Thiophanate-methyl induces severe hepatotoxicity in zebrafish
Graphical abstract
Introduction
Pesticide residue has won increasing attention for daily life. Due to the overuse of pesticides, its contamination has been harmful to aquatic ecosystems as well as human health (Menchen et al., 2017; Cao et al., 2019a). Thiophanate-methyl (TM) is a broad-spectrum internal absorption fungicide that can effectively prevent and control diseases of various crops (Nagai and Fukazu, 1969; Narisawa and Suzuki, 1969), so it is widely used as a bactericide in the agricultural industry. The bactericidal mechanism of TM is that TM can be converted to Methyl 2-benzim-idazolecarbamate (MBC) which has fungicidal properties in animal and plant tissues as well as in water (Seiler, 1975; Cycoń et al., 2011). TM can be easily transferred to the aquatic environment, and also produce pollution and enrichment to relevant crop such as previous work has reported that about 0.02 μg/mL TM was tested in vegetables, specifically Cabbages and Tomatoes by high-performance liquid chromatography–mass spectrometry (HPLC–MS) (Singh et al., 2007). Meanwhile, TM was found at concentrations ranging from 0.050 to 2.510 mg/kg in Bananas from Latin America and 0.24–1.97 mg/kg in grapes from China by the same method (Veneziano et al., 2004; Dong et al., 2018).
It has been reported that the 24 h lethal concentration of 50% (LC50) was 5.02 mg/L in rotifer Brachionus calyciflorus Pallas which is essential in a freshwater ecosystem after TM exposed, and the asexual and sexual reproduction of rotifers were severely damaged by TM (Xi and Feng, 2004). Moreover, TM can produce toxicity in four freshwater organisms, in which the 2 days LC50 was 8.5 mg/L in Chlorella pyrenoidosa, 16 mg/L in Daphnia magna, 130 mg/L in Lebistes reticulatus and 7.8 mg/L in Salmo gairdneri (Canton, 1976). In addition, the adverse effects of TM on the adrenal gland of the newt Triturus carnifex is that it can significantly decrease the number of secretory vesicles in the chromaffin cells (Capaldo et al., 2006). Multiple species of mammals such as mice, Wistar rats, guinea pigs, rabbits, and dogs (beagles) suffer from acute toxicity after the treatment of TM (Hashimoto et al., 1972). TM specifically can induce histological changes in the blood, liver, and kidneys of rats (Ibtissem et al., 2017). Taken together all these findings, the toxicity of TM has a broad spectrum and can endanger most species.
However, current studies have not been in-depth and there is still a lack of reports about the specific molecular mechanism of TM toxicity, especially with regards to the liver metabolism and over-evaluation perspectives. The liver is an extremely important organ in vertebrate body, and it is also the center of metabolism (Bui-Nguyen et al., 2015; Adeva-Andany et al., 2016). When xenobiotic chemicals such as pesticides enter into the body, some special cells of the liver contain various metabolic enzymes and bile which take part in the biotransformation of drugs and poisons through catabolism in zebrafish, similar to those in mammals (He et al., 2013a; Qiu et al., 2019). Because of this, drugs easily cause hepatotoxicity. The activation of caspase-3 through apoptotic pathways and oxidative stress are two characteristic reasons of drug-induced hepatotoxicity in animals (Nagai et al., 2015; El-Bakry et al., 2017; Liem et al., 2018). However, the information of TM itself on hepatotoxicity has been limited and should be considered.
A zebrafish (Danio rerio) liver develops rapidly into an accomplish liver morphogenesis at 48 h post-fertilization (hpf), enters growth spurts at 50 hpf, and has a fully developed metabolic function by 72 hpf, in which drug metabolism becomes active (He et al., 2013b; Zhang et al., 2016; Qiu et al., 2019). Furthermore, zebrafish have many additional advantages and have been widely used to estimate all kinds of toxicity (Garcia-Reyero et al., 2014; Cao et al., 2019b; Jiang et al., 2019; Wang et al., 2019a, 2019b). In this study, we used zebrafish to evaluate the hepatotoxicity of TM by validating a whole zebrafish phenotypic assay and analyzing the morphological and substance metabolism abnormalities of the liver. Indeed, the activation of caspase-3 and oxidative stress have been included in the main consideration. Our results have indicated that TM may induce severe disorder in material metabolism of the liver in zebrafish by mediating activation of caspase-3 and oxidative stress, indicating severe hepatotoxicity.
Section snippets
Chemicals
Thiophanate-methyl (70%, CAS:23,564-05-8) was purchased from Shandong haixun biochemistry co. LTD., (Shandong, China). The TM was diluted into 200 mg/L as a stock solution in embryo culture medium (0.3325 g/L baysalt, 4.2 mM NaHCO3; PH = 7.2) and stored in room temperature. PAS stain Kit, glutamic-pyruvic transaminase (GPT), 6-phosphate-glucose dehydrogenase (G6PDH) and Glycogen assay Kits were purchased from Solarbio (Beijing, China). Malondialdehyde (MDA), catalase (CAT), superoxide dismutase
The survival rate of zebrafish exposed in TM
Graph abstract shows the molecular formula of TM. The 24 h LC50 was 57.45 mg/L while both 48 h and 72 h LC50 were 50.45 mg/L (Fig. 1B). The adult zebrafish were exposed to 2, 4, 6 mg/L of TM or fresh fish water as vehicle control and the survival rate was calculated at 24, 48, 72 and 96 h (Supplementary Fig. 1S). The above results indicate that acute toxicity is induced after the treatment of TM in zebrafish.
TM specifically induces hepatotoxicity
To determine the hepatotoxicity of TM in zebrafish, we used Tg (fabp10a: DsRed)
Discussion
TM is widely used all over the world and is a typical example of pesticide residues. Some previous reports showed that TM had the potential to induce the changes of blood, liver and kidney histomorphology (Ben Amara et al., 2014; Ibtissem et al., 2017), as well as the toxicity of reproduction and immunity (Capaldo et al., 2006; Weis et al., 2019). However, the molecular mechanisms underlying the hepatotoxicity of TM are not well understood. Thus, we used a viable model animal zebrafish to
Conclusions
In summary, we found that TM induces serious hepatotoxicity but not the developmental toxicity of other organs in zebrafish larvae and adults. In addition, we explored the specific molecular mechanism of TM-induced hepatotoxicity and found that TM might triggered hepatotoxicity via activating caspase-3 through apoptotic pathways and oxidative stress in zebrafish, which subsequently resulted in a metabolic imbalance in zebrafish liver. These findings provide new evidence for a more comprehensive
CRediT authorship contribution statement
Kun Jia: Conceptualization, Methodology, Investigation, Writing - original draft. Bo Cheng: Validation, Formal analysis, Visualization, Data curation. Lirong Huang: Validation, Formal analysis, Visualization, Software. Juhua Xiao: Software, Data curation. Zhonghui Bai: Formal analysis, Data curation. Xinjun Liao: Writing - review & editing. Zigang Cao: Writing - review & editing. Tianzhu Shen: Software, Data curation. Chunping Zhang: Writing - review & editing. Chengyu Hu: Supervision, Project
Declaration of competing interest
The authors declare that there is no conflict of interest associated with this study.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81560109, 31771606, 31900597, 81860282), the Natural Science Foundation Project of Jiangxi Province (2018ACB21033, 20192ACB21013), the Science and Technology Foundation of the Education Department of Jiangxi Province (GJJ150759), China Postdoctoral Foundation (2019M652269), and the Jinggangshan University grant (JZB15006 and JFD1802).
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These authors contributed equally to this work.