Thiophanate-methyl induces severe hepatotoxicity in zebrafish

Highlights

• TM induced specifically hepatotoxicity.

• TM impaired the tissue morphology of the liver in zebrafish.

• TM induced severe hepatotoxicity via activation of caspase-3 and oxidative stress.

• TM resulted in glucose and lipid metabolism disorder in zebrafish liver.

Abstract

Thiophanate-methyl (TM) is widely used all over the world and is a typical example of pesticide residues, which can be detected in the soil, and even in vegetables and fruits. However, the molecular mechanisms underlying the hepatotoxicity of TM are not well understood. In this study, we utilized zebrafish to comprehensively evaluate the hepatotoxicity of TM and explore how the molecular mechanisms of hepatotoxicity are induced. The zebrafish larvae were exposed in 6.25, 12.5 and 25 mg/L TM from 72 to 144 hpf, while the adults were exposed in 2, 4 and 6 mg/L TM for 28 days. Here, we found that 12.5 and 25 mg/L TM induces specifically serious hepatotoxicity but not the toxicity of other organs in zebrafish larvae and adults. Moreover, it might triggered hepatotoxicity by activating the caspase-3 through apoptotic pathways and oxidative stress in zebrafish. Subsequently, this resulted in a metabolic imbalance in the zebrafish's liver. In conclusion, our results disclosed the fact that TM may induce severe hepatotoxicity by mediating activation of caspase-3 and oxidative stress in zebrafish.

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.