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Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice
Environmental Pollution xxx (xxxx) xxx
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Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice* Zhiyuan Meng a, Li Liu b, Yexun Xi a, Ming Jia a, Sen Yan a, Sinuo Tian a, Wei Sun a, Wentao Zhu a, Xuefeng Li a, Zhiqiang Zhou a, * a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Applied Chemistry, China Agricultural University, Beijing 100193, China School of Food Science and Engineering, Yangzhou University, Yangzhou 225127, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2019 Received in revised form 29 October 2019 Accepted 31 October 2019 Available online xxx
(±) - PEN is a chiral fungicide widely used to control powdery mildew in agriculture. Currently, only a few studies have investigated the toxic effects of (±) e penconazole ((±) e PEN) on non-target organisms, and whether (±) - PEN from the enantiomeric level have toxic effects remains unclear. In this study, we systematically evaluated the effects of exposure to (±) e PEN, (þ) e PEN and () e PEN on liver function in mice. Biochemical and histopathological analyses showed that exposure to (±) e PEN and () e PEN led to significant liver damage and inflammation. However, exposure to (þ) e PEN treatment did not cause no adverse effects on liver function and inflammation. 1H-NMR-based metabolomics revealed that exposure to (±) e PEN, (þ) e PEN and () e PEN led to the animals developing liver metabolic disorder that was caused by changes in glycolipid metabolism. Quantitative analysis of genes regulating glycolipid metabolism revealed that expression of gluconeogenesis and glycolytic pathway genes were altered in individuals exposed to (±) e PEN, (þ) e PEN and () e PEN. We also found that (±) e PEN, (þ) e PEN and () e PEN have different effects on lipid metabolism of the liver. Exposure to (±) e PEN and () e PEN resulted in significant accumulation of lipids by regulating fatty acid synthesis, triglyceride synthesis, and fatty acid b oxidation pathways. In summary, we found different toxicological effects in individuals exposed to (±) e PEN, (þ) e PEN and () e PEN. The results of this study are important for assessing the potential health risks of (±) e PEN. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Penconazole Enantiomers Metabolomics Glycolipid metabolism
1. Introduction As a typical triazole fungicide, (±) - penconazole ((±) - PEN, CAS: 66246-88-6) inhibits the biosynthesis of fungal ergosterols and is widely applied to fruits, vegetables and tea plants to control powdery mildew (Bicchi et al., 2001; Pose-Juan et al., 2010). In agricultural production, (±) - PEN is sprayed directly into the environment, and much of the sprayed substance is absorbed by the surrounding environment (Husak et al., 2017). Previous studies have shown that (±) - PEN and its major metabolites (±) - PEN-OH and (±) - PEN-COOH were detected in human urine (Mercadante et al., 2016; Sams et al., 2016). Moreover, exposure to (±) - PEN has been reported to have adverse effects on various organisms.
* This paper has been recommended for acceptance by Dr. Da Chen. * Corresponding author. E-mail address: [email protected] (Z. Zhou).
Specifically, exposure to (±) - PEN affects the growth and protein content of Scenedesmus acutus (Agirman et al., 2015), and exposure to (±) - PEN alters the survival of zebrafish (Danio rerio) embryos and causes dysplasia (Aksakal and Ciltas, 2018). Similarly, exposure to (±) - PEN disrupts the antioxidant system of the carp tissue of goldfish (Husak et al., 2017). For mammals, (±) - PEN has a lower acute toxicity, but it significantly affects testicular morphology and function, as well as the redox status of brain tissue in rats (Chaabane et al., 2017; El-Sharkawy and El-Nisr, 2013). In addition, the endocrine disruptions of (±) - PEN in human T-47D cells were confirmed, suggesting that (±) e PEN may have adverse effects on humans (Perdichizzi et al., 2014). The liver regulates metabolism of both exogenous and endogenous compounds (Sozen et al., 2015). The liver disorders can arise from various environmental factors, such as diet and exposure to toxic compounds (Meng et al., 2019a,b; Sampey et al., 2011). Recent studies have shown that exposure to some pesticides can affect liver metabolic functions. Specifically, oral exposure to
https://doi.org/10.1016/j.envpol.2019.113555 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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carbendazim can induce liver lipid metabolic disorders, which increases lipid accumulation in the liver of mice (Jin et al., 2015). Amino acid and glycolipid metabolism in the liver are negatively affected in zebrafish exposed to imazalil (Jin et al., 2017). In addition, myclobutanol significantly affects liver metabolic functions by altering the relative expression level of genes involved in metabolism in male Mongolia Racerunner (Zhu et al., 2018). In particular, the World Health Organization (WHO) has raised concern that exposure to (±) - PEN may cause liver toxicity (WHO/FAO, 2016). However, despite these concerns, the mechanism of effects of (±) PEN on liver functions of organisms are unclear. Therefore, it is necessary to systematically study the effects of (±) - PEN exposure on liver function using a vertebrate model, such as mice. PEN is a chiral pesticide that has two enantiomers: (þ) e penconazole ((þ) e PEN) and () - penconazole (() e PEN) (Fig. S1). Some chiral pesticides are stereoselective in its biological activity (Dong et al., 2013; Kurihara et al., 1999), toxicity (Huang et al., 2012; Kenneke et al., 2009) and degradation (Buerge et al., 2006; Wang et al., 2012). The processing of (±) - PEN in vegetables can be stereoselective. For example, ()- PEN is preferentially degraded and (þ)-PEN is enriched in cabbage and pakchoi, while cucumber and tomato do not display stereoselectivity when metabolizing (±) PEN (Wang et al., 2014). In addition, recent studies have shown that (±) e PEN, (þ) e PEN and () e PEN could have different effects on the gut microbiota and metabolic profile in mice (Meng et al., 2019a,b). In summary, some studies have shown that (±) e PEN has adverse ecotoxicological effects and provides useful information for environmental risk assessment. Differently, this study aimed to investigate the effects of (±) e PEN (þ) e PEN and () e PEN on liver function in mice. The results of the study aimed to elucidate the intrinsic mechanism of the effects of (±) e PEN (þ) e PEN and () e PEN on liver function in mice and provide more comprehensive toxicological risk assessment data. In this study, the different toxic effects of (±) - PEN, (þ) e PEN and () - PEN on the liver of mice were systematically assessed. The effects of exposure to (±) - PEN, (þ) e PEN and () - PEN on liver injury were assessed by biochemical and histopathology analyses. Furthermore, changes in endogenous metabolites that are stored in the liver were determined using 1H Nuclear Magnetic Resonance (1H NMR) non-targeted metabolomics. Furthermore, whether exposure to (±) - PEN, (þ) e PEN and () - PEN have different toxic effects on the liver were elucidated by quantifying relative expression levels of genes involved in glycolipid metabolism. Taken together, we provide new insights into the toxicological effects of (±) - PEN on mammals and further clarify the potential health risks of (±) - PEN. 2. Materials and methods 2.1. Animal and treatments A total of 32 male ICR mouse at 5 weeks of age were obtained from the Peking University Health Science Center (Beijing, China). All mice were maintained on a 12 h light and dark cycle in a barrier facility without pathogens. Test mice were given free access to water and a normal diet that was purchased from Keao Xieli Feed Co., Ltd. (Beijing, China). In order to assess the toxic effects of penconazole and its enantiomers, mice were randomly divided into four groups after a week of adaptation (n ¼ 8). Each group of mice was housed in one cage. Six week old ICR mice were expose to (±) PEN, (þ) - PEN and () - PEN at dosage of 30 mg/L through their drinking water for four weeks. The dosage was selected according to the allowable daily intake (ADI) (0.03 mg/kg bw/day) and the no observed effect level (NOEL) (0.71 mg/kg bw/day) (WHO/FAO, 2016) for long-term toxicity in mice. The control group (Vehicle)
Fig. 1. Experimental design. The ICR mice (6 weeks old) were expose to (±) - PEN, (þ) e PEN and () - PEN at dosage of 30 mg/L added to drinking water for a duration of 4 weeks. At the end of the experiment, the different effects of exposure to penconazole and its enantiomers on liver function of male mice were studied.
was treated with deionized water. Body weights were measured weekly in the morning. In addition, at the end of the experiment, the weights of liver tissue and epididymal white adipose tissue (epididymal WAT) were weighed. The experimental design is shown in Fig. 1. The liver tissue and serum samples from each mouse were collected at the end of the experiment and stored at 80 C. All animal experiments were carried out according to the independent Animal Ethical Committee of China Agricultural University. 2.2. Biochemical analysis and histopathology analysis The activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and contents of liver triglyceride (TG) and total cholesterol (T-Cho) were measured using assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions. Liver tissue samples were randomly selected from each treatment groups for histopathology analysis. Liver tissue was fixed in 10% neutral buffered formalin and embedded in paraffin for histological examination. Tissue sections were stained with hematoxylineeosin (H&E) and analyzed under light microscopy at 100 and 400 magnification. 2.3.
1
H NMR based liver metabolomics analysis
The liver samples of six mice from each group were randomly selected for 1H NMR based metabolomics analysis. Briefly, 50 mg liver tissue were directly extracted using methanol-water mixture (v/v ¼ 2/1) and homogenized with a MM 400 (Retsch GmbH, Germany). The extraction method was described in detail in previous studies (Meng et al., 2018). The 1H NMR analysis of liver extracts were analyzed using a Bruker Avance III 600 NMR spectrometer (MA, USA) following protocols from a previous study (Wang et al., 2018). Finally, multivariate statistical analysis of the spectral data were performed using SIMCA P (Umetrics, Sweden). Principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were used to identify metabolites that showed significant changes in all treatment groups. 2.4. Gene expression analysis Total RNA was extracted from liver tissue of the different treatment groups (Vehicle, (±) e PEN, (þ) e PEN and () e PEN) using the TRIzol reagent. Then, 1.5 mg RNA samples were used to generate cDNA using Fast Quant RT Kit (with gDNase) following the manufacturers’ instructions. TRIzol reagent and Fast Quant RT Kit were obtained from Tiangen Biochemical Technology (Beijing, China). RT-qPCR was performed using SuperReal PreMix Plus (SYBR Green) and the Bio-Rad CFX 96 PCR system. The relative abundance
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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of gene expression was normalized using the deltaedelta cycle threshold (Ct) method, with GAPDH as the reference gene. Primers were obtained from Sangon Biochemical Technology (Shanghai, China) and are listed in Table S1. 2.5. Statistical analysis All experimental values are expressed as the mean ± SEM and the data were assessed by one-way ANOVA and a Tukey’s post-hoc test followed by significant difference test using SPSS 19.0 (IBM, USA). The normality of the variables was confirmed by the ShapiroeWilk test and homogeneity of variance by the Levene’s test. A non-parametric KruskaleWallis was used when data did not meet the parametric assumptions. GraphPad Prism version 6.0 (GraphPad Software, Inc., USA) was used for Graphical illustrations. Asterisk (*) indicates a statistically significant difference between the control and treatment groups (p < 0:05). A hashtag (#) indicates a statistically significant difference between the (±) e PEN treatment group and (þ) - PEN or () - PEN treatment groups (p < 0.05). 3. Results 3.1. Effects of exposure to penconazole and its enantiomers on growth phenotypes We first analyzed the effects of (±)-PEN, (þ) - PEN and () - PEN exposure on growth of mice (Fig. 2). There were no adverse effects on body weight in animals exposed to (±)-PEN, (þ) - PEN and () e PEN compared to the control group treated with vehicle (water) (Fig. 2A). Liver weight was significantly reduced after 28 days of exposure to (þ) e PEN (P < 0.05) (Fig. 2B). In addition, the ratios of liver weight to body weight were significantly higher in individuals exposed to (±) e PEN and () e PEN compared to the control group (P < 0.05) (Fig. 2C). Treatment with (±)-PEN, (þ) - PEN and () e PEN did not affect epididymal WAT weight and the ratios of epididymal WAT weight to body weight (Fig. 2D and E). These
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observations suggest that exposure to (±) e PEN, (þ) - PEN and () e PEN may affect liver metabolic function. 3.2. Effects of exposure to penconazole and its enantiomers on liver function and inflammation The significant change in the liver weight and the ratio of liver weight to body weight in animals exposed to (±) e PEN, (þ) - PEN and () e PEN prompted us to explore the effects of penconazole and its enantiomers on liver tissue. The liver TG and T-Cho contents and the activities of serum AST and ALT were quantified. We found that TG and T-Cho contents significantly decreased in the (þ) e PEN treatment group. However, compare to the control vehicle group, TG and T-Cho contents were significantly higher in individuals exposed to () e PEN (P < 0.05). In the (±) - PEN treatment group, there was no significant changes in TG and T-Cho contents (p > 0.05) (Fig. 3A and B). Furthermore, serum ALT and AST activity levels were significantly increased in animals exposed to (±) e PEN and () e PEN compared to the vehicle group (P < 0.05). Exposure to (þ) e PEN did not affect serum ALT and AST activity levels (p > 0.05) (Fig. 3C and D). The activity of serum ALT and AST are associated with hepatic injuries, therefore we next conducted histopathological analyses of the liver samples (Fig. 3E). A large number of massive micro or macro vesicular intracellular lipid droplets were apparent in the (±) - PEN and () e PEN treatment groups. Interestingly, exposure to (þ) e PEN did not cause significant changes in hepatic sections. This suggested that exposure to (±) - PEN and () e PEN significantly deteriorated steatosis and caused hepatic injuries. We next quantified the relative mRNA expressions of the inflammation related genes Tnf-a, IL-1b and IL-6 (Fig. 3F). Tnf-a and IL-6 expression levels were up-regulated in the (±) e PEN and () e PEN treatment groups compared to the vehicle group (P < 0.05). However, expression levels of Tnf-a, IL-1b and IL-6 were similar for animals exposed to (þ) e PEN and control vehicle group (p > 0.05). These results indicated that exposure to (±) e PEN and () e PEN may cause inflammation of the liver.
Fig. 2. Effects of penconazole and its enantiomers exposure on growth phenotypes. A: Body weight. BeC: Liver weight and %Liver weight/Body weight. DeE: Epididymal WAT weight and %epiWAT weight/Body weight. (n ¼ 6 mouse per group). Data are expressed as mean ± SD. *p < 0.05 compared with the Vehicle treatment group. #p < 0.05 compared with (±) e PEN treatment group.
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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Fig. 3. Effects of penconazole and its enantiomers exposure on hepatic steatosis and injury. AeB: The contents of liver triglycerides (TG) and total cholesterol (T-Cho). CeD: The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). E: Representative images of H&E staining of hepatic sections from different groups. F: Relative mRNA expression of inflammatory related genes in liver tissue. (n ¼ 3e6 mouse per group). Data are expressed as mean ± SD. *p < 0.05 compared with the Vehicle treatment group. #p < 0.05 compared with (±) e PEN treatment group.
3.3. Effects of exposure to penconazole and its enantiomers on metabolic profiles of the liver The effects of exposure to (±) e PEN, (þ) e PEN and () - PEN on the metabolic profile of the liver were studied by using 1H NMR based metabolomics analysis. A representative 1H NMR spectrum of liver samples is shown in Fig. S2. A total of 31 metabolites showed a chemical shift and peak intensity in the liver samples. The PCA score plots were made using NMR data of liver samples, which shored a clear separation between the (±) ePEN, (þ) e PEN and () e PEN treatment groups and the vehicle group (Fig. 4A), suggesting that the metabolic profiles of the liver were significant altered in
the three treatment group. Metabolites with significant changes in relative abundance were identified by OPLS-DA scored plots and Splot models (Fig. 4B, C and D). The metabolites that were disrupted upon exposure to (±) ePEN, (þ) e PEN and () e PEN are shown in Table 1 and Fig. 5. There were 15 metabolites that significantly changed in animals exposed to (±) ePEN, (þ) e PEN and () e PEN compared to the control vehicle group (Table 1). A heatmap was made and cluster analyses were performed to test differences in the relative abundances of the target 17 metabolites (Fig. 5A). Interestingly, changes in metabolic profiles caused by (±) ePEN, (þ) e PEN and () e PEN exposure were highly similar. The relative abundances of the following 15 metabolites were significantly
Fig. 4. The score plot of PCA and OPLS-DA models constructed using 1H NMR data of liver samples. A: PCA score plot of all treatment groups. BeD: OPLS-DA scored plots and corresponding S-plots were obtained from pairwise comparisons between three treatments groups and vehicle group.
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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Table 1 Identification of significant changed metabolites based on VIP scores and P values of one way ANOVA.
different in the three treatment groups compared to the control: bile acid, lipid, choline, phosphocholine (PC), glycogen, a-glucose, b-glucose, lactate, acetate, isoleucine, leucine, valine, lysine, alanine and glutamate. In addition, the relative abundances of glycerophosphocholine (GPC) and glutamine were significant changed in the (±) ePEN and () e PEN groups (P < 0.05). Subsequently, the metabolic pathways were analyzed using the MetaboAnalyst 4.0 to identify metabolic pathways that were significantly affected by exposure to penconazole in the liver, which identified three metabolic pathways involved in glycolipid metabolism, amino acid metabolism and glutamine and glutamate metabolism as significantly disrupted (Table 1). Importantly, the relative abundances of 10 metabolites related to liver glycolipid metabolism were significantly altered (Fig. 5B). Among them, the relative abundance of lipids increased significantly in the (±) e PEN and () e PEN treatment groups and significantly decreased in the (þ) e PEN treatment group compared to vehicle group. Moreover, the relative abundances of glycogen, a-glucose and b-glucose significantly decreased in animals exposed to (±) ePEN, (þ) e PEN and () e PEN. Taken together, the metabolomics data demonstrate that exposure to (±) ePEN, (þ) e PEN and () e PEN significantly affects liver metabolic homeostasis in mice.
glycogen synthesis (Gys2), glycolysis (Gck and Pklr) and glucose transport (Glut2) (Fig. 6, Fig. S4). Srebp-1c, Acaca and Dgat1, which are involved in lipid metabolism, were significantly up-regulated in the (±) ePEN treatment group (P < 0.05). In addition, the relative expression level of Ppara significantly decreased in livers of animals exposed to (±) ePEN (P < 0.05). In the (þ) e PEN treatment group, the expression of one lipid metabolism related gene, Cpt-1a, was significantly down-regulated compared to the vehicle group (P < 0.05). In contrast, exposure to () e PEN significantly upregulated the expression of Srebp-1c, Fasn, Pparg, Dgat1, Migat1 and Cpt-1a compared to the control (P < 0.05). Ppara expression was significantly decreased in animals exposed to (þ) ePEN (P < 0.05) (Fig. 6B). For genes involved in glucose metabolism, exposure to (±) ePEN, (þ) e PEN and () e PEN significantly increased expression G6pase and decreased expression of Pklr (P < 0.05). Exposure to () e PEN also led to a significant decrease in Gck expression and a significant increase in Glut2 expression (P < 0.05) (Fig. 6C). Taken together, the expression patterns of genes involved in glycolipid metabolism related genes were disrupted to varying degrees in animals exposed to (±) e PEN, (þ) e PEN and () e PEN.
4. Discussions 3.4. Effects of exposure to penconazole and its enantiomers on expression of genes involved in glycolipid metabolism To elucidate the mechanism of glycolipid metabolism disorders which was induced by (±) ePEN, (þ) e PEN and () e PEN exposure, we further quantified the relative expression of several glycolipid metabolism related genes involved in TG and fatty acid synthesis (Srebp-1c, Fasn, Scd1, Acaca, Dgat1, Dgat2 and Mogat1), fatty acid uptake and transport (Pparg, Cd36 and Mttp), fatty acid boxidation (Ppara and Cpt-1a), gluconeogenesis (G6Pase and Pepck),
In recent years, the toxic effects of (±) e PEN on different organisms have been reported. (±) e PEN disrupts the functions of testis and brain tissue in rats (Mercadante et al., 2016; Sams et al., 2016), and includes developmental toxicity of zebrafish embryos (Aksakal and Ciltas, 2018). However, the effects of (±) e PEN on liver function remains poorly understood. In addition, studies on the different toxic effects of (±) e PEN and its enantiomers have not been reported. Therefore, we studied the effects of exposure to (±) e PEN, (þ) e PEN and () e PEN on liver metabolic function in
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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Fig. 5. The heat map generated by hierarchical clustering of the potential discriminating metabolites obtained from the vehicle, (±) ePEN, (þ) e PEN and () e PEN treatment groups (A). The significant changed metabolites related to liver glycolipid metabolism in male mouse (B). (n ¼ 6 mouse per group). Data are expressed as mean ± SD. *p < 0.05 compared with the Vehicle treatment group.
mice. We found that the liver metabolic profile of mice were significantly altered upon exposure to (±) e PEN, (þ) e PEN and () e PEN. Among the pathways that regulate metabolism in the liver, glycolipid metabolism was particularly disrupted in our treatment conditions. In particular, the disruptions to liver metabolic function were stronger in animals exposed to (±) e PEN and () e PEN compared to (þ) - PEN. We first tested the effects of (±) e PEN, (þ) e PEN and () e PEN on growth phenotypes of mice. It is well known that liver tissue converted absorbed glucose into fatty acids and triglycerides, which were secreted as a major component of low density lipoproteins. Subsequently, it is absorbed by fat cells through the action of lipoprotein lipase and is mainly stored in white adipose tissue (Kuriyama et al., 2005). Due to the role of liver and white adipose tissue in maintaining the body’s glycolipid homeostasis, its weights were investigated. The results showed that the growth of the liver was most significantly affected in the three treatment groups. This prompted us to further explore the effects of penconazole and its enantiomers on liver metabolic functions. The activity levels of serum ALT and AST were significantly increased in (±) e PEN and
() e PEN treatment groups. Increased ALT and AST activity levels are sensitive indicators for liver dysfunction and liver damage (CoxNorth, 2009; Dube et al., 2014). Furthermore, the liver damage induced by (±) e PEN and () e PEN treatments were verified through histopathological alterations. We also found a significant increase in the relative expression levels of inflammation related genes Tnf-a and IL-6. This suggested that expose to (±) e PEN and () e PEN leads to increased inflammation of the liver in mice. Interestingly, adverse effects were observed in the (þ) e PEN treatment group. Inflammation is considered to accompany development of many diseases, and inflammation is closely linked to liver metabolic disorders (Egbuonu et al., 2013; Esser et al., 2014). We further studied the effects of (±) e PEN, (þ) e PEN and () e PEN exposure on the liver metabolic profile using 1H NMR metabolomics analysis. In recent years, metabolomics has been widely used in environmental toxicology studies to determine the effects of exogenous contaminants on the metabolism of endogenous pollutants (Lankadurai et al., 2013; Wang et al., 2018). Metabolomics analysis of the liver showed that exposure to (±) e PEN, (þ) e PEN and () e PEN significantly altered the metabolic
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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Fig. 6. Relative mRNA expression of glycolipid metabolism related genes in liver tissue of male mouse. A: The heat map generated by the relative expression level of glycolipid metabolism related genes. B: Significantly altered lipid metabolism related genes. C: Significantly altered glucose metabolism related genes. (n ¼ 6 mouse per group). Data are expressed as mean ± SD. *p < 0.05 compared with the Vehicle treatment group. #p < 0.05 compared with (±) e PEN treatment group.
profile of compounds involved in glycolipid metabolism, amino acid metabolism and glutamine and glutamate metabolism. Among these, the glycolipid metabolism pathway was significantly disturbed in the (±) e PEN, (þ) e PEN and () e PEN treatment groups. Similar results were observed exposed to fungicides propamocarb and carbendazim in mouse, where exposure induced hepatic glycolipid metabolism disorders by inducing gut microbiota dysbiosis (Jin et al., 2015; Wu et al., 2018). Consistently, our previous studies have shown that exposure to (±) e PEN, (þ) e PEN
and () e PEN could also cause disorders in gut microbiota (Meng et al., 2019a,b). To further understand the effects of exposure to (±) e PEN, (þ) e PEN and () e PEN on hepatic glycolipid metabolism, the relative expression levels of genes involved in glycolipid metabolism were quantified. Exposure to (±) e PEN, (þ) e PEN and () e PEN altered the expression levels of genes involved in glycolipid metabolism. However, changes in expression pattern were different in animals treated with (±) e PEN, (þ) e PEN and () e PEN. Exposure to (±) e PEN and () e PEN led to significant up-
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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regulation of Srebp-1c and Dgat1 and down-regulation of Ppara. Srebp-1c is an important transcriptional regulator that can induce changes in the expression levels of fatty acid synthesis, genes such as Acaca and Fasn (Ho et al., 2019). Dgat1 plays an important role in the synthesis of triglycerides, where can esterify exogenous fatty acids into triglycerides (Chitraju et al., 2017; Chitraju et al., 2019). In addition, PPARa, as a key enzyme in the fatty acid b oxidation of liver, plays a very important role in liver lipid accumulation (Pawlak et al., 2015; Souza-Mello, 2015). In agreement to these findings, exposure to (±) e PEN and () e PEN promoted the biosynthesis of fatty acids and triglycerides, inhibited fatty acid oxidation, and led to the accumulation of lipids in the liver. However, exposure to (þ) e PEN only changed the expression pattern of Cpt-1a, which is involved in fatty acid oxidation (Tian et al., 2018). For hepatic glucose metabolism, the relative expression levels of G6pase and Pklr were significantly altered with the exposure to (±) e PEN, (þ) e PEN and () e PEN, suggesting that both gluconeogenesis and glycolysis are significantly disturbed in the (±) e PEN, (þ) e PEN and () e PEN treatment groups. In addition, exposure to () e PEN significantly increased the expression of Glut2, which is involved in glycogen transport (Bektur et al., 2019). The above results confirmed that liver glycolipid homeostasis was disrupted in animals exposed to (±) e PEN, (þ) e PEN and () e PEN. As the main source of energy supply for organisms, glycolipid metabolism plays a key role in life activities. Glycolipid metabolism disorders contribute to the development of complications associated with diabetes and metabolic syndrome (Perry et al., 2014). Long-term abnormal glucose and lipid levels could cause damage to the organs of the body and lead to a gradual decline in function (Lan et al., 2015). Consistently, in mouse studies, we found that exposure to penconazole and its enantiomers interfered with liver glycolipid metabolism and further affected liver function. Consistently, in mouse studies, we found that exposure to penconazole and its enantiomers interfered with liver glycolipid metabolism and further affected liver function. This moaned that it may pose a potential threat to human health. Further, comparing the toxicity differences between the two enantiomers of penconazole, we found that (þ) - PEN had lesser effects on glycolipid metabolism. This suggested that the application of (þ) - PEN could reduce the risks to human health. Of course, this hypothesis still needs to be proven through a large number of studies involving target activity, metabolic behavior, and the like. In general, exposure to (±) e PEN, (þ) e PEN and () e PEN resulted in an imbalance in liver glycolipid metabolism. In particular, (±) e PEN and () e PEN significantly promoted the accumulation of lipids in the liver by interfering with fatty acid synthesis, triglyceride synthesis and fatty acid b oxidation pathways. These changes may be the main mechanism underlying liver damage and inflammation caused by (±) e PEN and () e PEN exposure. Furthermore, both gluconeogenesis and glycolysis pathways were disturbed, which likely contributed to the significant reduction in glycogen, a-glucose and b-glucose in the (±) e PEN, (þ) e PEN and () e PEN treatment groups. 5. Conclusion In this study, the effects of exposure to (±) e PEN, (þ) e PEN and () e PEN on liver function of mice were investigated, specifically focusing on liver damage and metabolic profiles. Metabolomics analyses revealed that exposure to (±) e PEN, (þ) e PEN and () e PEN disrupted metabolic function of the liver, including glycolipid metabolism, amino acid metabolism and glutamine and glutamate metabolism. Mainly, glycolipid metabolism pathways were the most affected by exposure to (±) e PEN, (þ) e PEN and () e PEN. Consistent with this finding, exposure to (±) e PEN, (þ) e PEN and
() e PEN significantly reduced glycogen, a-glucose and b-glucose in the liver by interfering with gluconeogenesis and glycolysis pathways. Unlike (þ) e PEN, exposure to (±) e PEN and () e PEN significantly affected lipid metabolism pathways, including fatty acid synthesis, triglyceride synthesis and fatty acid b oxidation, and these changes likely underlie lipid accumulation in the liver. Moreover, (±) e PEN and () e PEN also induced liver damage and inflammation. Although we observed different toxicological effects between (±) e PEN, (þ) e PEN and () e PEN, the underlying molecular mechanisms for their toxicity requires further investigation. Declaration of competing interest I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously. The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgments We gratefully acknowledge the financial support from National Key Research and Development Program of China (2016YFD0200202), and the National Natural Science Foundation of China (21577171). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113555. References Agirman, N., Bedil, B., Kendirlioglu, G., Cetin, A.K., 2015. Toxic effects of fungicides (penconazole and triadimenol) on growth and protein amount of Scenedesmus acutus. J. Chem. Soc. Pak. 37, 1220e1225. Aksakal, F.I., Ciltas, A., 2018. Developmental toxicity of penconazole in Zebrafish (Danio rerio) embryos. Chemosphere 200, 8e15. Bektur, E., Sahin, E., Baycu, C., 2019. Mirtazapine may show anti-hyperglycemic effect by decreasing GLUT2 through leptin and galanin expressions in the liver of type 1 diabetic rats. Iranian Journal of Basic Medical Sciences 22, 676e682. Bicchi, C., Cordero, C., Rubiolo, P., Occelli, A., 2001. Simultaneous determination of six triazolic pesticide residues in apple and pear pulps by liquid chromatography with ultraviolet diode array detection. J. AOAC Int. 84, 1543e1550. Buerge, I.J., Poiger, T., Muller, M.D., Buser, H.R., 2006. Influence of pH on the stereoselective degradation of the fungicides epoxiconazole and cyproconazole in soils. Environ. Sci. Technol. 40, 5443e5450. Chaabane, M., Ghorbel, I., Elwej, A., Mnif, H., Boudawara, T., Chaabouni, S.E., Zeghal, N., Soudani, N., 2017. Penconazole alters redox status, cholinergic function, and membrane-bound ATPases in the cerebrum and cerebellum of adult rats. Hum. Exp. Toxicol. 36, 854e866. Chitraju, C., Mejhert, N., Haas, J.T., Diaz-Ramirez, L.G., Grueter, C.A., Imbriglio, J.E., Pinto, S., Koliwad, S.K., Walther, T.C., Farese Jr., R.V., 2017. Triglyceride synthesis by DGAT1 protects adipocytes from lipid-induced ER stress during lipolysis. Cell Metabol. 26, 407e418. Chitraju, C., Walther, T.C., Farese Jr., R.V., 2019. The triglyceride synthesis enzymes DGAT1 and DGAT2 have distinct and overlapping functions in adipocytes. JLR (J. Lipid Res.) 60, 1112e1120. Cox-North, P.P., 2009. The relationship of hepatitis antibodies and elevated liver enzymes with impaired fasting glucose and undiagnosed diabetes. J. Am. Board Fam. Med. 22, 339-339. Dong, F., Li, J., Chankvetadze, B., Cheng, Y., Xu, J., Liu, X., Li, Y., Chen, X., Bertucci, C., Tedesco, D., Zanasi, R., Zheng, Y., 2013. Chiral triazole fungicide difenoconazole: absolute stereochemistry, stereoselective bioactivity, aquatic toxicity, and environmental behavior in vegetables and soil. Environ. Sci. Technol. 47, 3386e3394. Dube, P.N., Shwetha, A., Hosetti, B.B., 2014. Impact of copper cyanide on the key metabolic enzymes of freshwater fish Catla catla (Hamilton). Biotechnol. Anim. Husb. 30, 499e508. Egbuonu, A.C.C., Ezeanyika, L.U.S., Ijeh, I.I., 2013. Alterations in the liver histology and markers of metabolic syndrome associated with inflammation and liver
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Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice* Zhiyuan Meng a, Li Liu b, Yexun Xi a, Ming Jia a, Sen Yan a, Sinuo Tian a, Wei Sun a, Wentao Zhu a, Xuefeng Li a, Zhiqiang Zhou a, * a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Applied Chemistry, China Agricultural University, Beijing 100193, China School of Food Science and Engineering, Yangzhou University, Yangzhou 225127, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2019 Received in revised form 29 October 2019 Accepted 31 October 2019 Available online xxx
(±) - PEN is a chiral fungicide widely used to control powdery mildew in agriculture. Currently, only a few studies have investigated the toxic effects of (±) e penconazole ((±) e PEN) on non-target organisms, and whether (±) - PEN from the enantiomeric level have toxic effects remains unclear. In this study, we systematically evaluated the effects of exposure to (±) e PEN, (þ) e PEN and () e PEN on liver function in mice. Biochemical and histopathological analyses showed that exposure to (±) e PEN and () e PEN led to significant liver damage and inflammation. However, exposure to (þ) e PEN treatment did not cause no adverse effects on liver function and inflammation. 1H-NMR-based metabolomics revealed that exposure to (±) e PEN, (þ) e PEN and () e PEN led to the animals developing liver metabolic disorder that was caused by changes in glycolipid metabolism. Quantitative analysis of genes regulating glycolipid metabolism revealed that expression of gluconeogenesis and glycolytic pathway genes were altered in individuals exposed to (±) e PEN, (þ) e PEN and () e PEN. We also found that (±) e PEN, (þ) e PEN and () e PEN have different effects on lipid metabolism of the liver. Exposure to (±) e PEN and () e PEN resulted in significant accumulation of lipids by regulating fatty acid synthesis, triglyceride synthesis, and fatty acid b oxidation pathways. In summary, we found different toxicological effects in individuals exposed to (±) e PEN, (þ) e PEN and () e PEN. The results of this study are important for assessing the potential health risks of (±) e PEN. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Penconazole Enantiomers Metabolomics Glycolipid metabolism
1. Introduction As a typical triazole fungicide, (±) - penconazole ((±) - PEN, CAS: 66246-88-6) inhibits the biosynthesis of fungal ergosterols and is widely applied to fruits, vegetables and tea plants to control powdery mildew (Bicchi et al., 2001; Pose-Juan et al., 2010). In agricultural production, (±) - PEN is sprayed directly into the environment, and much of the sprayed substance is absorbed by the surrounding environment (Husak et al., 2017). Previous studies have shown that (±) - PEN and its major metabolites (±) - PEN-OH and (±) - PEN-COOH were detected in human urine (Mercadante et al., 2016; Sams et al., 2016). Moreover, exposure to (±) - PEN has been reported to have adverse effects on various organisms.
* This paper has been recommended for acceptance by Dr. Da Chen. * Corresponding author. E-mail address: [email protected] (Z. Zhou).
Specifically, exposure to (±) - PEN affects the growth and protein content of Scenedesmus acutus (Agirman et al., 2015), and exposure to (±) - PEN alters the survival of zebrafish (Danio rerio) embryos and causes dysplasia (Aksakal and Ciltas, 2018). Similarly, exposure to (±) - PEN disrupts the antioxidant system of the carp tissue of goldfish (Husak et al., 2017). For mammals, (±) - PEN has a lower acute toxicity, but it significantly affects testicular morphology and function, as well as the redox status of brain tissue in rats (Chaabane et al., 2017; El-Sharkawy and El-Nisr, 2013). In addition, the endocrine disruptions of (±) - PEN in human T-47D cells were confirmed, suggesting that (±) e PEN may have adverse effects on humans (Perdichizzi et al., 2014). The liver regulates metabolism of both exogenous and endogenous compounds (Sozen et al., 2015). The liver disorders can arise from various environmental factors, such as diet and exposure to toxic compounds (Meng et al., 2019a,b; Sampey et al., 2011). Recent studies have shown that exposure to some pesticides can affect liver metabolic functions. Specifically, oral exposure to
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Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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carbendazim can induce liver lipid metabolic disorders, which increases lipid accumulation in the liver of mice (Jin et al., 2015). Amino acid and glycolipid metabolism in the liver are negatively affected in zebrafish exposed to imazalil (Jin et al., 2017). In addition, myclobutanol significantly affects liver metabolic functions by altering the relative expression level of genes involved in metabolism in male Mongolia Racerunner (Zhu et al., 2018). In particular, the World Health Organization (WHO) has raised concern that exposure to (±) - PEN may cause liver toxicity (WHO/FAO, 2016). However, despite these concerns, the mechanism of effects of (±) PEN on liver functions of organisms are unclear. Therefore, it is necessary to systematically study the effects of (±) - PEN exposure on liver function using a vertebrate model, such as mice. PEN is a chiral pesticide that has two enantiomers: (þ) e penconazole ((þ) e PEN) and () - penconazole (() e PEN) (Fig. S1). Some chiral pesticides are stereoselective in its biological activity (Dong et al., 2013; Kurihara et al., 1999), toxicity (Huang et al., 2012; Kenneke et al., 2009) and degradation (Buerge et al., 2006; Wang et al., 2012). The processing of (±) - PEN in vegetables can be stereoselective. For example, ()- PEN is preferentially degraded and (þ)-PEN is enriched in cabbage and pakchoi, while cucumber and tomato do not display stereoselectivity when metabolizing (±) PEN (Wang et al., 2014). In addition, recent studies have shown that (±) e PEN, (þ) e PEN and () e PEN could have different effects on the gut microbiota and metabolic profile in mice (Meng et al., 2019a,b). In summary, some studies have shown that (±) e PEN has adverse ecotoxicological effects and provides useful information for environmental risk assessment. Differently, this study aimed to investigate the effects of (±) e PEN (þ) e PEN and () e PEN on liver function in mice. The results of the study aimed to elucidate the intrinsic mechanism of the effects of (±) e PEN (þ) e PEN and () e PEN on liver function in mice and provide more comprehensive toxicological risk assessment data. In this study, the different toxic effects of (±) - PEN, (þ) e PEN and () - PEN on the liver of mice were systematically assessed. The effects of exposure to (±) - PEN, (þ) e PEN and () - PEN on liver injury were assessed by biochemical and histopathology analyses. Furthermore, changes in endogenous metabolites that are stored in the liver were determined using 1H Nuclear Magnetic Resonance (1H NMR) non-targeted metabolomics. Furthermore, whether exposure to (±) - PEN, (þ) e PEN and () - PEN have different toxic effects on the liver were elucidated by quantifying relative expression levels of genes involved in glycolipid metabolism. Taken together, we provide new insights into the toxicological effects of (±) - PEN on mammals and further clarify the potential health risks of (±) - PEN. 2. Materials and methods 2.1. Animal and treatments A total of 32 male ICR mouse at 5 weeks of age were obtained from the Peking University Health Science Center (Beijing, China). All mice were maintained on a 12 h light and dark cycle in a barrier facility without pathogens. Test mice were given free access to water and a normal diet that was purchased from Keao Xieli Feed Co., Ltd. (Beijing, China). In order to assess the toxic effects of penconazole and its enantiomers, mice were randomly divided into four groups after a week of adaptation (n ¼ 8). Each group of mice was housed in one cage. Six week old ICR mice were expose to (±) PEN, (þ) - PEN and () - PEN at dosage of 30 mg/L through their drinking water for four weeks. The dosage was selected according to the allowable daily intake (ADI) (0.03 mg/kg bw/day) and the no observed effect level (NOEL) (0.71 mg/kg bw/day) (WHO/FAO, 2016) for long-term toxicity in mice. The control group (Vehicle)
Fig. 1. Experimental design. The ICR mice (6 weeks old) were expose to (±) - PEN, (þ) e PEN and () - PEN at dosage of 30 mg/L added to drinking water for a duration of 4 weeks. At the end of the experiment, the different effects of exposure to penconazole and its enantiomers on liver function of male mice were studied.
was treated with deionized water. Body weights were measured weekly in the morning. In addition, at the end of the experiment, the weights of liver tissue and epididymal white adipose tissue (epididymal WAT) were weighed. The experimental design is shown in Fig. 1. The liver tissue and serum samples from each mouse were collected at the end of the experiment and stored at 80 C. All animal experiments were carried out according to the independent Animal Ethical Committee of China Agricultural University. 2.2. Biochemical analysis and histopathology analysis The activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and contents of liver triglyceride (TG) and total cholesterol (T-Cho) were measured using assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions. Liver tissue samples were randomly selected from each treatment groups for histopathology analysis. Liver tissue was fixed in 10% neutral buffered formalin and embedded in paraffin for histological examination. Tissue sections were stained with hematoxylineeosin (H&E) and analyzed under light microscopy at 100 and 400 magnification. 2.3.
1
H NMR based liver metabolomics analysis
The liver samples of six mice from each group were randomly selected for 1H NMR based metabolomics analysis. Briefly, 50 mg liver tissue were directly extracted using methanol-water mixture (v/v ¼ 2/1) and homogenized with a MM 400 (Retsch GmbH, Germany). The extraction method was described in detail in previous studies (Meng et al., 2018). The 1H NMR analysis of liver extracts were analyzed using a Bruker Avance III 600 NMR spectrometer (MA, USA) following protocols from a previous study (Wang et al., 2018). Finally, multivariate statistical analysis of the spectral data were performed using SIMCA P (Umetrics, Sweden). Principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were used to identify metabolites that showed significant changes in all treatment groups. 2.4. Gene expression analysis Total RNA was extracted from liver tissue of the different treatment groups (Vehicle, (±) e PEN, (þ) e PEN and () e PEN) using the TRIzol reagent. Then, 1.5 mg RNA samples were used to generate cDNA using Fast Quant RT Kit (with gDNase) following the manufacturers’ instructions. TRIzol reagent and Fast Quant RT Kit were obtained from Tiangen Biochemical Technology (Beijing, China). RT-qPCR was performed using SuperReal PreMix Plus (SYBR Green) and the Bio-Rad CFX 96 PCR system. The relative abundance
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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of gene expression was normalized using the deltaedelta cycle threshold (Ct) method, with GAPDH as the reference gene. Primers were obtained from Sangon Biochemical Technology (Shanghai, China) and are listed in Table S1. 2.5. Statistical analysis All experimental values are expressed as the mean ± SEM and the data were assessed by one-way ANOVA and a Tukey’s post-hoc test followed by significant difference test using SPSS 19.0 (IBM, USA). The normality of the variables was confirmed by the ShapiroeWilk test and homogeneity of variance by the Levene’s test. A non-parametric KruskaleWallis was used when data did not meet the parametric assumptions. GraphPad Prism version 6.0 (GraphPad Software, Inc., USA) was used for Graphical illustrations. Asterisk (*) indicates a statistically significant difference between the control and treatment groups (p < 0:05). A hashtag (#) indicates a statistically significant difference between the (±) e PEN treatment group and (þ) - PEN or () - PEN treatment groups (p < 0.05). 3. Results 3.1. Effects of exposure to penconazole and its enantiomers on growth phenotypes We first analyzed the effects of (±)-PEN, (þ) - PEN and () - PEN exposure on growth of mice (Fig. 2). There were no adverse effects on body weight in animals exposed to (±)-PEN, (þ) - PEN and () e PEN compared to the control group treated with vehicle (water) (Fig. 2A). Liver weight was significantly reduced after 28 days of exposure to (þ) e PEN (P < 0.05) (Fig. 2B). In addition, the ratios of liver weight to body weight were significantly higher in individuals exposed to (±) e PEN and () e PEN compared to the control group (P < 0.05) (Fig. 2C). Treatment with (±)-PEN, (þ) - PEN and () e PEN did not affect epididymal WAT weight and the ratios of epididymal WAT weight to body weight (Fig. 2D and E). These
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observations suggest that exposure to (±) e PEN, (þ) - PEN and () e PEN may affect liver metabolic function. 3.2. Effects of exposure to penconazole and its enantiomers on liver function and inflammation The significant change in the liver weight and the ratio of liver weight to body weight in animals exposed to (±) e PEN, (þ) - PEN and () e PEN prompted us to explore the effects of penconazole and its enantiomers on liver tissue. The liver TG and T-Cho contents and the activities of serum AST and ALT were quantified. We found that TG and T-Cho contents significantly decreased in the (þ) e PEN treatment group. However, compare to the control vehicle group, TG and T-Cho contents were significantly higher in individuals exposed to () e PEN (P < 0.05). In the (±) - PEN treatment group, there was no significant changes in TG and T-Cho contents (p > 0.05) (Fig. 3A and B). Furthermore, serum ALT and AST activity levels were significantly increased in animals exposed to (±) e PEN and () e PEN compared to the vehicle group (P < 0.05). Exposure to (þ) e PEN did not affect serum ALT and AST activity levels (p > 0.05) (Fig. 3C and D). The activity of serum ALT and AST are associated with hepatic injuries, therefore we next conducted histopathological analyses of the liver samples (Fig. 3E). A large number of massive micro or macro vesicular intracellular lipid droplets were apparent in the (±) - PEN and () e PEN treatment groups. Interestingly, exposure to (þ) e PEN did not cause significant changes in hepatic sections. This suggested that exposure to (±) - PEN and () e PEN significantly deteriorated steatosis and caused hepatic injuries. We next quantified the relative mRNA expressions of the inflammation related genes Tnf-a, IL-1b and IL-6 (Fig. 3F). Tnf-a and IL-6 expression levels were up-regulated in the (±) e PEN and () e PEN treatment groups compared to the vehicle group (P < 0.05). However, expression levels of Tnf-a, IL-1b and IL-6 were similar for animals exposed to (þ) e PEN and control vehicle group (p > 0.05). These results indicated that exposure to (±) e PEN and () e PEN may cause inflammation of the liver.
Fig. 2. Effects of penconazole and its enantiomers exposure on growth phenotypes. A: Body weight. BeC: Liver weight and %Liver weight/Body weight. DeE: Epididymal WAT weight and %epiWAT weight/Body weight. (n ¼ 6 mouse per group). Data are expressed as mean ± SD. *p < 0.05 compared with the Vehicle treatment group. #p < 0.05 compared with (±) e PEN treatment group.
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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Fig. 3. Effects of penconazole and its enantiomers exposure on hepatic steatosis and injury. AeB: The contents of liver triglycerides (TG) and total cholesterol (T-Cho). CeD: The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). E: Representative images of H&E staining of hepatic sections from different groups. F: Relative mRNA expression of inflammatory related genes in liver tissue. (n ¼ 3e6 mouse per group). Data are expressed as mean ± SD. *p < 0.05 compared with the Vehicle treatment group. #p < 0.05 compared with (±) e PEN treatment group.
3.3. Effects of exposure to penconazole and its enantiomers on metabolic profiles of the liver The effects of exposure to (±) e PEN, (þ) e PEN and () - PEN on the metabolic profile of the liver were studied by using 1H NMR based metabolomics analysis. A representative 1H NMR spectrum of liver samples is shown in Fig. S2. A total of 31 metabolites showed a chemical shift and peak intensity in the liver samples. The PCA score plots were made using NMR data of liver samples, which shored a clear separation between the (±) ePEN, (þ) e PEN and () e PEN treatment groups and the vehicle group (Fig. 4A), suggesting that the metabolic profiles of the liver were significant altered in
the three treatment group. Metabolites with significant changes in relative abundance were identified by OPLS-DA scored plots and Splot models (Fig. 4B, C and D). The metabolites that were disrupted upon exposure to (±) ePEN, (þ) e PEN and () e PEN are shown in Table 1 and Fig. 5. There were 15 metabolites that significantly changed in animals exposed to (±) ePEN, (þ) e PEN and () e PEN compared to the control vehicle group (Table 1). A heatmap was made and cluster analyses were performed to test differences in the relative abundances of the target 17 metabolites (Fig. 5A). Interestingly, changes in metabolic profiles caused by (±) ePEN, (þ) e PEN and () e PEN exposure were highly similar. The relative abundances of the following 15 metabolites were significantly
Fig. 4. The score plot of PCA and OPLS-DA models constructed using 1H NMR data of liver samples. A: PCA score plot of all treatment groups. BeD: OPLS-DA scored plots and corresponding S-plots were obtained from pairwise comparisons between three treatments groups and vehicle group.
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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Table 1 Identification of significant changed metabolites based on VIP scores and P values of one way ANOVA.
different in the three treatment groups compared to the control: bile acid, lipid, choline, phosphocholine (PC), glycogen, a-glucose, b-glucose, lactate, acetate, isoleucine, leucine, valine, lysine, alanine and glutamate. In addition, the relative abundances of glycerophosphocholine (GPC) and glutamine were significant changed in the (±) ePEN and () e PEN groups (P < 0.05). Subsequently, the metabolic pathways were analyzed using the MetaboAnalyst 4.0 to identify metabolic pathways that were significantly affected by exposure to penconazole in the liver, which identified three metabolic pathways involved in glycolipid metabolism, amino acid metabolism and glutamine and glutamate metabolism as significantly disrupted (Table 1). Importantly, the relative abundances of 10 metabolites related to liver glycolipid metabolism were significantly altered (Fig. 5B). Among them, the relative abundance of lipids increased significantly in the (±) e PEN and () e PEN treatment groups and significantly decreased in the (þ) e PEN treatment group compared to vehicle group. Moreover, the relative abundances of glycogen, a-glucose and b-glucose significantly decreased in animals exposed to (±) ePEN, (þ) e PEN and () e PEN. Taken together, the metabolomics data demonstrate that exposure to (±) ePEN, (þ) e PEN and () e PEN significantly affects liver metabolic homeostasis in mice.
glycogen synthesis (Gys2), glycolysis (Gck and Pklr) and glucose transport (Glut2) (Fig. 6, Fig. S4). Srebp-1c, Acaca and Dgat1, which are involved in lipid metabolism, were significantly up-regulated in the (±) ePEN treatment group (P < 0.05). In addition, the relative expression level of Ppara significantly decreased in livers of animals exposed to (±) ePEN (P < 0.05). In the (þ) e PEN treatment group, the expression of one lipid metabolism related gene, Cpt-1a, was significantly down-regulated compared to the vehicle group (P < 0.05). In contrast, exposure to () e PEN significantly upregulated the expression of Srebp-1c, Fasn, Pparg, Dgat1, Migat1 and Cpt-1a compared to the control (P < 0.05). Ppara expression was significantly decreased in animals exposed to (þ) ePEN (P < 0.05) (Fig. 6B). For genes involved in glucose metabolism, exposure to (±) ePEN, (þ) e PEN and () e PEN significantly increased expression G6pase and decreased expression of Pklr (P < 0.05). Exposure to () e PEN also led to a significant decrease in Gck expression and a significant increase in Glut2 expression (P < 0.05) (Fig. 6C). Taken together, the expression patterns of genes involved in glycolipid metabolism related genes were disrupted to varying degrees in animals exposed to (±) e PEN, (þ) e PEN and () e PEN.
4. Discussions 3.4. Effects of exposure to penconazole and its enantiomers on expression of genes involved in glycolipid metabolism To elucidate the mechanism of glycolipid metabolism disorders which was induced by (±) ePEN, (þ) e PEN and () e PEN exposure, we further quantified the relative expression of several glycolipid metabolism related genes involved in TG and fatty acid synthesis (Srebp-1c, Fasn, Scd1, Acaca, Dgat1, Dgat2 and Mogat1), fatty acid uptake and transport (Pparg, Cd36 and Mttp), fatty acid boxidation (Ppara and Cpt-1a), gluconeogenesis (G6Pase and Pepck),
In recent years, the toxic effects of (±) e PEN on different organisms have been reported. (±) e PEN disrupts the functions of testis and brain tissue in rats (Mercadante et al., 2016; Sams et al., 2016), and includes developmental toxicity of zebrafish embryos (Aksakal and Ciltas, 2018). However, the effects of (±) e PEN on liver function remains poorly understood. In addition, studies on the different toxic effects of (±) e PEN and its enantiomers have not been reported. Therefore, we studied the effects of exposure to (±) e PEN, (þ) e PEN and () e PEN on liver metabolic function in
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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Fig. 5. The heat map generated by hierarchical clustering of the potential discriminating metabolites obtained from the vehicle, (±) ePEN, (þ) e PEN and () e PEN treatment groups (A). The significant changed metabolites related to liver glycolipid metabolism in male mouse (B). (n ¼ 6 mouse per group). Data are expressed as mean ± SD. *p < 0.05 compared with the Vehicle treatment group.
mice. We found that the liver metabolic profile of mice were significantly altered upon exposure to (±) e PEN, (þ) e PEN and () e PEN. Among the pathways that regulate metabolism in the liver, glycolipid metabolism was particularly disrupted in our treatment conditions. In particular, the disruptions to liver metabolic function were stronger in animals exposed to (±) e PEN and () e PEN compared to (þ) - PEN. We first tested the effects of (±) e PEN, (þ) e PEN and () e PEN on growth phenotypes of mice. It is well known that liver tissue converted absorbed glucose into fatty acids and triglycerides, which were secreted as a major component of low density lipoproteins. Subsequently, it is absorbed by fat cells through the action of lipoprotein lipase and is mainly stored in white adipose tissue (Kuriyama et al., 2005). Due to the role of liver and white adipose tissue in maintaining the body’s glycolipid homeostasis, its weights were investigated. The results showed that the growth of the liver was most significantly affected in the three treatment groups. This prompted us to further explore the effects of penconazole and its enantiomers on liver metabolic functions. The activity levels of serum ALT and AST were significantly increased in (±) e PEN and
() e PEN treatment groups. Increased ALT and AST activity levels are sensitive indicators for liver dysfunction and liver damage (CoxNorth, 2009; Dube et al., 2014). Furthermore, the liver damage induced by (±) e PEN and () e PEN treatments were verified through histopathological alterations. We also found a significant increase in the relative expression levels of inflammation related genes Tnf-a and IL-6. This suggested that expose to (±) e PEN and () e PEN leads to increased inflammation of the liver in mice. Interestingly, adverse effects were observed in the (þ) e PEN treatment group. Inflammation is considered to accompany development of many diseases, and inflammation is closely linked to liver metabolic disorders (Egbuonu et al., 2013; Esser et al., 2014). We further studied the effects of (±) e PEN, (þ) e PEN and () e PEN exposure on the liver metabolic profile using 1H NMR metabolomics analysis. In recent years, metabolomics has been widely used in environmental toxicology studies to determine the effects of exogenous contaminants on the metabolism of endogenous pollutants (Lankadurai et al., 2013; Wang et al., 2018). Metabolomics analysis of the liver showed that exposure to (±) e PEN, (þ) e PEN and () e PEN significantly altered the metabolic
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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Fig. 6. Relative mRNA expression of glycolipid metabolism related genes in liver tissue of male mouse. A: The heat map generated by the relative expression level of glycolipid metabolism related genes. B: Significantly altered lipid metabolism related genes. C: Significantly altered glucose metabolism related genes. (n ¼ 6 mouse per group). Data are expressed as mean ± SD. *p < 0.05 compared with the Vehicle treatment group. #p < 0.05 compared with (±) e PEN treatment group.
profile of compounds involved in glycolipid metabolism, amino acid metabolism and glutamine and glutamate metabolism. Among these, the glycolipid metabolism pathway was significantly disturbed in the (±) e PEN, (þ) e PEN and () e PEN treatment groups. Similar results were observed exposed to fungicides propamocarb and carbendazim in mouse, where exposure induced hepatic glycolipid metabolism disorders by inducing gut microbiota dysbiosis (Jin et al., 2015; Wu et al., 2018). Consistently, our previous studies have shown that exposure to (±) e PEN, (þ) e PEN
and () e PEN could also cause disorders in gut microbiota (Meng et al., 2019a,b). To further understand the effects of exposure to (±) e PEN, (þ) e PEN and () e PEN on hepatic glycolipid metabolism, the relative expression levels of genes involved in glycolipid metabolism were quantified. Exposure to (±) e PEN, (þ) e PEN and () e PEN altered the expression levels of genes involved in glycolipid metabolism. However, changes in expression pattern were different in animals treated with (±) e PEN, (þ) e PEN and () e PEN. Exposure to (±) e PEN and () e PEN led to significant up-
Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555
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regulation of Srebp-1c and Dgat1 and down-regulation of Ppara. Srebp-1c is an important transcriptional regulator that can induce changes in the expression levels of fatty acid synthesis, genes such as Acaca and Fasn (Ho et al., 2019). Dgat1 plays an important role in the synthesis of triglycerides, where can esterify exogenous fatty acids into triglycerides (Chitraju et al., 2017; Chitraju et al., 2019). In addition, PPARa, as a key enzyme in the fatty acid b oxidation of liver, plays a very important role in liver lipid accumulation (Pawlak et al., 2015; Souza-Mello, 2015). In agreement to these findings, exposure to (±) e PEN and () e PEN promoted the biosynthesis of fatty acids and triglycerides, inhibited fatty acid oxidation, and led to the accumulation of lipids in the liver. However, exposure to (þ) e PEN only changed the expression pattern of Cpt-1a, which is involved in fatty acid oxidation (Tian et al., 2018). For hepatic glucose metabolism, the relative expression levels of G6pase and Pklr were significantly altered with the exposure to (±) e PEN, (þ) e PEN and () e PEN, suggesting that both gluconeogenesis and glycolysis are significantly disturbed in the (±) e PEN, (þ) e PEN and () e PEN treatment groups. In addition, exposure to () e PEN significantly increased the expression of Glut2, which is involved in glycogen transport (Bektur et al., 2019). The above results confirmed that liver glycolipid homeostasis was disrupted in animals exposed to (±) e PEN, (þ) e PEN and () e PEN. As the main source of energy supply for organisms, glycolipid metabolism plays a key role in life activities. Glycolipid metabolism disorders contribute to the development of complications associated with diabetes and metabolic syndrome (Perry et al., 2014). Long-term abnormal glucose and lipid levels could cause damage to the organs of the body and lead to a gradual decline in function (Lan et al., 2015). Consistently, in mouse studies, we found that exposure to penconazole and its enantiomers interfered with liver glycolipid metabolism and further affected liver function. Consistently, in mouse studies, we found that exposure to penconazole and its enantiomers interfered with liver glycolipid metabolism and further affected liver function. This moaned that it may pose a potential threat to human health. Further, comparing the toxicity differences between the two enantiomers of penconazole, we found that (þ) - PEN had lesser effects on glycolipid metabolism. This suggested that the application of (þ) - PEN could reduce the risks to human health. Of course, this hypothesis still needs to be proven through a large number of studies involving target activity, metabolic behavior, and the like. In general, exposure to (±) e PEN, (þ) e PEN and () e PEN resulted in an imbalance in liver glycolipid metabolism. In particular, (±) e PEN and () e PEN significantly promoted the accumulation of lipids in the liver by interfering with fatty acid synthesis, triglyceride synthesis and fatty acid b oxidation pathways. These changes may be the main mechanism underlying liver damage and inflammation caused by (±) e PEN and () e PEN exposure. Furthermore, both gluconeogenesis and glycolysis pathways were disturbed, which likely contributed to the significant reduction in glycogen, a-glucose and b-glucose in the (±) e PEN, (þ) e PEN and () e PEN treatment groups. 5. Conclusion In this study, the effects of exposure to (±) e PEN, (þ) e PEN and () e PEN on liver function of mice were investigated, specifically focusing on liver damage and metabolic profiles. Metabolomics analyses revealed that exposure to (±) e PEN, (þ) e PEN and () e PEN disrupted metabolic function of the liver, including glycolipid metabolism, amino acid metabolism and glutamine and glutamate metabolism. Mainly, glycolipid metabolism pathways were the most affected by exposure to (±) e PEN, (þ) e PEN and () e PEN. Consistent with this finding, exposure to (±) e PEN, (þ) e PEN and
() e PEN significantly reduced glycogen, a-glucose and b-glucose in the liver by interfering with gluconeogenesis and glycolysis pathways. Unlike (þ) e PEN, exposure to (±) e PEN and () e PEN significantly affected lipid metabolism pathways, including fatty acid synthesis, triglyceride synthesis and fatty acid b oxidation, and these changes likely underlie lipid accumulation in the liver. Moreover, (±) e PEN and () e PEN also induced liver damage and inflammation. Although we observed different toxicological effects between (±) e PEN, (þ) e PEN and () e PEN, the underlying molecular mechanisms for their toxicity requires further investigation. Declaration of competing interest I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously. The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgments We gratefully acknowledge the financial support from National Key Research and Development Program of China (2016YFD0200202), and the National Natural Science Foundation of China (21577171). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113555. References Agirman, N., Bedil, B., Kendirlioglu, G., Cetin, A.K., 2015. Toxic effects of fungicides (penconazole and triadimenol) on growth and protein amount of Scenedesmus acutus. J. Chem. Soc. Pak. 37, 1220e1225. Aksakal, F.I., Ciltas, A., 2018. Developmental toxicity of penconazole in Zebrafish (Danio rerio) embryos. Chemosphere 200, 8e15. Bektur, E., Sahin, E., Baycu, C., 2019. Mirtazapine may show anti-hyperglycemic effect by decreasing GLUT2 through leptin and galanin expressions in the liver of type 1 diabetic rats. Iranian Journal of Basic Medical Sciences 22, 676e682. Bicchi, C., Cordero, C., Rubiolo, P., Occelli, A., 2001. Simultaneous determination of six triazolic pesticide residues in apple and pear pulps by liquid chromatography with ultraviolet diode array detection. J. AOAC Int. 84, 1543e1550. Buerge, I.J., Poiger, T., Muller, M.D., Buser, H.R., 2006. Influence of pH on the stereoselective degradation of the fungicides epoxiconazole and cyproconazole in soils. Environ. Sci. Technol. 40, 5443e5450. Chaabane, M., Ghorbel, I., Elwej, A., Mnif, H., Boudawara, T., Chaabouni, S.E., Zeghal, N., Soudani, N., 2017. Penconazole alters redox status, cholinergic function, and membrane-bound ATPases in the cerebrum and cerebellum of adult rats. Hum. Exp. Toxicol. 36, 854e866. Chitraju, C., Mejhert, N., Haas, J.T., Diaz-Ramirez, L.G., Grueter, C.A., Imbriglio, J.E., Pinto, S., Koliwad, S.K., Walther, T.C., Farese Jr., R.V., 2017. Triglyceride synthesis by DGAT1 protects adipocytes from lipid-induced ER stress during lipolysis. Cell Metabol. 26, 407e418. Chitraju, C., Walther, T.C., Farese Jr., R.V., 2019. The triglyceride synthesis enzymes DGAT1 and DGAT2 have distinct and overlapping functions in adipocytes. JLR (J. Lipid Res.) 60, 1112e1120. Cox-North, P.P., 2009. The relationship of hepatitis antibodies and elevated liver enzymes with impaired fasting glucose and undiagnosed diabetes. J. Am. Board Fam. Med. 22, 339-339. Dong, F., Li, J., Chankvetadze, B., Cheng, Y., Xu, J., Liu, X., Li, Y., Chen, X., Bertucci, C., Tedesco, D., Zanasi, R., Zheng, Y., 2013. Chiral triazole fungicide difenoconazole: absolute stereochemistry, stereoselective bioactivity, aquatic toxicity, and environmental behavior in vegetables and soil. Environ. Sci. Technol. 47, 3386e3394. Dube, P.N., Shwetha, A., Hosetti, B.B., 2014. Impact of copper cyanide on the key metabolic enzymes of freshwater fish Catla catla (Hamilton). Biotechnol. Anim. Husb. 30, 499e508. Egbuonu, A.C.C., Ezeanyika, L.U.S., Ijeh, I.I., 2013. Alterations in the liver histology and markers of metabolic syndrome associated with inflammation and liver
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Please cite this article as: Meng, Z et al., Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113555