, malondialdehyde (MDA) and reactive oxygen species (ROS) level, as well as the reduction of intracellular calcium ions (Ca2+) and mitochondrial membrane potential (MMP), which indicated that strobilurins could cause mitochondrial dysfunction and cell apoptosis. The present study was performed a systematic analysis of strobilurins to zebrafish at multi-levels, which provided suggestions for further investigation of molecular mechanisms underlying the toxicity induced by strobilurins on aquatic organisms.

Introduction

Fungicides are used in agriculture to control fungal pathogens during crop production. As outstanding new fungicides exhibiting excellent activity against most major fungal pathogens, strobilurins are the largest part of the commercial Quinone outside inhibitor (QoI) fungicides (Bartlett et al., 2002). The mode of action of strobilurins is the inhibition of mitochondrial respiration by blocking electron transfer at the ubiquinol oxidizing (Qo) site of the complex III (mitochondrial cytochrome bc1 complex), thus inhibiting ATP synthesis and inducing cellular oxidative stress (Balba, 2007; Bartlett et al., 2002; Gisi et al., 2002). With their increasing use, the residues of strobilurins can leach to aquatic ecosystem via the surface and groundwater, ultimately endanger aquatic organisms and human health.

Studies had demonstrated that the strobilurins were frequently detected in different aquatic ecosystems. It was reported that the concentrations of azoxystrobin (AZ) in run-off waters ranged from 0.15 to 34 μg/L in US or Germany (Cao et al., 2016; Garanzini and Menone, 2015), and the detected concentration in paddy water was up to 0.183 mg/L after AZ sprayed for 10 days in China (Cao et al., 2018). The maximum concentrations of trifloxystrobin (TR) and pyraclostrobin (PY) were 0.73 and 1.61 μg/L in surface water around farmland, respectively (Mimbs et al., 2016; Wightwick et al., 2012). In China, the concentrations of PY and TR in paddy water were 17.24  and 170 μg/L after 3 and 5 days of post-application, respectively (Li et al., 2018). The concentration of kresoxim-methyl (KM) was up to 2.9 μg/L in different streams in European (Liess and Von Der Ohe, 2005). Although strobilurins are considered to have low toxicity to mammals, birds and bees (Bartlett et al., 2002), recent studies have clearly showed that strobilurins even at μg/L level could cause lethal and teratogenic effects on non-target aquatic organisms (Belden et al., 2010; Hartman et al., 2014; Hooser et al., 2012).

Literatures about the adverse effects of strobilurins on aquatic organisms were mainly focused on lethal effects, oxidative stress or genotoxicity. The median lethal concentration (LC50) values of strobilurins for different aquatic organisms ranged from 0.0114 to 3.9 mg/L, such as Lepomis macrochirus, Daphnia magna, Oncorhynchus mykiss, Ctenopharyngodon idella, Chlorella vulgaris and Danio rerio (Cui et al., 2017; Jia et al., 2018; Li et al., 2018; Liu et al., 2013; Liu et al., 2015; Ochoa-Acuna et al., 2009; Rodrigues et al., 2015; Zhang et al., 2017). It was reported that strobilurins could induce DNA damage and oxidative stress by regulating the activity or the transcription of key enzymes involved in antioxidant system, such as catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD), or inducing the reactive oxygen species (ROS) and malonaldehyde (MDA) in many aquatic organisms (Garanzini and Menone, 2015; Jia et al., 2018; Li et al., 2018; Liu et al., 2013; Liu et al., 2015; Zhang et al., 2017). Previous researches showed that strobilurins inhibited mitochondrial respiration and induced apoptosis through triggering the mitochondrial pathway in mammalian or fish cell lines (Flampouri et al., 2016; Gao et al., 2014; Regueiro et al., 2015; Rodrigues et al., 2015; Shi et al., 2017). The toxicological effects of strobilurins on mitochondrial respiration of aquatic organisms are still not fully elucidated, and hence in-depth studies on the potential mechanisms of strobilurins are extraordinarily exigent.

Zebrafish have been widely used as an ideal model organism in toxicological research and environmental safety assessment because of its short life cycle, high fecundity and transparency. Given the conservation of many molecular and cellular processes between zebrafish and human, the observed influence and mechanism of toxicity on zebrafish provides opportunities to accelerate the process of drug discovery and the possibility to extrapolate the potential health risk to human (Tat et al., 2013; Zon and Peterson, 2005). Compared to traditional toxicological endpoints, transcriptomics can provide high-throughput data of numerous biomolecules and reflect integrated responses within an organism, give more insight into the mechanisms of toxicity (Simmons et al., 2015). To better understand the toxicity of strobilurins, we investigated the transcriptomics and developmental toxicity of a group of four strobilurins TR, KM, AZ and PY on zebrafish embryos after 72 h exposure. The results of our study will provide a systematic understanding of the mechanism and pathway responsible for the toxicity of strobilurins in zebrafish.

Section snippets

Chemical and reagents

Pyraclostrobin (98% purity) (CAS number: 175013-18-0) was provided by Shandong Hailir Chemical Co., Ltd. (Shandong, China). Azoxystrobin (96% purity) (CAS number: 131860-33-8) was obtained from Shanghai Taihe (Cacch) Co. Ltd. (China). Kresoxim-methyl (97% purity) (CAS number: 143390-89-0) was provided by Jiangsu Frey Agrochemicals Co., Ltd. (Jiangsu, China). Trifloxystrobin (97% purity) (CAS number: 141517-21-7) was obtained from China Agriculture University. Stock solutions were prepared in

Acute toxicity of strobilurins on developmental stages of zebrafish

Table 1 showed the acute toxicities of strobilurins at various life stages of zebrafish. The results demonstrated that these four tested strobilurins possessed different toxic sensitivities to embryo, larvae, juvenile and adult of zebrafish. According to the 96 h-LC50 values, PY demonstrated the highest acute toxicity to embryos, larvae, juvenile and adult zebrafish, with 96 h-LC50 at 0.048 mg/L, 0.029 mg/L, 0.039 mg/L, 0.031 mg/L, when compared with the acute toxicity of TR, KM and AZ at the

Discussion

The most sensitive developmental stage in zebrafish is the early life stage (Tat et al., 2013; Zon and Peterson, 2005), zebrafish larval stage begins at 2 or 3 dpf when the organism hatches from its chorion (Stephens et al., 2016). The present study demonstrated that the larval stage was the most sensitive stage in zebrafish, and PY showed the highest acute toxicity to embryos, larvae, juvenile and adult zebrafish among the four strobilurins, which was over 10-fold higher than the toxicity

Conclusions

The present study demonstrated that the acute toxicity of PY to zebrafish at embryo, larvae, juvenile and adult stages was higher than those of TR, KM and AZ, and the larval stage was the most susceptible among the four life stages. Transcriptomic analysis demonstrated that strobilurins disturbed the transcription of organelle membrane and mitochondrion components, as well as the pathways directly or indirectly relating to cell apoptosis and cancer. The results were further evidenced by the

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgments

This study was supported by grants from the State Key Laboratory for Quality and Safety of Agro-products (2010DS700124-ZZ1702), Public Technology Application Research of Zhejiang Province (2016C32098), Zhejiang Provincial Major Scientific Research Project for Agriculture (Grant No. ZJNY2017001).

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