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Comparison of subacute effects of two types of pyrethroid insecticides using metabolomics methods
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Comparison of subacute effects of two types of pyrethroid insecticides using metabolomics methods Jiyan Miao, Dezhen Wang, Jin Yan, Yao Wang, Miaomiao Teng, Zhiqiang Zhou, Wentao Zhu⁎ Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Applied Chemistry, China Agricultural University, Beijing 100193, P.R. China
A R T I C L E I N F O
A B S T R A C T
Keywords: Bifenthrin Lambda-cyhalothrin Metabolomics 1 H NMR LC-MS/MS
In this study, 1H NMR based metabolomics analysis, LC-MS/MS based serum metabolomics and histopathology techniques were used to investigate the toxic effects of subacute exposure to two types of pyrethroid insecticides bifenthrin and lambda-cyhalothrin in mice. Metabolomic analysis of tissues extracts and serum showed that these two types of pyrethroid insecticides resulted in alterations of metabolites in the liver, kidney and serum of mice. Based on the altered metabolites, several significant pathways were identified, which are associated with gut microbial metabolism, lipid metabolism, nucleotide catabolism, tyrosine metabolism and energy metabolism. The results showed that bifenthrin and lambda-cyhalothrin have similarities in disruption of metabolic pathways in kidney, indicating that the toxicological mechanisms of these two types of insecticides have some likeness to each other. This study may provide novel insight into revealing differences of toxicological mechanisms between these two types of pyrethroid insecticides.
1. Introduction Pyrethroids are botanical insecticides which are synthetic derivatives of pyrethrins and have been used for many years. However, most of pyrethroids are defined as moderately hazardous (Class II) by the World Health Organization (WHO, 2009) [1]. The residues of pyrethroids have been detected in fruits, vegetables, tea, pasteurized milk and porcine muscle [2]. Pyrethroids' widely use has posed a serious risk to environment and human. Therefore, it may be an urgent need to evaluate the possible adverse effects of their use. Pyrethroids can be assorted into two types. Type II pyrethroids have an alpha-cyano moiety at the α-position whereas type I don't [3]. Type I pyrethroids affect the peripheral nerves and type II pyrethroids poison the central nervous system. Therefore, type II pyrethroids are more toxic than type I [4]. Bifenthrin (BF) as one of type I pyrethroid pesticides has great photostability and insecticidal activity, and is widely used as a miticide in orchards, homes and nurseries [5]. Previous studies reported bifenthrin could induce hepatotoxicity [6], neurotoxicity [7], oxidative stress [8]. Liu et al. demonstrated that BF had estrogenlike effect as an endocrine-disrupting chemical and exposure to BF may increase the risk of ovulatory dysfunction in females [9]. Velisek et al. reported that the exposure of rainbow trout to BF caused alterations in haematological and biochemical indices as well as in tissue enzymes, which resulted in stress to the organism [10]. Lambda-cyhalothrin (LCT) is the most commonly used type II pyrethroid pesticide which can
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induce neurotoxicity, nephrotoxicity, hepatotoxicity, reproductive toxicity and oxidative damages [11–14]. El-Demerdash found that LCT had the capability to induce oxidative damage as evidenced in terms of increased perturbations in various antioxidant enzymes and lipid peroxidation [15]. In addition, it was predicted by Celik et al. that LCT had a great potential to cause genetic toxicity and cytotoxicity to mammals [16]. In recent years, metabolomics has been widely used in toxicity risk assessment and biomarker discovery. Metabolomics is defined as the quantitative measurement of the dynamic metabolic response of living systems to pathophysiological stimuli or genetic modification [17]. 1H nuclear magnetic resonance (1H NMR) and mass spectrometry (MS) are two primary investigative techniques applied in metabolomics [18]. As suitable techniques for metabolomics analysis, they have different analytical strengths and weaknesses which can give complementary information [19]. NMR measurements, combined with multivariate statistical analysis, chemometrics methods for the purpose of latent-information extraction and sample classifications, allows simultaneous detection of hundreds of low-molecular weight species within a biological matrix. In addition, it has been proven to be a quantitative, noninvasive, nondestructive, nonequilibrium perturbing technique of obtaining high quality, detailed information on solution-state molecular structures [20,21]. Compared to NMR spectroscopy, MS combined with chromatographic separation can quantify a large number of metabolites in a
Corresponding author. E-mail addresses: [email protected] (J. Miao), [email protected] (W. Zhu).
http://dx.doi.org/10.1016/j.pestbp.2017.08.002 Received 26 March 2017; Received in revised form 6 June 2017; Accepted 4 August 2017 0048-3575/ © 2017 Published by Elsevier Inc.
Please cite this article as: Miao, J., Pesticide Biochemistry and Physiology (2017), http://dx.doi.org/10.1016/j.pestbp.2017.08.002
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biological sample with a broad range of concentrations. It allows for the detection of thousands of compounds in a global manner and also can be used to identify unknown metabolites. MS has the advantages of high sensitivity and selectivity, that is particularly true for targeted analyses [22,23]. In this study, we conducted study to evaluate the toxicity of LCT and BF on mice using metabolomics methods. This study was designed to determine the biochemical effects of exposure to type I and type II pyrethroids in mice by identifying, characterizing and comparing endogenous metabolites in the liver, kidney and serum of mice treated with BF and LCT. To our knowledge, no metabolomics assessment for BF or LCT toxicity has been conducted. Previous reports indicated that both BF and LCT can induce neurotoxicity, hepatotoxicity, reproductive toxicity and oxidative damages. Therefore, we speculated that the toxicological mechanisms of these two types of insecticides may have some likeness to each other.
(2000 ×g, 4 °C), the supernatant was transferred into a clean centrifuge tube. Such procedure was repeated twice to ensure near complete extraction. The supernatant was then dried by nitrogen and individually reconstituted in 600 μL of phosphate buffer, the mixture was then centrifuged at 12,000 rpm for 5 min to obtain the supernatant. The supernatant was added with 50 μL of 3-trimethylsilyl-1-[2,2,3,3-2H4] propionate (TSP)-D2O solution (1 mM, final concentration) and pipetted into 5 mm NMR tubes for NMR analysis.
2. Materials and methods
2.6.1. 1H NMR spectroscopic measurement of tissues 1 H NMR spectra of tissue samples were measured on a Bruker AV600 Spectrometer (Bruker Biospin, Germany) at 298 K. For each sample, one 1H NMR spectrum was acquired with a standard NOESY pulse sequence using 64 free induction decay (FIDs), 128 transients were collected into 64 K data points with the spectral width of 20 ppm. The FIDs were weighted by an exponential function with a 0.3 Hz linebroadening factor prior to Fourier transformation.
2.5. Histopathology The formalin-fixed tissues were processed into paraffin wax blocks. Thin sections of 5–6 μm were cut, and stained with hematoxylin and eosin for histopathological assessment under the microscope. 2.6. 1H NMR metabolic analysis
2.1. Chemicals The pyrethroid pesticides of BF (≥98% purity) and LCT (≥ 98% purity) were purchased from Institute for the Control of Agrochemicals (Ministry of Agriculture, China). 20 types of amino acids were purchased from Aladdin. [U-13C, U-15N] labeled cell free amino acids used as internal standards were provided by Sigma. All other chemicals and reagents were analytical grade and commercially available.
2.6.2. Data reduction and multivariate statistical analysis After Fourier transformation, all 1H NMR spectra were baseline and phase corrected using TopSpin2.1 (Bruker Biospin, Germany).The region (6.1–4.7 ppm) was excluded prior to statistical analysis to eliminate the effects of water resonance, and then the NMR spectra of δ0.2–10.0 were segmented into the region of 0.01 ppm width using AMIX (version 3.9.11). The integral regions were normalized to the total spectra area, then the resulted data sets were processed for multivariate statistical analysis using SIMCA package (Version 11, Umetrics, Sweden). Principle component analysis (PCA) was initially used to analyze the NMR data set and visualize inherent clustering between controls, low-dosed and high-dosed group. Partial least squares-discriminate analysis (PLS-DA) was further applied to clarify the difference between control and exposure groups. Prior to PLS-DA, the values of all NMR data were mean-centered and pareto scaled. The score plots and loading plots were generated from PLS-DA. Variable influence on projection (VIP) score, estimating the importance of each variable in the projection, was used to discriminate special regions. The significantly changed metabolites (SCMs) between the control and treated groups were identified with p values of ANOVA < 0.05 and the VIP score > 1. If significant metabolite was identified, post hoc analysis was performed using the Fisher's LSD. The metabolite resonances were identified according to the information from the Human Metabolome Database (HMDB). Metabolic pathways analysis was conducted on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database.
2.2. Animals and treatment Eight-week-old male CD-1 mice were purchased from Vital River Laboratory Animal Company (Beijing, China). Animals were housed in stainless-steel, wire-mesh cages after acclimatization for at least one week. During the experiment, the room was kept at a controlled temperature of 22 °C with a humidity of 50 ± 5% and the light cycle was 12 h light and 12 h dark. All animals were housed with free access to water and food. All animal experiments were performed in accordance with the current Chinese legislation and approved by the independent Animal Ethical committee at China Agricultural University. A total of 30 mice were randomly assigned to five groups (6 mice per group), a control group, plus low and high treatment groups for each of the following treatments, BF and LCT. Based on the report of Environmental Protection Agency (US), the acute half-lethal doses (LD50) of BF and LCT were 43 mg/kg and 20 mg/kg respectively. We chose 1/100 and 1/10 LD50 of each pesticide as low and high treatment group. Consequently, the low exposure dose of BF and LCT was 0.43 and 0.2 mg/kg respectively while the high dose was 4.3 and 2 mg/kg respectively. All pesticides were suspended in corn oil via primary solution in acetone, and then evaporated. The control group was given an equivalent volume of corn oil. Animals were orally administered for consecutive 28 days. 2.3. Sample collection and preparation
2.7. LC/MS/MS metabolic analysis After 28 days exposure, blood was collected into centrifuge tubes from the eye and centrifuged for 10 min at 3000 rpm to obtain serum samples, which was stored at − 80 °C for further LC-MS/MS analysis. Livers and kidneys of mice were dissected and then cut a part which was fixed in 10% formalin. The rest tissues were immediately frozen for NMR analysis.
2.7.1. Determination of 20 amino acids 20 μL of the internal standard amino acids mixture was mixed with 50 μL of serum sample. The mixture was diluted to 200 μL containing 0.1% formic acid in water. Next, the sample was mixed with 80 μL reagent I (1-propanol/3-picoline 77/23) and 50 μL reagent II (chloroform/propyl-chloroformate/isooctane 71.6/17.4/11), and then vortexed for 1 min. 200 μL ethyl acetate was added to extract the derivatives. The supernatant was carefully transferred into a new centrifuge tube and dried under a stream of nitrogen and redissolved in 200 μL 0.1% formic acid in water. Chromatography was conducted on an UltiMate 3000 systems
2.4. Preparation of kidney and liver sample Liver or kidney tissues (about 100 mg) were homogenized with 400 μL of methanol and 200 μL of water in a homogenization tube. The samples were then vortexed for 60 s and centrifuged for 5 min 2
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presented in Fig. 3. PLS-DA score plots suggested that aqueous liver and kidney extracts in treatment groups were clearly separated from control groups, indicating that whether BF or LCT affected the metabolic profiles of mice. The corresponding loading plots indicated the significance of the metabolite contribution to the class separation. p values of ANOVA < 0.05 and the VIP score > 1 was considered to be the significantly changed metabolites.
coupled with TSQ Quantum Access Max mass spectrometer. MRM and positive ion mode were used for detection. EZ:faast 4u AAA-MS (Phenomenex) column was used to separate 20 amino acids at 25 °C. The mobile phase was comprised with eluent A (0.1% formic acid in acetonitrile) and eluent B (0.1% formic acid in water). The starting mobile phase was 62% A and 38% B and then was ramped to 79% A and 21% B linearly in 12 min, then returned to the starting state in 1 min. The final composition was used to equilibrate for 3 min. Sample injection volume was 20 μL and the flow rate was 0.3 mL/min.
3.3. Metabolomic changes in liver 2.7.2. Statistical analysis One-way analysis of variance (ANOVA) followed by post hoc Fisher's LSD was used to evaluate the changes of amino acids between control group and treatment groups. A value of p < 0.05 was considered significantly different.
The results revealed that BF caused a hepatic increase in isoleucine, valine, threonine, lactate, glutamate, trimethylamine oxide, betaine and succinate together with a decrease in glutamine in low-dose group. Mice in high-dose group showed high levels of threonine, lactate, succinate accompanied by low levels of glutamine. Metabolomic analysis indicates that LCT display an increase in hepatic trimethylamine oxide in low-dose group, decrease in glycine together with increased hypoxanthine in high-dose group. Comparing these two pyrethroids, BF altered more metabolites than LCT and they both induced an increase in TMAO in liver.
3. Results 3.1. Histopathology Microscopy did not show histological changes in the liver in all treatment groups. Histopathological studies showed that BF and LCT induced mild edema of renal tubular epithelium, increased cell volume, blurring cell boundary. Eosinophilic red small particles were found in the cell cytoplasm (Fig. 1). However, there was no difference between low-dose and high-dose groups.
3.4. Metabolomic changes in kidney Metabolomic analysis of aqueous kidney extracts of BF indicted that acetate, tyrosine, phenylalanine increased in low-dose group, and this increase was together with a decrease in proline. Increased acetate, tyrosine, isoleucine, valine, alanine, arginine, methanol, glycine was observed in high-dose group. Treatment with LCT was followed by an increase in methanol, glycine, tyrosine together with a decrease in pyruvate in low-dose group in the kidney of mice. Mice in high-dose group showed high levels of tyrosine, acetate and phenylalanine. To sum up the above, BF and LCT both caused an increase in methanol, glycine, tyrosine, acetate, phenylalanine in the kidney of mice.
3.2. 1H NMR spectroscopy and pattern recognition Aqueous liver and kidney extract samples were measured by 600 MHz 1H NMR in this research. The results of metabolomics analysis of aqueous liver extract from control group and each treatment group of BF and LCT are shown in Fig. 2. PLS-DA score plots and corresponding loading plots based on 1H NMR spectra of aqueous kidney extract are
Fig. 1. (a) Representative histopathological sections of kidney after treated with BF low-dose group (L) and high-dose group (H) versus control group (C). (b) Representative histopathological sections of kidney after treated with LCT low-dose group (L) and high-dose group (H) versus control group (C). The black arrows point out eosinophilic red small particles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. (a) PLS-DA score plots and loading plots based on 1H NMR spectra (δ0.2–10.0) of aqueous liver extract from mice orally treated with BF. (A) Control group (♦), (B) low-dose group (●), (C) high-dose group (■). R2 = 0.698, Q2 = 0.507. (b) PLS-DA score plots and loading plots based on 1H NMR spectra (δ0.2–10.0) of aqueous liver extract from mice orally treated with LCT. (A) Control group (♦), (B) low-dose group (●), (C) high-dose group (■). R2 = 0.710, Q2 = 0.275.
Fig. 3. (a) PLS-DA score plots and loading plots based on 1H NMR spectra (δ0.2–10.0) of aqueous kidney extract from mice orally treated with BF. (A) Control group (♦), (B) low-dose group (●), (C) high-dose group (■). R2 = 0.711, Q2 = 0.579. (b) PLS-DA score plots and loading plots based on 1H NMR spectra (δ0.2–10.0) of aqueous kidney extract from mice orally treated with LCT. (A) Control group (♦), (B) low-dose group (●), (C) high-dose group (■). R2 = 0.743, Q2 = 0.574.
exposure caused depletion of 13 amino acids in low-dosed group including serine, asparagine, glycine, alanine, histidine, lysine, proline, methionine, aspartic acid, glutamic acid, phenylalanine, leucine, and isoleucine. In high-dose group, there is a decrease of aspartic acid, tryptophan, glutamic acid and isoleucine accompanied with elevation for asparagine and isoleucine.
3.5. Analysis of 20 amino acids To correct for drift in instrument performance and sample preparation variations during extraction and chemical derivatization (control matrix effect), isotopic internal standard LC-MS/MS method has been established for the determination of 20 amino acids. 20 amino acids were accurate quantitated in serum using stable isotope-labeled internal standards by LC-MS/MS (Fig. 4 and Fig. 5). Through the ANOVA analysis, we found that as many as 11 amino acids including serine, threonine, alanine, lysine, proline, methionine, aspartic acid, glutamic acid, phenylalanine, leucine, isoleucine are decreased in low-dosed BF-treated mice while threonine, alanine, proline, aspartic acid, tryptophan, glutamic acid, phenylalanine and isoleucine are increased in high-dosed BF-treated mice. We also found LCT
4. Discussion The above findings indicated that the exposure caused comprehensive metabolic alterations in liver, kidney and serum. Therefore, an integrative view of the major altered pathways induced by BF and LCT is presented in Fig. 6, which included the main affected metabolites and perturbed KEGG pathways. 4
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Fig. 4. Quantification of 20 amino acids by LC-MS/MS in BF treated groups versus control group (*P < 0.05).
interruption of gut microflora [28]. The liver maintains the whole-body energy homeostasis as a major source of energy. The imbalance of gut microflora and energy metabolism induced by BF may result in disruption of energy supply. Previous studies reported that hypoxanthine was derived from inosine and further transformed into uric acid and xanthine in the presence of xanthine oxidase [29]. Our observations of the LCT-induced elevation of hepatic hypoxanthine probably indicated that LCT decreased nucleotide catabolism in mice [30]. Conjugation of free bile acids with glycine can enhance the solubility of lipids [31], the decrease of glycine may cause a perturbation of lipid metabolism due to LCT exposure. As discussed above, LCT exposure induced disruption of lipid metabolism as well as nucleotide catabolism in the liver and disturbance of gut microflora. Paliwal et al. reported that the level of liver enzymes aspartate transaminase (AST) and alanine transaminase (ALT) increased under the stress of LCT, which suggested that LCT can induce
4.1. Metabolomic alterations in liver The metabolomic profiling revealed that isoleucine, valine, and threonine were significantly up-regulated due to BF exposure in the liver of mice. The results suggested that BF exposure caused obvious disorder of amino acid metabolism in mice, which might affect protein synthesis and trigger inflammation in the liver [24,25]. Lactate is the end-product of glycolysis, succinate is an important intermediate for TCA cycle, which is crucial for energy metabolism. The increase of lactate and succinate accompanied by decrease of glutamine in the aqueous liver extracts indicated that the energy metabolism has been disturbed [26]. 1H NMR of liver extracts of BF-treated mice showed increase in TMAO and betaine, the latter metabolite having been identified as arising from gut microflora. TMAO is derived from the oxidation of TMA which is a product of the gut microbial breakdown of choline [27]. Although the results did not show a significant change of choline, the increase of TMAO and betaine can also be indicators of
Fig. 5. Quantification of 20 amino acids by LC-MS/MS in LCT treated groups versus control group (*P < 0.05).
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Fig. 6. Perturbed pathways and fluctuant metabolites induced by BF and LCT exposure. The blue arrow represents differential metabolites in LCT treated groups. The black arrow represents differential metabolites in BF treated groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
liver injury [32]. Fetoui et al. suggested that LCT induce oxidative stress and modify biochemical parameters such as increase of malondialdehyde (MDA) levels and decrease of glutathione (GSH) content in the liver [33].On the other hand, BF exposure disturbed amino acid metabolism and energy metabolism in the liver and caused an interruption of gut microflora. Jin et al. showed that the MDA and GSH content both increased in the liver significantly in the BF treated groups thus concluded that BF can induce oxidative stress in the liver [34]. However, we did not find evident metabolites as sensitive indicators of hepatotoxicity, which suggest that these two types of insecticides did not cause a significant adverse impact in the liver. This is consistent with the histological results.
kidney of rat [11]. In conclusion, BF and LCT exposure both altered the content of glycine, tyrosine, acetate, phenylalanine and methanol in the kidney, which represent that energy metabolism, tyrosine metabolism, lipid metabolism have been affected by both BF and LCT. This indicates that there are some similarities in toxicological mechanism between these two insecticides. Nonetheless, BF also changed the content of other amino acids, including proline, isoleucine, valine and arginine. This may suggests that, at least at subacute exposure levels, BF can cause more metabolites disturbation than LCT.
4.2. Metabolomic alterations in kidney
The content of amino acids was significantly decreased in serum, including five essential amino acids (Thr, Lys, Phe, Try and Leu) in BFtreated mice. In LCT-treated mice, there was also five essential amino acids (Lys, Phe, Leu, Ile, Try) decreased in serum. Essential amino acids in the liver only can be supplied from the diet, coherent with the increase of amino acids observed in the liver, we speculate that 28 day exposure to BF or LCT activated the key enzymes related to the catabolism of amino acids thereby promoting absorption of nutrients, especially that of amino acids in the liver and kidney, resulting in the observed decreases of amino acids in serum [4].
4.3. Change of amino acids in serum
BF intakes clearly caused significant content changes for many amino acids in the kidney of mice highlighted with elevation of tyrosine, phenylalanine, isoleucine, valine, alanine, arginine and glycine together with depletion of proline. Phenylalanine and tyrosine are all essential amino acids, the former is a precursor of the latter and phenylalanine always converts to tyrosine in the existence of phenylalanine hydroxylase and biopterin cofactor. Increased phenylalanine and tyrosine means disruption of tyrosine metabolism in mice which is likely to disrupt neurotransmitters synthesis as tyrosine is a material to produce neurotransmitters catecholamine [26,35]. Arginine is metabolically interconvertible with proline. As it has multiple metabolic fates, thus is one of the most versatile amino acids [36]. The increase of glycine could be related to perturbation of lipid metabolism [31]. Moreover, energy metabolism related pathways were also altered. Alanine was increased, which is related to pyruvate metabolism. Acetate is also connected with energy metabolism as reported that hepatic acetate is associated with the content of hepatic glucose since acetate can be converted into acetyl-CoA attending gluconeogenesis and TCA cycle [37]. LCT exposure caused variation of phenylalanine, tyrosine, and glycine in the kidney extracts. As discussed earlier, these changes perturbed tyrosine metabolism and lipid metabolism in the kidney of mice. Moreover, the depletion of pyruvate together with acetate decrease which played an important role in TCA cycle indicated perturbation of energy metabolism. Fetoui et al. also reported that exposure of rats to lambda-cyhalothrin caused a significant increase in the kidney MDA and protein carbonyl levels, and the activities of antioxidant enzymes were significantly decreased, resulting significant dysfunction in the
5. Conclusions This study revealed the toxic effects of BF and LCT on mice and identified primary perturbed metabolic pathways based on metabolomic method. Our results indicate that both BF and LCT did not cause a significant adverse impact in the liver whereas BF may be a more potent nephrotoxin than LCT. With regard to kidney, the toxicological mechanisms of these two types of insecticides have some likeness as they both disturbed energy metabolism, tyrosine metabolism, lipid metabolism. In addition, the insecticide exposure promoted absorption of amino acids from blood to liver and kidney. This approach may provide new insights into the toxicological mechanisms of pyrethroid insecticides. Acknowledgments We gratefully acknowledge the financial support from National Key Research and Development Program of China (2016YFD0200202), and Young Elite Scientists Sponsorship Program by CAST (YESS20150164). 6
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References [20] [1] H.K. Jensen, F. Konradsen, A. Dalsgaard, Pesticide use and self-reported symptoms of acute pesticide poisoning among aquatic farmers in Phnom Penh, Cambodia, Int. J. Toxicol. 2011 (1687-8191) (2011) 639814. [2] Y. Nakamura, Y. Tonogai, Y. Tsumura, Y. Ito, Determination of pyrethroid residues in vegetables, fruits, grains, beans, and green tea leaves: applications to pyrethroid residue monitoring studies, J. AOAC Int. 76 (1993) 1348–1361. [3] H.Z. Yang, M. Nishimura, K. Nishimura, S. Kuroda, T. Fujita, Quantitative structureactivity studies of pyrethroids, Pestic. Biochem. Physiol. 29 (1987) 217–232. [4] Y.J. Liang, H.P. Wang, D.X. Long, W. Li, Y.J. Wu, A metabonomic investigation of the effects of 60 days exposure of rats to two types of pyrethroid insecticides, Chem. Biol. Interact. 206 (2013) 302–308. [5] M. Jin, X. Zhang, L. Wang, C. Huang, Y. Zhang, M. Zhao, Developmental toxicity of bifenthrin in embryo-larval stages of zebrafish, Aquat. Toxicol. 95 (2009) 347–354. [6] S.A. Memon, N. Memon, S.A. Shaikh, Z. Butt, B. Mal, Patho-biochemical biomarkers of hepatotoxicity on exposure to bifenthrin insecticide in birds (Columba livia), Pure Appl. Biol. 4 (2015) 597–604. [7] E.J. Scollon, J.M. Starr, K.M. Crofton, M.J. Wolansky, M.J. Devito, M.F. Hughes, Correlation of tissue concentrations of the pyrethroid bifenthrin with neurotoxicity in the rat, Toxicology 290 (2011) 1–6. [8] I. Sadowska-Woda, D. Popowicz, A. Karowicz-Bilińska, Bifenthrin-induced oxidative stress in human erythrocytes in vitro and protective effect of selected flavonols, Toxicol. in Vitro 24 (2010) 460–464. [9] J. Liu, Y. Yang, Y. Yang, Y. Zhang, W. Liu, Disrupting effects of bifenthrin on ovulatory gene expression and prostaglandin synthesis in rat ovarian granulosa cells, Toxicology 282 (2011) 47–55. [10] J. Velisek, Z. Svobodova, V. Piackova, Effects of acute exposure to bifenthrin on some haematological, biochemical and histopathological parameters of rainbow trout (Oncorhynchus mykiss), Vet. Med. 54 (2009) 131–137. [11] H. Fetoui, M. Makni, E.M. Garoui, N. Zeghal, Toxic effects of lambda-cyhalothrin, a synthetic pyrethroid pesticide, on the rat kidney: involvement of oxidative stress and protective role of ascorbic acid, Exp. Toxicol. Pathol. 62 (2010) 593–599. [12] D.A. Righi, J. Palermo-Neto, Effects of type II pyrethroid cyhalothrin on peritoneal macrophage activity in rats, Toxicology 212 (2005) 98–106. [13] M.I. Yousef, Vitamin E modulates reproductive toxicity of pyrethroid lambda-cyhalothrin in male rabbits, Food Chem. Toxicol. 48 (2010) 1152–1159. [14] H.K. Oularbi, Biochemical and histopathological changes in the kidney and adrenal gland of rats following repeated exposure to lambda-cyhalothrin, J. Xenobiotics 4 (2014). [15] F. El-Demerdash, Lambda-cyhalothrin-induced changes in oxidative stress biomarkers in rabbit erythrocytes and alleviation effect of some antioxidants, Toxicol. in Vitro 21 (2007) 392–397. [16] A. Celik, B. Mazmanci, Y. Camlica, U. Cömelekoğlu, A. Aşkin, Evaluation of cytogenetic effects of lambda-cyhalothrin on Wistar rat bone marrow by gavage administration, Ecotoxicol. Environ Saf. 61 (2005) 128–133. [17] J.K. Nicholson, J.C. Lindon, E. Holmes, ‘Metabonomics’: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data, Xenobiotica 29 (1999) 1181–1189. [18] T.P. Sangster, J.E. Wingate, L. Burton, F. Teichert, I.D. Wilson, Investigation of analytical variation in metabonomic analysis using liquid chromatography/mass spectrometry, Rapid Commun. Mass Spectrom. 21 (2007) 2965–2970. [19] O. Beckonert, H.C. Keun, T.M. Ebbels, J. Bundy, E. Holmes, J.C. Lindon, J.K. Nicholson, Metabolic profiling, metabolomic and metabonomic procedures for
[21] [22] [23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34] [35]
[36] [37]
7
NMR spectroscopy of urine, plasma, serum and tissue extracts, Nat. Protoc. 2 (2007) 2692–2703. H.P. Wang, Y.J. Liang, Y.J. Sun, J.X. Chen, W.Y. Hou, D.X. Long, Y.J. Wu, 1H NMRbased metabonomic analysis of the serum and urine of rats following subchronic exposure to dichlorvos, deltamethrin, or a combination of these two pesticides, Chem. Biol. Interact. 203 (3) (2013) 588–596. J.K. Nicholson, J. Connelly, J.C. Lindon, E. Holmes, Metabonomics: a platform for studying drug toxicity and gene function, Nat. Rev. Drug Discov. 1 (2002) 153–161. W.B. Dunn, D.I. Ellis, Metabolomics: current analytical platforms and methodologies, TrAC Trends Anal. Chem. 24 (2005) 285–294. R. Kaddurah-Daouk, B.S. Kristal, R.M. Weinshilboum, Metabolomics: a global biochemical approach to drug response and disease, Annu. Rev. Pharmacol. 48 (2008) 653–683. O. Cloarec, M.E. Dumas, J. Trygg, A. Craig, R.H. Barton, J.C. Lindon, J.K. Nicholson, E. Holmes, Evaluation of the orthogonal projection on latent structure model limitations caused by chemical shift variability and improved visualization of biomarker changes in 1H NMR spectroscopic metabonomic studies, Anal. Chem. 77 (2) (2005) 517–526. N.A. Robinson, G.A. Miura, C.F. Matson, R.E. Dinterman, J.G. Pace, Characterization of chemically tritiated microcystin-LR and its distribution in mice, Toxicon 27 (1989) 1035–1042. Y. Zhang, Z. Zhang, Y. Zhao, S. Cheng, H. Ren, Identifying health effects of exposure to trichloroacetamide using transcriptomics and metabonomics in mice (Mus musculus), Environ. Sci. Technol. 47 (2013) 2918–2924. M.E. Dumas, R.H. Barton, A. Toye, O. Cloarec, C. Blancher, A. Rothwell, J. Fearnside, R. Tatoud, V. Blanc, J.C. Lindon, Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 12511–12516. Z. Wang, E. Klipfell, B.J. Bennett, R. Koeth, B.S. Levison, B. Dugar, A.E. Feldstein, E.B. Britt, X. Fu, Y.M. Chung, Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease, Nature 472 (2011) 57–63. Y. Ohta, M. Kongo-Nishimura, Y. Imai, T. Kishikawa, Contribution of xanthine oxidase-derived oxygen free radicals to the development of carbon tetrachlorideinduced acute liver injury in rats, J. Clin. Biochem. Nutr. 33 (2003) 83–93. L. Jiang, J. Huang, Y. Wang, H. Tang, Metabonomic analysis reveals the CCl4-induced systems alterations for multiple rat organs, J. Proteome Res. 11 (2012) 3848–3859. J. He, J. Chen, L. Wu, G. Li, P. Xie, Metabolic response to oral microcystin-LR exposure in the rat by NMR-based metabonomic study, J. Proteome Res. 11 (2012) 5934–5946. A. Paliwal, R.K. Gurjar, H.N. Sharma, Analysis of liver enzymes in albino rat under stress of λ-cyhalothrin and nuvan toxicity, Biol. Med. 1 (2009) 70–73. H. Fetoui, E.M. Garoui, N. Zeghal, Lambda-cyhalothrin-induced biochemical and histopathological changes in the liver of rats: ameliorative effect of ascorbic acid, Exp. Toxicol. Pathol. 61 (2009) 189–196. Y. Jin, J. Wang, X. Pan, L. Wang, Z. Fu, cis‑Bifenthrin enantioselectively induces hepatic oxidative stress in mice, Pestic. Biochem. Physiol. 107 (2013) 61–67. S. Ruppert, G. Kelsey, A. Schedl, E. Schmid, E. Thies, G. Schütz, Deficiency of an enzyme of tyrosine metabolism underlies altered gene expression in newborn liver of lethal albino mice, Genes Dev. 6 (1992) 1430–1443. M.S. Jr, Arginine metabolism: boundaries of our knowledge, J. Nutr. 137 (2007) 1602S–1609S. Y. An, W. Xu, H. Li, H. Lei, L. Zhang, F. Hao, Y. Duan, X. Yan, Y. Zhao, J. Wu, Highfat diet induces dynamic metabolic alterations in multiple biological matrices of rats, J. Proteome Res. 12 (2013) 3755–3768.
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Comparison of subacute effects of two types of pyrethroid insecticides using metabolomics methods Jiyan Miao, Dezhen Wang, Jin Yan, Yao Wang, Miaomiao Teng, Zhiqiang Zhou, Wentao Zhu⁎ Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Applied Chemistry, China Agricultural University, Beijing 100193, P.R. China
A R T I C L E I N F O
A B S T R A C T
Keywords: Bifenthrin Lambda-cyhalothrin Metabolomics 1 H NMR LC-MS/MS
In this study, 1H NMR based metabolomics analysis, LC-MS/MS based serum metabolomics and histopathology techniques were used to investigate the toxic effects of subacute exposure to two types of pyrethroid insecticides bifenthrin and lambda-cyhalothrin in mice. Metabolomic analysis of tissues extracts and serum showed that these two types of pyrethroid insecticides resulted in alterations of metabolites in the liver, kidney and serum of mice. Based on the altered metabolites, several significant pathways were identified, which are associated with gut microbial metabolism, lipid metabolism, nucleotide catabolism, tyrosine metabolism and energy metabolism. The results showed that bifenthrin and lambda-cyhalothrin have similarities in disruption of metabolic pathways in kidney, indicating that the toxicological mechanisms of these two types of insecticides have some likeness to each other. This study may provide novel insight into revealing differences of toxicological mechanisms between these two types of pyrethroid insecticides.
1. Introduction Pyrethroids are botanical insecticides which are synthetic derivatives of pyrethrins and have been used for many years. However, most of pyrethroids are defined as moderately hazardous (Class II) by the World Health Organization (WHO, 2009) [1]. The residues of pyrethroids have been detected in fruits, vegetables, tea, pasteurized milk and porcine muscle [2]. Pyrethroids' widely use has posed a serious risk to environment and human. Therefore, it may be an urgent need to evaluate the possible adverse effects of their use. Pyrethroids can be assorted into two types. Type II pyrethroids have an alpha-cyano moiety at the α-position whereas type I don't [3]. Type I pyrethroids affect the peripheral nerves and type II pyrethroids poison the central nervous system. Therefore, type II pyrethroids are more toxic than type I [4]. Bifenthrin (BF) as one of type I pyrethroid pesticides has great photostability and insecticidal activity, and is widely used as a miticide in orchards, homes and nurseries [5]. Previous studies reported bifenthrin could induce hepatotoxicity [6], neurotoxicity [7], oxidative stress [8]. Liu et al. demonstrated that BF had estrogenlike effect as an endocrine-disrupting chemical and exposure to BF may increase the risk of ovulatory dysfunction in females [9]. Velisek et al. reported that the exposure of rainbow trout to BF caused alterations in haematological and biochemical indices as well as in tissue enzymes, which resulted in stress to the organism [10]. Lambda-cyhalothrin (LCT) is the most commonly used type II pyrethroid pesticide which can
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induce neurotoxicity, nephrotoxicity, hepatotoxicity, reproductive toxicity and oxidative damages [11–14]. El-Demerdash found that LCT had the capability to induce oxidative damage as evidenced in terms of increased perturbations in various antioxidant enzymes and lipid peroxidation [15]. In addition, it was predicted by Celik et al. that LCT had a great potential to cause genetic toxicity and cytotoxicity to mammals [16]. In recent years, metabolomics has been widely used in toxicity risk assessment and biomarker discovery. Metabolomics is defined as the quantitative measurement of the dynamic metabolic response of living systems to pathophysiological stimuli or genetic modification [17]. 1H nuclear magnetic resonance (1H NMR) and mass spectrometry (MS) are two primary investigative techniques applied in metabolomics [18]. As suitable techniques for metabolomics analysis, they have different analytical strengths and weaknesses which can give complementary information [19]. NMR measurements, combined with multivariate statistical analysis, chemometrics methods for the purpose of latent-information extraction and sample classifications, allows simultaneous detection of hundreds of low-molecular weight species within a biological matrix. In addition, it has been proven to be a quantitative, noninvasive, nondestructive, nonequilibrium perturbing technique of obtaining high quality, detailed information on solution-state molecular structures [20,21]. Compared to NMR spectroscopy, MS combined with chromatographic separation can quantify a large number of metabolites in a
Corresponding author. E-mail addresses: [email protected] (J. Miao), [email protected] (W. Zhu).
http://dx.doi.org/10.1016/j.pestbp.2017.08.002 Received 26 March 2017; Received in revised form 6 June 2017; Accepted 4 August 2017 0048-3575/ © 2017 Published by Elsevier Inc.
Please cite this article as: Miao, J., Pesticide Biochemistry and Physiology (2017), http://dx.doi.org/10.1016/j.pestbp.2017.08.002
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biological sample with a broad range of concentrations. It allows for the detection of thousands of compounds in a global manner and also can be used to identify unknown metabolites. MS has the advantages of high sensitivity and selectivity, that is particularly true for targeted analyses [22,23]. In this study, we conducted study to evaluate the toxicity of LCT and BF on mice using metabolomics methods. This study was designed to determine the biochemical effects of exposure to type I and type II pyrethroids in mice by identifying, characterizing and comparing endogenous metabolites in the liver, kidney and serum of mice treated with BF and LCT. To our knowledge, no metabolomics assessment for BF or LCT toxicity has been conducted. Previous reports indicated that both BF and LCT can induce neurotoxicity, hepatotoxicity, reproductive toxicity and oxidative damages. Therefore, we speculated that the toxicological mechanisms of these two types of insecticides may have some likeness to each other.
(2000 ×g, 4 °C), the supernatant was transferred into a clean centrifuge tube. Such procedure was repeated twice to ensure near complete extraction. The supernatant was then dried by nitrogen and individually reconstituted in 600 μL of phosphate buffer, the mixture was then centrifuged at 12,000 rpm for 5 min to obtain the supernatant. The supernatant was added with 50 μL of 3-trimethylsilyl-1-[2,2,3,3-2H4] propionate (TSP)-D2O solution (1 mM, final concentration) and pipetted into 5 mm NMR tubes for NMR analysis.
2. Materials and methods
2.6.1. 1H NMR spectroscopic measurement of tissues 1 H NMR spectra of tissue samples were measured on a Bruker AV600 Spectrometer (Bruker Biospin, Germany) at 298 K. For each sample, one 1H NMR spectrum was acquired with a standard NOESY pulse sequence using 64 free induction decay (FIDs), 128 transients were collected into 64 K data points with the spectral width of 20 ppm. The FIDs were weighted by an exponential function with a 0.3 Hz linebroadening factor prior to Fourier transformation.
2.5. Histopathology The formalin-fixed tissues were processed into paraffin wax blocks. Thin sections of 5–6 μm were cut, and stained with hematoxylin and eosin for histopathological assessment under the microscope. 2.6. 1H NMR metabolic analysis
2.1. Chemicals The pyrethroid pesticides of BF (≥98% purity) and LCT (≥ 98% purity) were purchased from Institute for the Control of Agrochemicals (Ministry of Agriculture, China). 20 types of amino acids were purchased from Aladdin. [U-13C, U-15N] labeled cell free amino acids used as internal standards were provided by Sigma. All other chemicals and reagents were analytical grade and commercially available.
2.6.2. Data reduction and multivariate statistical analysis After Fourier transformation, all 1H NMR spectra were baseline and phase corrected using TopSpin2.1 (Bruker Biospin, Germany).The region (6.1–4.7 ppm) was excluded prior to statistical analysis to eliminate the effects of water resonance, and then the NMR spectra of δ0.2–10.0 were segmented into the region of 0.01 ppm width using AMIX (version 3.9.11). The integral regions were normalized to the total spectra area, then the resulted data sets were processed for multivariate statistical analysis using SIMCA package (Version 11, Umetrics, Sweden). Principle component analysis (PCA) was initially used to analyze the NMR data set and visualize inherent clustering between controls, low-dosed and high-dosed group. Partial least squares-discriminate analysis (PLS-DA) was further applied to clarify the difference between control and exposure groups. Prior to PLS-DA, the values of all NMR data were mean-centered and pareto scaled. The score plots and loading plots were generated from PLS-DA. Variable influence on projection (VIP) score, estimating the importance of each variable in the projection, was used to discriminate special regions. The significantly changed metabolites (SCMs) between the control and treated groups were identified with p values of ANOVA < 0.05 and the VIP score > 1. If significant metabolite was identified, post hoc analysis was performed using the Fisher's LSD. The metabolite resonances were identified according to the information from the Human Metabolome Database (HMDB). Metabolic pathways analysis was conducted on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database.
2.2. Animals and treatment Eight-week-old male CD-1 mice were purchased from Vital River Laboratory Animal Company (Beijing, China). Animals were housed in stainless-steel, wire-mesh cages after acclimatization for at least one week. During the experiment, the room was kept at a controlled temperature of 22 °C with a humidity of 50 ± 5% and the light cycle was 12 h light and 12 h dark. All animals were housed with free access to water and food. All animal experiments were performed in accordance with the current Chinese legislation and approved by the independent Animal Ethical committee at China Agricultural University. A total of 30 mice were randomly assigned to five groups (6 mice per group), a control group, plus low and high treatment groups for each of the following treatments, BF and LCT. Based on the report of Environmental Protection Agency (US), the acute half-lethal doses (LD50) of BF and LCT were 43 mg/kg and 20 mg/kg respectively. We chose 1/100 and 1/10 LD50 of each pesticide as low and high treatment group. Consequently, the low exposure dose of BF and LCT was 0.43 and 0.2 mg/kg respectively while the high dose was 4.3 and 2 mg/kg respectively. All pesticides were suspended in corn oil via primary solution in acetone, and then evaporated. The control group was given an equivalent volume of corn oil. Animals were orally administered for consecutive 28 days. 2.3. Sample collection and preparation
2.7. LC/MS/MS metabolic analysis After 28 days exposure, blood was collected into centrifuge tubes from the eye and centrifuged for 10 min at 3000 rpm to obtain serum samples, which was stored at − 80 °C for further LC-MS/MS analysis. Livers and kidneys of mice were dissected and then cut a part which was fixed in 10% formalin. The rest tissues were immediately frozen for NMR analysis.
2.7.1. Determination of 20 amino acids 20 μL of the internal standard amino acids mixture was mixed with 50 μL of serum sample. The mixture was diluted to 200 μL containing 0.1% formic acid in water. Next, the sample was mixed with 80 μL reagent I (1-propanol/3-picoline 77/23) and 50 μL reagent II (chloroform/propyl-chloroformate/isooctane 71.6/17.4/11), and then vortexed for 1 min. 200 μL ethyl acetate was added to extract the derivatives. The supernatant was carefully transferred into a new centrifuge tube and dried under a stream of nitrogen and redissolved in 200 μL 0.1% formic acid in water. Chromatography was conducted on an UltiMate 3000 systems
2.4. Preparation of kidney and liver sample Liver or kidney tissues (about 100 mg) were homogenized with 400 μL of methanol and 200 μL of water in a homogenization tube. The samples were then vortexed for 60 s and centrifuged for 5 min 2
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presented in Fig. 3. PLS-DA score plots suggested that aqueous liver and kidney extracts in treatment groups were clearly separated from control groups, indicating that whether BF or LCT affected the metabolic profiles of mice. The corresponding loading plots indicated the significance of the metabolite contribution to the class separation. p values of ANOVA < 0.05 and the VIP score > 1 was considered to be the significantly changed metabolites.
coupled with TSQ Quantum Access Max mass spectrometer. MRM and positive ion mode were used for detection. EZ:faast 4u AAA-MS (Phenomenex) column was used to separate 20 amino acids at 25 °C. The mobile phase was comprised with eluent A (0.1% formic acid in acetonitrile) and eluent B (0.1% formic acid in water). The starting mobile phase was 62% A and 38% B and then was ramped to 79% A and 21% B linearly in 12 min, then returned to the starting state in 1 min. The final composition was used to equilibrate for 3 min. Sample injection volume was 20 μL and the flow rate was 0.3 mL/min.
3.3. Metabolomic changes in liver 2.7.2. Statistical analysis One-way analysis of variance (ANOVA) followed by post hoc Fisher's LSD was used to evaluate the changes of amino acids between control group and treatment groups. A value of p < 0.05 was considered significantly different.
The results revealed that BF caused a hepatic increase in isoleucine, valine, threonine, lactate, glutamate, trimethylamine oxide, betaine and succinate together with a decrease in glutamine in low-dose group. Mice in high-dose group showed high levels of threonine, lactate, succinate accompanied by low levels of glutamine. Metabolomic analysis indicates that LCT display an increase in hepatic trimethylamine oxide in low-dose group, decrease in glycine together with increased hypoxanthine in high-dose group. Comparing these two pyrethroids, BF altered more metabolites than LCT and they both induced an increase in TMAO in liver.
3. Results 3.1. Histopathology Microscopy did not show histological changes in the liver in all treatment groups. Histopathological studies showed that BF and LCT induced mild edema of renal tubular epithelium, increased cell volume, blurring cell boundary. Eosinophilic red small particles were found in the cell cytoplasm (Fig. 1). However, there was no difference between low-dose and high-dose groups.
3.4. Metabolomic changes in kidney Metabolomic analysis of aqueous kidney extracts of BF indicted that acetate, tyrosine, phenylalanine increased in low-dose group, and this increase was together with a decrease in proline. Increased acetate, tyrosine, isoleucine, valine, alanine, arginine, methanol, glycine was observed in high-dose group. Treatment with LCT was followed by an increase in methanol, glycine, tyrosine together with a decrease in pyruvate in low-dose group in the kidney of mice. Mice in high-dose group showed high levels of tyrosine, acetate and phenylalanine. To sum up the above, BF and LCT both caused an increase in methanol, glycine, tyrosine, acetate, phenylalanine in the kidney of mice.
3.2. 1H NMR spectroscopy and pattern recognition Aqueous liver and kidney extract samples were measured by 600 MHz 1H NMR in this research. The results of metabolomics analysis of aqueous liver extract from control group and each treatment group of BF and LCT are shown in Fig. 2. PLS-DA score plots and corresponding loading plots based on 1H NMR spectra of aqueous kidney extract are
Fig. 1. (a) Representative histopathological sections of kidney after treated with BF low-dose group (L) and high-dose group (H) versus control group (C). (b) Representative histopathological sections of kidney after treated with LCT low-dose group (L) and high-dose group (H) versus control group (C). The black arrows point out eosinophilic red small particles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. (a) PLS-DA score plots and loading plots based on 1H NMR spectra (δ0.2–10.0) of aqueous liver extract from mice orally treated with BF. (A) Control group (♦), (B) low-dose group (●), (C) high-dose group (■). R2 = 0.698, Q2 = 0.507. (b) PLS-DA score plots and loading plots based on 1H NMR spectra (δ0.2–10.0) of aqueous liver extract from mice orally treated with LCT. (A) Control group (♦), (B) low-dose group (●), (C) high-dose group (■). R2 = 0.710, Q2 = 0.275.
Fig. 3. (a) PLS-DA score plots and loading plots based on 1H NMR spectra (δ0.2–10.0) of aqueous kidney extract from mice orally treated with BF. (A) Control group (♦), (B) low-dose group (●), (C) high-dose group (■). R2 = 0.711, Q2 = 0.579. (b) PLS-DA score plots and loading plots based on 1H NMR spectra (δ0.2–10.0) of aqueous kidney extract from mice orally treated with LCT. (A) Control group (♦), (B) low-dose group (●), (C) high-dose group (■). R2 = 0.743, Q2 = 0.574.
exposure caused depletion of 13 amino acids in low-dosed group including serine, asparagine, glycine, alanine, histidine, lysine, proline, methionine, aspartic acid, glutamic acid, phenylalanine, leucine, and isoleucine. In high-dose group, there is a decrease of aspartic acid, tryptophan, glutamic acid and isoleucine accompanied with elevation for asparagine and isoleucine.
3.5. Analysis of 20 amino acids To correct for drift in instrument performance and sample preparation variations during extraction and chemical derivatization (control matrix effect), isotopic internal standard LC-MS/MS method has been established for the determination of 20 amino acids. 20 amino acids were accurate quantitated in serum using stable isotope-labeled internal standards by LC-MS/MS (Fig. 4 and Fig. 5). Through the ANOVA analysis, we found that as many as 11 amino acids including serine, threonine, alanine, lysine, proline, methionine, aspartic acid, glutamic acid, phenylalanine, leucine, isoleucine are decreased in low-dosed BF-treated mice while threonine, alanine, proline, aspartic acid, tryptophan, glutamic acid, phenylalanine and isoleucine are increased in high-dosed BF-treated mice. We also found LCT
4. Discussion The above findings indicated that the exposure caused comprehensive metabolic alterations in liver, kidney and serum. Therefore, an integrative view of the major altered pathways induced by BF and LCT is presented in Fig. 6, which included the main affected metabolites and perturbed KEGG pathways. 4
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Fig. 4. Quantification of 20 amino acids by LC-MS/MS in BF treated groups versus control group (*P < 0.05).
interruption of gut microflora [28]. The liver maintains the whole-body energy homeostasis as a major source of energy. The imbalance of gut microflora and energy metabolism induced by BF may result in disruption of energy supply. Previous studies reported that hypoxanthine was derived from inosine and further transformed into uric acid and xanthine in the presence of xanthine oxidase [29]. Our observations of the LCT-induced elevation of hepatic hypoxanthine probably indicated that LCT decreased nucleotide catabolism in mice [30]. Conjugation of free bile acids with glycine can enhance the solubility of lipids [31], the decrease of glycine may cause a perturbation of lipid metabolism due to LCT exposure. As discussed above, LCT exposure induced disruption of lipid metabolism as well as nucleotide catabolism in the liver and disturbance of gut microflora. Paliwal et al. reported that the level of liver enzymes aspartate transaminase (AST) and alanine transaminase (ALT) increased under the stress of LCT, which suggested that LCT can induce
4.1. Metabolomic alterations in liver The metabolomic profiling revealed that isoleucine, valine, and threonine were significantly up-regulated due to BF exposure in the liver of mice. The results suggested that BF exposure caused obvious disorder of amino acid metabolism in mice, which might affect protein synthesis and trigger inflammation in the liver [24,25]. Lactate is the end-product of glycolysis, succinate is an important intermediate for TCA cycle, which is crucial for energy metabolism. The increase of lactate and succinate accompanied by decrease of glutamine in the aqueous liver extracts indicated that the energy metabolism has been disturbed [26]. 1H NMR of liver extracts of BF-treated mice showed increase in TMAO and betaine, the latter metabolite having been identified as arising from gut microflora. TMAO is derived from the oxidation of TMA which is a product of the gut microbial breakdown of choline [27]. Although the results did not show a significant change of choline, the increase of TMAO and betaine can also be indicators of
Fig. 5. Quantification of 20 amino acids by LC-MS/MS in LCT treated groups versus control group (*P < 0.05).
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Fig. 6. Perturbed pathways and fluctuant metabolites induced by BF and LCT exposure. The blue arrow represents differential metabolites in LCT treated groups. The black arrow represents differential metabolites in BF treated groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
liver injury [32]. Fetoui et al. suggested that LCT induce oxidative stress and modify biochemical parameters such as increase of malondialdehyde (MDA) levels and decrease of glutathione (GSH) content in the liver [33].On the other hand, BF exposure disturbed amino acid metabolism and energy metabolism in the liver and caused an interruption of gut microflora. Jin et al. showed that the MDA and GSH content both increased in the liver significantly in the BF treated groups thus concluded that BF can induce oxidative stress in the liver [34]. However, we did not find evident metabolites as sensitive indicators of hepatotoxicity, which suggest that these two types of insecticides did not cause a significant adverse impact in the liver. This is consistent with the histological results.
kidney of rat [11]. In conclusion, BF and LCT exposure both altered the content of glycine, tyrosine, acetate, phenylalanine and methanol in the kidney, which represent that energy metabolism, tyrosine metabolism, lipid metabolism have been affected by both BF and LCT. This indicates that there are some similarities in toxicological mechanism between these two insecticides. Nonetheless, BF also changed the content of other amino acids, including proline, isoleucine, valine and arginine. This may suggests that, at least at subacute exposure levels, BF can cause more metabolites disturbation than LCT.
4.2. Metabolomic alterations in kidney
The content of amino acids was significantly decreased in serum, including five essential amino acids (Thr, Lys, Phe, Try and Leu) in BFtreated mice. In LCT-treated mice, there was also five essential amino acids (Lys, Phe, Leu, Ile, Try) decreased in serum. Essential amino acids in the liver only can be supplied from the diet, coherent with the increase of amino acids observed in the liver, we speculate that 28 day exposure to BF or LCT activated the key enzymes related to the catabolism of amino acids thereby promoting absorption of nutrients, especially that of amino acids in the liver and kidney, resulting in the observed decreases of amino acids in serum [4].
4.3. Change of amino acids in serum
BF intakes clearly caused significant content changes for many amino acids in the kidney of mice highlighted with elevation of tyrosine, phenylalanine, isoleucine, valine, alanine, arginine and glycine together with depletion of proline. Phenylalanine and tyrosine are all essential amino acids, the former is a precursor of the latter and phenylalanine always converts to tyrosine in the existence of phenylalanine hydroxylase and biopterin cofactor. Increased phenylalanine and tyrosine means disruption of tyrosine metabolism in mice which is likely to disrupt neurotransmitters synthesis as tyrosine is a material to produce neurotransmitters catecholamine [26,35]. Arginine is metabolically interconvertible with proline. As it has multiple metabolic fates, thus is one of the most versatile amino acids [36]. The increase of glycine could be related to perturbation of lipid metabolism [31]. Moreover, energy metabolism related pathways were also altered. Alanine was increased, which is related to pyruvate metabolism. Acetate is also connected with energy metabolism as reported that hepatic acetate is associated with the content of hepatic glucose since acetate can be converted into acetyl-CoA attending gluconeogenesis and TCA cycle [37]. LCT exposure caused variation of phenylalanine, tyrosine, and glycine in the kidney extracts. As discussed earlier, these changes perturbed tyrosine metabolism and lipid metabolism in the kidney of mice. Moreover, the depletion of pyruvate together with acetate decrease which played an important role in TCA cycle indicated perturbation of energy metabolism. Fetoui et al. also reported that exposure of rats to lambda-cyhalothrin caused a significant increase in the kidney MDA and protein carbonyl levels, and the activities of antioxidant enzymes were significantly decreased, resulting significant dysfunction in the
5. Conclusions This study revealed the toxic effects of BF and LCT on mice and identified primary perturbed metabolic pathways based on metabolomic method. Our results indicate that both BF and LCT did not cause a significant adverse impact in the liver whereas BF may be a more potent nephrotoxin than LCT. With regard to kidney, the toxicological mechanisms of these two types of insecticides have some likeness as they both disturbed energy metabolism, tyrosine metabolism, lipid metabolism. In addition, the insecticide exposure promoted absorption of amino acids from blood to liver and kidney. This approach may provide new insights into the toxicological mechanisms of pyrethroid insecticides. Acknowledgments We gratefully acknowledge the financial support from National Key Research and Development Program of China (2016YFD0200202), and Young Elite Scientists Sponsorship Program by CAST (YESS20150164). 6
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References [20] [1] H.K. Jensen, F. Konradsen, A. Dalsgaard, Pesticide use and self-reported symptoms of acute pesticide poisoning among aquatic farmers in Phnom Penh, Cambodia, Int. J. Toxicol. 2011 (1687-8191) (2011) 639814. [2] Y. Nakamura, Y. Tonogai, Y. Tsumura, Y. Ito, Determination of pyrethroid residues in vegetables, fruits, grains, beans, and green tea leaves: applications to pyrethroid residue monitoring studies, J. AOAC Int. 76 (1993) 1348–1361. [3] H.Z. Yang, M. Nishimura, K. Nishimura, S. Kuroda, T. Fujita, Quantitative structureactivity studies of pyrethroids, Pestic. Biochem. Physiol. 29 (1987) 217–232. [4] Y.J. Liang, H.P. Wang, D.X. Long, W. Li, Y.J. Wu, A metabonomic investigation of the effects of 60 days exposure of rats to two types of pyrethroid insecticides, Chem. Biol. Interact. 206 (2013) 302–308. [5] M. Jin, X. Zhang, L. Wang, C. Huang, Y. Zhang, M. Zhao, Developmental toxicity of bifenthrin in embryo-larval stages of zebrafish, Aquat. Toxicol. 95 (2009) 347–354. [6] S.A. Memon, N. Memon, S.A. Shaikh, Z. Butt, B. Mal, Patho-biochemical biomarkers of hepatotoxicity on exposure to bifenthrin insecticide in birds (Columba livia), Pure Appl. Biol. 4 (2015) 597–604. [7] E.J. Scollon, J.M. Starr, K.M. Crofton, M.J. Wolansky, M.J. Devito, M.F. Hughes, Correlation of tissue concentrations of the pyrethroid bifenthrin with neurotoxicity in the rat, Toxicology 290 (2011) 1–6. [8] I. Sadowska-Woda, D. Popowicz, A. Karowicz-Bilińska, Bifenthrin-induced oxidative stress in human erythrocytes in vitro and protective effect of selected flavonols, Toxicol. in Vitro 24 (2010) 460–464. [9] J. Liu, Y. Yang, Y. Yang, Y. Zhang, W. Liu, Disrupting effects of bifenthrin on ovulatory gene expression and prostaglandin synthesis in rat ovarian granulosa cells, Toxicology 282 (2011) 47–55. [10] J. Velisek, Z. Svobodova, V. Piackova, Effects of acute exposure to bifenthrin on some haematological, biochemical and histopathological parameters of rainbow trout (Oncorhynchus mykiss), Vet. Med. 54 (2009) 131–137. [11] H. Fetoui, M. Makni, E.M. Garoui, N. Zeghal, Toxic effects of lambda-cyhalothrin, a synthetic pyrethroid pesticide, on the rat kidney: involvement of oxidative stress and protective role of ascorbic acid, Exp. Toxicol. Pathol. 62 (2010) 593–599. [12] D.A. Righi, J. Palermo-Neto, Effects of type II pyrethroid cyhalothrin on peritoneal macrophage activity in rats, Toxicology 212 (2005) 98–106. [13] M.I. Yousef, Vitamin E modulates reproductive toxicity of pyrethroid lambda-cyhalothrin in male rabbits, Food Chem. Toxicol. 48 (2010) 1152–1159. [14] H.K. Oularbi, Biochemical and histopathological changes in the kidney and adrenal gland of rats following repeated exposure to lambda-cyhalothrin, J. Xenobiotics 4 (2014). [15] F. El-Demerdash, Lambda-cyhalothrin-induced changes in oxidative stress biomarkers in rabbit erythrocytes and alleviation effect of some antioxidants, Toxicol. in Vitro 21 (2007) 392–397. [16] A. Celik, B. Mazmanci, Y. Camlica, U. Cömelekoğlu, A. Aşkin, Evaluation of cytogenetic effects of lambda-cyhalothrin on Wistar rat bone marrow by gavage administration, Ecotoxicol. Environ Saf. 61 (2005) 128–133. [17] J.K. Nicholson, J.C. Lindon, E. Holmes, ‘Metabonomics’: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data, Xenobiotica 29 (1999) 1181–1189. [18] T.P. Sangster, J.E. Wingate, L. Burton, F. Teichert, I.D. Wilson, Investigation of analytical variation in metabonomic analysis using liquid chromatography/mass spectrometry, Rapid Commun. Mass Spectrom. 21 (2007) 2965–2970. [19] O. Beckonert, H.C. Keun, T.M. Ebbels, J. Bundy, E. Holmes, J.C. Lindon, J.K. Nicholson, Metabolic profiling, metabolomic and metabonomic procedures for
[21] [22] [23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34] [35]
[36] [37]
7
NMR spectroscopy of urine, plasma, serum and tissue extracts, Nat. Protoc. 2 (2007) 2692–2703. H.P. Wang, Y.J. Liang, Y.J. Sun, J.X. Chen, W.Y. Hou, D.X. Long, Y.J. Wu, 1H NMRbased metabonomic analysis of the serum and urine of rats following subchronic exposure to dichlorvos, deltamethrin, or a combination of these two pesticides, Chem. Biol. Interact. 203 (3) (2013) 588–596. J.K. Nicholson, J. Connelly, J.C. Lindon, E. Holmes, Metabonomics: a platform for studying drug toxicity and gene function, Nat. Rev. Drug Discov. 1 (2002) 153–161. W.B. Dunn, D.I. Ellis, Metabolomics: current analytical platforms and methodologies, TrAC Trends Anal. Chem. 24 (2005) 285–294. R. Kaddurah-Daouk, B.S. Kristal, R.M. Weinshilboum, Metabolomics: a global biochemical approach to drug response and disease, Annu. Rev. Pharmacol. 48 (2008) 653–683. O. Cloarec, M.E. Dumas, J. Trygg, A. Craig, R.H. Barton, J.C. Lindon, J.K. Nicholson, E. Holmes, Evaluation of the orthogonal projection on latent structure model limitations caused by chemical shift variability and improved visualization of biomarker changes in 1H NMR spectroscopic metabonomic studies, Anal. Chem. 77 (2) (2005) 517–526. N.A. Robinson, G.A. Miura, C.F. Matson, R.E. Dinterman, J.G. Pace, Characterization of chemically tritiated microcystin-LR and its distribution in mice, Toxicon 27 (1989) 1035–1042. Y. Zhang, Z. Zhang, Y. Zhao, S. Cheng, H. Ren, Identifying health effects of exposure to trichloroacetamide using transcriptomics and metabonomics in mice (Mus musculus), Environ. Sci. Technol. 47 (2013) 2918–2924. M.E. Dumas, R.H. Barton, A. Toye, O. Cloarec, C. Blancher, A. Rothwell, J. Fearnside, R. Tatoud, V. Blanc, J.C. Lindon, Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 12511–12516. Z. Wang, E. Klipfell, B.J. Bennett, R. Koeth, B.S. Levison, B. Dugar, A.E. Feldstein, E.B. Britt, X. Fu, Y.M. Chung, Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease, Nature 472 (2011) 57–63. Y. Ohta, M. Kongo-Nishimura, Y. Imai, T. Kishikawa, Contribution of xanthine oxidase-derived oxygen free radicals to the development of carbon tetrachlorideinduced acute liver injury in rats, J. Clin. Biochem. Nutr. 33 (2003) 83–93. L. Jiang, J. Huang, Y. Wang, H. Tang, Metabonomic analysis reveals the CCl4-induced systems alterations for multiple rat organs, J. Proteome Res. 11 (2012) 3848–3859. J. He, J. Chen, L. Wu, G. Li, P. Xie, Metabolic response to oral microcystin-LR exposure in the rat by NMR-based metabonomic study, J. Proteome Res. 11 (2012) 5934–5946. A. Paliwal, R.K. Gurjar, H.N. Sharma, Analysis of liver enzymes in albino rat under stress of λ-cyhalothrin and nuvan toxicity, Biol. Med. 1 (2009) 70–73. H. Fetoui, E.M. Garoui, N. Zeghal, Lambda-cyhalothrin-induced biochemical and histopathological changes in the liver of rats: ameliorative effect of ascorbic acid, Exp. Toxicol. Pathol. 61 (2009) 189–196. Y. Jin, J. Wang, X. Pan, L. Wang, Z. Fu, cis‑Bifenthrin enantioselectively induces hepatic oxidative stress in mice, Pestic. Biochem. Physiol. 107 (2013) 61–67. S. Ruppert, G. Kelsey, A. Schedl, E. Schmid, E. Thies, G. Schütz, Deficiency of an enzyme of tyrosine metabolism underlies altered gene expression in newborn liver of lethal albino mice, Genes Dev. 6 (1992) 1430–1443. M.S. Jr, Arginine metabolism: boundaries of our knowledge, J. Nutr. 137 (2007) 1602S–1609S. Y. An, W. Xu, H. Li, H. Lei, L. Zhang, F. Hao, Y. Duan, X. Yan, Y. Zhao, J. Wu, Highfat diet induces dynamic metabolic alterations in multiple biological matrices of rats, J. Proteome Res. 12 (2013) 3755–3768.