Although the use of diquat is limited in the European Union, it consumed in high levels in many countries and is often abused (Yuan et al. 2021). The tested 20% diquat aqua is a relatively common and widely used preparation product. Diquat is one of the few herbicides that can be directly used in aquatic systems, has been found in the surface water of many rivers and reservoirs, but no clear conclusions could be drawn in the risk assessment of aquatic organisms (Hiltibran 1972; Rosic et al. 2020). It is important to understand the sublethal effects of exposure to ecological related concentrations of diquat on fish. As a multifunctional important organ of fish, the liver plays a key role in metabolism and detoxification. The habitat of fish determines that the toxic and harmful substances in the water body are more likely to damage the liver, although the liver possesses an extraordinary repair ability. This work illustrated the liver changes of zebrafish exposed to diquat for a long time.
The high concentration of diquat we used was approximately equivalent to the concentration applied in the water body. The zebrafish liver cells showed obvious enlargement and vacuolization, blurred cell boundaries, cell deformation and disappearance of nucleus, which showed that the liver cells were damaged, the cell membrane system was destroyed, and there was a precursor to cell necrosis or apoptosis. Exposure to herbicide diquat lead to the increase of redox cycling compounds which generate the formation of active oxygen radical, and mediates systemic toxicity (Eklöw-Låstbom et al. 1986). A large number of relevant research results have demonstrated that oxidative stress is the common pathophysiological basis for the pathogenesis of various chronic liver diseases in fish (Stewart et al. 2004; Gonzalez et al. 2005). The changes of biochemical indexes also showed that diquat exposure destroyed the redox state of zebrafish liver and affected liver function. High dosage of diquat treatment significantly increased the MDA content in zebrafish liver cells, indicated that the increased level of lipid peroxidation inducing membrane damage, which is consistent with the cell edema and blurred contour observed in histopathology. It was also observed that there was no significant change in antioxidant enzyme SOD activity and a compensatory increase in CAT activity. Similar to the results in a previous study that diquat exposure caused zebrafish embryos to show higher transcript levels of CAT compared to control fish and without altering SOD gene expression (Wang et al. 2018). Diquat induces oxidative stress and changes the activity of antioxidant enzymes in cell, the degree of damage depends on the exposure time and concentration (Fussell et al. 2011; Nisar et al. 2015). There are evidences that CAT gene expression can be induced by environmental stressors and poisons (Cupertino et al. 2017). Although increased the CAT activity represents a counter-regulatory reaction to toxicity caused by mild stress from diquat, this reaction is insufficient to prevent liver pathological damage. In this case, we also observed decrease of the GST activity at 35 days. Inconsistent with the research of Sanchez et al. (2006) that 222 µg·L−1 and 444 µg·L−1 diquat induced increase in activities of GST of Gasterosteus aculeatus for 21 days. GST catalyze the conversion of a variety of electrophilic metabolites with glutathione into hydrophilic compounds, and accelerate the detoxification and excretion of harmful compounds. Prolonged stimulation of diquat at high concentration impaired the detoxification ability of the liver and might lead to the accumulation of toxic substances in hepatocyte. Additionally, high dosage of diquat in this experiment induced the increase in liver ALT levels, and AST also increased but no significant difference, indicating that amino acid decomposition increased, liver synthesis function was hindered, metabolism and detoxification ability decreased. Small changes in AST suggested that there was no damage to the liver parenchyma (Eklöw-Låstbom et al. 1986). Significant histopathological injury and changes of biochemical indexes were not detected in zebrafish after 35 d exposure to 0.34 mg·L−1diquat. Environmental concentration of diquat may not elicit the same significant stress response on adult zebrafish as larvae (Wang et al. 2018).
Liver damage caused by any hepatotoxic agent will cause endogenous severely disordered metabolites (Xu et al, 2015; Wang et al, 2017; Liu et al, 2018). We used GC/MS metabolomics to understand the biological principle by identifying important metabolic differences, and discussed the potential mechanism of diquat induced hepatotoxicity based on metabolic networks to provide in-depth insights into the toxic effect. The metabolome approach is very sensitive as an early toxicity identifier based on metabolite changes and can provide useful information (Li et al. 2014; Xu et al. 2015; Wang et al. 2017). In this study, the metabolic profiling of two treatment groups discriminated themselves from the control. Histopathology and clinical chemistry can only observe the significant changes when the concentration of diquat is 1.69 mg·L−1. The current GC-MS analysis can prove the difference between diquat and normal group at the dose level of 0.34 mg·L−1. Similarly, it was found that goldfish after long-term exposure to glyphosate had slight changes in the level of metabolites in the brain, but no histopathological damage was observed (Li et al. 2017). Schoonen et al. (2007) used NMR spectroscopy to analyze mouse urine, which increased a 4- to 16-fold sensitivity versus histopathology and clinical chemistry to recognize early events of liver toxicity. Additionally, the present analysis results support that the response of aquatic species under low and high stress may be different, and the generation of compensatory response or serious toxic damage is affected by the dose of toxic substances. The potential response scheme of zebrafish is summarized in Fig. 6.
Noteworthy aspartic acid up-regulation and glutamate/glutamine down-regulation were in the two exposure groups (Table 1 and Table 2). Aspartic acid and glutamic acid are important intermediates in the alanine, aspartate and glutamate metabolism, that was the most influenced common pathway in the two exposure groups, play a role in energy storage (Fig. 5). As a major metabolic fuel source, the decreased glutamate content demonstrated the reduction of energy reserves. Aspartic acid is the precursor of oxaloacetic acid, an important intermediate in the tricarboxylic acid cycle, and its accumulation suggested that aerobic metabolism might be hindered. At low concentration diquat group, the increase of glycerol 3-phosphate, an intermediate in glycolysis, reflected the transition of energy metabolism from aerobic respiration to anaerobic respiration (Li et al. 2014). It has been reported that diquat can damage the gills, the respiratory organs vital to the survival of fish, and definitely cause breathing difficulties and hypoxia (Berry et al. 1984). In addition, diquat was effective at generating ROS in redox cycling assays with recombinant cytochrome P450 reductase, with the reduction of intracellular oxygen available for metabolic processes (Fussell et al. 2011). A significant decrease in oxygen induces a greater reliance on anaerobic glycolysis to obtain ATP supply (Speers-Roesch et al. 2010). It has been proved that the energy metabolism of goldfish exposed on λ-cyhalothrin is more dependent on anaerobic respiration to provide energy (Li et al. 2014). However, the decrease in lactic acid and inositol, as products of anaerobic respiration, were observed in the high concentration diquat group. The result suggested 1.69 mg·L−1 diquat caused obvious damage to liver cells, not only the aerobic respiration was restricted, but the anaerobic pathway was also affected and cannot played a good role in energy compensation. Moreover, this research found that creatine increased in the diquat low-concentration group and decreased in the high-concentration one. Creatine is a well-known energy booster in cellular energy homeostasis (Dworak et al. 2014). A few of studies have reported that the creatine-phosphocreatine system plays a key role in cellular energy metabolism (Oudman et al. 2013). Fluctuations of creatine in zebrafish liver caused by diquat exposure indicated that the disturbance of energy metabolism consistented with the above conclusions. In addition, since creatine also has an indirect antioxidant effect, the activation of creatine at low concentration of diquat could play a certain role in enhancing protection (Junior and Pereira 2008). Energy expenditure is essential for immune function (Eikenaar et al. 2019). The inhibitory effect of diquat exposure on oxygen and energy metabolism severely disrupted the metabolic balance, hindered the synthesis of substances and energy, and posed a challenge to the fish.
D-Ribose and 6-phosphogluconic acid, metabolites in the pentose phosphate pathway, increased in zebrafish liver after exposure to two concentrations of diquat. At the same time, the increase of uridine in the two exposure groups suggested that the enhancement of pentose phosphate pathway, which is the bypass of sugar metabolism provide raw materials for nucleic acid biosynthesis, was reasonable. According to the results of metabolomic analysis, as the raw material for purine and pyrimidine production, the change of pentose affected the downstream nucleotide metabolism. The results of McCuaig et al. (2020) regarding the proteomic of rainbow trout after exposure to diquat showed that the RNA process was enhanced. This imbalance in nucleotide levels is usually associated with immunodeficiency, energy metabolism, and multicellular functions (Aird and Zhang, 2015). In addition, the increased level of metabolism of the pentose phosphate pathway provides more NADPH with reducing power. NADPH provides electrons for the reductive biosynthesis of fatty acids and cholesterol. In the low-concentration diquat group, significant fluctuations in lipid metabolism were observed, and the content of various fatty acids and their derivatives increased. Studies have shown that the chronic exposure of some herbicides and fungicides disturbed the hepatic glycolipid metabolism at sublethal doses and the progression to fatty liver were confirmed (Mesnage et al. 2017; Bao et al. 2020). Diquat and paraquat rely on NADPH for enzymatic single-electron reduction during the redox cycle to form free radical cations (Fussell et al. 2011). The high concentration of diquat occupied a large amount of NADPH for oxidative cycle. Accordingly, the fluctuation of fatty acids observed in metabolomics was weaker than that in the low concentration group. Only one substance monoste arin was observed to increase, which was also included in the differences in the low concentration group. NADPH promotes the regeneration of reduced glutathione, and several studies have confirmed that maintaining glutathione can alleviate the toxicity of diquat to cells and animals (Awad et al. 1994; Rogers et al. 2006; Djurdjevic et al. 2013). Exposure to 1.69 mg·L−1 diquat reduced pantothenic acid in glutathione metabolism of zebrafish, and the content of precursor glutamate also decreased, indicating that glutathione synthesis was weakened. The decrease of GST activity observed in the detection of biochemical indexes were consistent with these results. Disturbance of the pentose phosphate pathway caused by diquat disrupts the physiological state of the body may be one of the reasons for the serious symptoms of fish.
According to the metabolic results, changes in a large number of amino acids and derivatives were observed in the high-concentration diquat exposure group. Among the 16 impacted pathways, 10 were related to amino acid metabolism, and two pathways were also present at low concentrations diquat group. Significant changes in amino acids are widely observed in the metabolic profiles of aquatic species exposed to various environmental pollutants (Xu et al. 2015; Wang et al. 2017; Li et al. 2018). In the 1.69 mg·L−1 diquat treatment group, the increase of liver transaminase level indicated the enhancement of amino acid catabolism, which was consistent with the downward trend of most amino acids. Free amino acids could function in energy storage and as molecular modulators involved in important physiological processes (Nagato et al. 2016; Zhang and Zhao 2017). Alanine, as a sugar-generating amino acid, together with glutamic acid generates succinic acid, an important intermediate in the tricarboxylic acid cycle. The reduction of alanine may be used to compensate for the lack of carbohydrates to reduce the dependence on oxygen in hypoxia. Glutamate is not only related to energy metabolism, but also a key modulator in the initiation and development of immune cells (Pacheco et al. 2007; Xue 2011). The decrease of its content will affect the regulation of immune function. Phenylalanine is the precursor of tyrosine, both of which are significantly reduced in the high-dose diquat group. They could be used as an energy substrate to cope with stress, and converted into the neurotransmitter dopamine (Salamanca et al. 2020). Some studies have shown that long-term exposure to diquat causes neurodegenerative diseases such as Parkinson's disease symptoms (Sechi et al. 1992; Baltazar et al. 2014). The decrease of dopamine uptake, one of the important factors of neurodegenerative diseases, could be explained by the lack of these precursor substances. When exposed to 1.69 mg·L−1 diquat, putrescine, the downstream substance of arginine and proline metabolism, decreased and guanidinoacetate, the upstream substance, accumulated, which indicated that the pathway might be inhibited in zebrafish liver. It has been reported that the obstruction of arginine and proline metabolism might be one of the reasons for liver injury mediated by reactive oxygen (Tzirogiannis et al. 2004; Tkachenko et al. 2012). Additionally, glycine aminotransferase catalyzed the amino transfer of arginine to glycine to produce glycocyamine, the direct precursor of creatine, decreased in this study and creatine showed an increase (Item et al. 2001). The research of Tachikawa et al. (2015) showed that significant change in creatine was also associated with abnormal behavior caused by neurotransmitter metabolism disorder in fish.
The accumulation of galactose at 1.69 mg·L−1 diquat exposure supported the hypothesis that anaerobic respiration was inhibited, and correspondingly, inositol, as a by-product of galactose metabolism, was observed to decrease. Inositol is not only related to energy metabolism, but also a precursor molecule for many secondary messengers, including inositol phosphates. Inositol phosphates mediates important intracellular signal transduction and play key roles in a variety of cell physiological processes, such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration. The metabolic analysis of liver showed that inositol decreased after exposure to diquat, which may lead to the weakening of signal pathway. Park and Koh (2019) proposed that the apoptosis of PC12 cells induced by diquat was related to the activation of caspase-3 and the inhibition of mTOR, which both are downstream molecules of the PI3K/Akt pathway. PI3K/Akt signaling pathway disorders are closely related to the onset and subsequent development of neurodegenerative diseases (Nakano et al. 2017). In addition, some studies have shown that supplementation of free inositol as an immune enhancer help increase feed conversion and reduce liver fat level for fish to cope with adverse conditions (Shiau and Su 2005). Therefore, low levels of inositol in 1.69 mg·L−1 diquat exposure group may be harmful to energy utilization, cause signal conduction to be blocked, and impair a variety of physiological functions.
In conclusion, the hepatotoxic effects of chronic exposure to sublethal doses of diquat were investigated. The results showed that diquat induced hepatic damage and dysregulation of various metabolic pathways involving energy metabolism, lipid metabolism, nucleotide metabolism and amino acid metabolism. The metabolome method is very sensitive as a toxicity identifier to the environmental concentration of diquat according to the change of metabolites. The difference in the effects of two exposure doses on metabolic characteristics reflected different mechanisms. This work provides new insights into the toxic effects of diquat, enabled a comprehensive evaluation of the toxicology beyond the physiological and biochemical changes on zebrafish upon diquat exposure and to propose a potential explanation regarding a series of metabolic pathways disruptions. The specific toxic effects observed in this study, such as neurotransmitter disorders and fat accumulation in the liver, should be further investigated to evaluate toxicity.