Microbiology Received: July 8, 2014 Accepted after revision: January 5, 2015 Published online: February 28, 2015
Chemotherapy 2014;60:135–142 DOI: 10.1159/000371837
Changes in Gut Microbiota May Be Early Signs of Liver Toxicity Induced by Epoxiconazole in Rats Cheng Xu a, b Qian Liu a, b Fei Huan c Jianhua Qu a, b Wei Liu a, b Aihua Gu a, b Yubang Wang a, c Zhaoyan Jiang d
a
State Key Laboratory of Reproductive Medicine, Institute of Toxicology, b Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, and c Safety Assessment and Research Center for Drugs, Pesticides and Veterinary Drugs of the Jiangsu Province, Nanjing Medical University, Nanjing, d Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, China
Abstract Objective: The gut microbiome is essential for human health due to its effects on disease development, drug metabolism and the immune system. It may also play a role in the interaction with environmental toxicants. However, the effect of epoxiconazole, a fungicide active ingredient from the class of azoles developed to protect crops, on the abundance and composition of the gut microbiome has never been studied. We put forward the hypothesis that changes in gut microbiota may be early signs of toxicity induced by epoxiconazole. Methods: In this study, female rats were fed with epoxiconazole-adulterated diets (0, 4 and 100 mg/kg/day) for 90 days. The gut microbiome was determined by 16S rRNA gene sequencing. Body and organ weight, and blood biochemistry were also measured after 90 days of oral epoxiconazole exposure. Results: Interestingly, the abundance of gut Firmicutes decreased, and Bacteroidetes and ProteobacC. Xu, Q. Liu and F. Huan contributed equally to this study and should be regarded as joint first authors.
© 2015 S. Karger AG, Basel 0009–3157/15/0602–0135$39.50/0 E-Mail [email protected] www.karger.com/che
teria increased. At family level, Lachnospiraceae and Enterobacteriaceae were selectively enriched following epoxiconazole exposure. Our results indicate that epoxiconazole exposure may induce changes in the gut microbiome and potential liver toxicity. Conclusion: Changes in the gut microbiome may be used as early indicators for monitoring the health risk of the host. © 2015 S. Karger AG, Basel
Introduction
The microbiome is found all over the biosphere; it is even included in a portion of the alimentary system of the host. Approximately 100 trillion microbes exist as common microbiota in the gastrointestinal tract of hosts, with bacteria being the major participants of the human microbiome [1]. In the human gut microbiome, there are >300,000 gene-encoding genes [2], whereas in the host, the human genome encodes only 20,000–25,000 proteincoding genes. Recent investigations into the human gut microbiome found that nearly 98% of bacterial phyla Aihua Gu, Yubang Wang State Key Laboratory of Reproductive Medicine Institute of Toxicology, Nanjing Medical University Nanjing 210029 (China) E-Mail aihuagu @ njmu.edu.cn, wyb @ njmu.edu.cn Zhaoyan Jiang Department of Surgery, Shanghai Institute of Digestive Surgery Ruijin Hospital, Shanghai JiaoTong University School of Medicine Shanghai 200025 (China) E-Mail zhaoyanjiang @ gmail.com
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Key Words Epoxiconazole · Gut microbiota · Host health · Liver toxicity · Microarray analysis
Materials and Methods
Epoxiconazole-adulterated diets were prepared according to a previous report [20] with some modifications. The following adulterated feeds were prepared: epoxiconazole (0, 40 and 1,000 ppm) with phytoestrogen-free food (Shuangshi Animal Food Inc., Suzhou, China). The feeding concentrations were selected based on the feeding levels used in our preliminary 14-day acute toxicity study (data not shown). Based on the preliminary study, 4 and 100 mg/ kg body weight/day were chosen as low- and high-concentration diets, respectively. These doses, 4 and 100 mg/kg/day, were chosen as the lower dose (less than no observed adverse effect level) and higher dose (more than the lowest observed adverse effect level), respectively (http://enfo.agt.bme.hu/drupal/sites/default/ files/epoxiconazole_0.pdf). The rats were allowed to acclimate for 1 week before study commencement and were then given the above adulterated feeds for 90 days. The rats were randomly divided into three groups (10 rats/ group and 2 rats/metabolic cage): the control group (CG), the lowepoxiconazole group (LDG) and the high-epoxiconazole group (HDG) and fed adulterated feeds and water ad libitum. All animal procedures were approved by the local Animal Care and Use Committee of the Nanjing Medical University. Collection of Biological Samples, Determination of Serum Biochemical Parameters and Histological Analysis Twenty-four hours after completion of the final task session, the rats were euthanized by CO2 asphyxiation. Blood was collected from all rats by cardiac puncture using a 23-gauge needle into BD Vacutainer serum blood collection tubes and centrifuged immediately at 1,200 g for 20 min at 4 ° C. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, albumin, total bilirubin (TBIL), alkaline phosphatase, glucose, blood urea nitrogen, creatinine, cholesterol and cholinesterase (CHE) of all animals were measured by automatic biochemical analyzer in the Center for Hygienic Analysis and Detection, Nanjing Medical University. The initial and final body weights were recorded. The relevant primary organs were excised and weighed. The liver was prepared for histological studies: tissues were fixed in 4% (w/v) paraformaldehyde, embedded in paraffin, stained with hematoxylin-eosin (HE) and observed under a light microscope. Feces were collected from the bottom of the metabolic cage on day 90, transferred to 15-ml Corning tubes and stored at –80 ° C until analysis.
Genomic DNA Extraction and PCR Amplification DNA Extraction and PCR Amplification. Microbial DNA was extracted from fecal samples using the E.Z.N.A.® stool DNA kit (Omega Bio-Tek, Norcross, Ga., USA) according to the manufacturer’s protocols. The V4–V5 region of the bacterial 16S ribosomal RNA gene was amplified by PCR (95 ° C for 2 min, followed by 25 cycles at 95 ° C for 30 s, 55 ° C for 30 s and 72 ° C for 30 s, and a final extension at 72 ° C for 5 min) using primers 515F 5′-barcodeGTG CCA GCM GCC GCG G-3′ and 907R 5′-CCG TCA ATT CMT TTR AGT TT-3′ [21], where the barcode is an 8-base sequence unique to each sample. PCR reactions were performed in triplicate using 20 μl of mixture containing 4 μl of 5× FastPfu buffer, 2 μl of the 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl of FastPfu polymerase and 10 ng of template DNA. Illumina MiSeq Sequencing. Amplicons were extracted from 2% agarose gel and purified using the AxyPrep DNA gel extraction kit (Axygen Biosciences, Union City, Calif., USA) according
Animals and Epoxiconazole Treatment Epoxiconazole (97% purity; molecular formula: C17H13ClFN3O; CAS No. 133855-98-8) was obtained from Hebei Chinally International Trade Co., Ltd. (Hebei, China). Thirty female Sprague-Dawley rats (weight: 83.4 ± 4.38 g, age: 55–65 days) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) under license No. SCXK (HU) 2012-0002. They were housed in a barrier environment at controlled temperature (22.5 ± 2.5 ° C) and humidity (30–70%) on a 12-hour light/12-hour dark cycle.
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were Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria [3], with the dominant phyla in the human gut being Firmicutes and Bacteroidetes. Gut bacteria can provide several trophic factors for the host via the metabolism of substances such as bile salts [4] and short-chain fatty acids, which can be involved in the communication of gut-liver recycling and gut-brain axis [5]. Associations between the gut microbiome and diseases such as obesity [6], necrotizing enterocolitis [7], diabetes [8], irritable bowel syndrome [9], nonalcoholic fatty liver disease [10] and colon cancer [11] have been reported. Epoxiconazole, a broad-spectrum azole fungicide, is widely used as antifungal in agriculture [12], where it is effective in preventing leaf blotch (Septoria tritici) [13] and rust (Puccinia triticina) in wheat [14], which ensues the possibility of human exposure due to the consumption of azole fungicide-contaminated food. Epoxiconazole may antagonize the androgen receptor and inhibit testosterone formation in vitro [15]. It has fetotoxic effects in rats following exposure in utero or during pregnancy [16, 17], which is related with increased maternal plasma testosterone and reduced estradiol levels [17]. Epoxiconazole may also affect key enzymes such as cytochrome P450 17A1 or 17α-hydroxylase/17,20-lyase (CYP17), sequentially affecting the synthesis of steroid hormones [16]. A carcinogenic dose of epoxiconazole could reduce serum cholesterol levels and all-trans-retinoic acid levels, increase response to CYP and glutathione S-transferases genes, and up-regulate overexpression of transforming growth factor-α in mice [18]. Oral exposure to environmental chemicals may influence systemic toxicity by altering the gut microbiome [19]. However, little is known on the effects of pesticides on gut microbiota. Therefore, in this study, we aimed to evaluate whether exposure to epoxiconazole may impact on the abundance and composition of the gut microbiome, as well as their adverse effects on key organ functions.
Statistical Analyses Data are expressed as means ± SD. One-way analysis of variance (ANOVA) with post hoc Bonferroni’s multiple comparison test and nonparametric Kruskal-Wallis test was performed across the groups. Significance was defined as p < 0.05.
Table 1. Body and organ weights of the rats
Parameters, g
CG
Epoxiconazole treatment LDG
Initial body weight1 79.5±4.38a 79.2±3.43a Final body weight2 336.5±24.0a 331.7±23.1a Brain1 1.89±0.1a 1.94±0.1a 2 a Heart 1.01±0.1 1.00±0.1a Lung1 1.33±0.2a 1.21±0.2a 2 a Liver 11.0±1.6 10.5±1.3a, b 2 a Spleen 0.60±0.07 0.59±0.06a Kidney2 2.2±0.2a 2.2±0.1a, b 1 a Paranephros 0.06±0.01 0.07±0.01a Ovary2 0.11±0.02a 0.12±0.03a
Effect of Epoxiconazole on Organ Weights The body weight and relative vital organ weights of rats before and after epoxiconazole exposure are shown in table 1. No difference existed regarding the weight of the body and vital organs such as the brain, heart, lung, spleen, paranephros or ovary after exposure to epoxiconazole. However, the weight of the liver was significantly increased (p < 0.001) in HDG as well as the kidney (p < 0.05). Effect of Epoxiconazole on Serum Biochemical Parameters The effect of epoxiconazole on serum biochemical parameters is summarized in table 2. Epoxiconazole significantly decreased serum levels of TBIL and CHE in HDG compared with LDG and CG. Glucose levels were also increased in HDG. No changes were observed in the levels of AST, ALT, total protein, albumin, alkaline phosphatase, blood urea nitrogen, creatinine and cholesterol.
78.9±2.81a 325.3±29.2a 1.80±0.2a 0.98±0.1a 1.31±0.2a 12.9±1.2c 0.59±0.10a 2.4±0.2b 0.08±0.02a 0.14±0.05a
Means ± SD (n = 10 rats/group). Values in the same row with different letters are significantly different at p < 0.05. 1 Data present nonnormal distribution (Kruskal-Wallis test). 2 Data present normal distribution (ANOVA).
Table 2. Serum biochemical parameters
Parameters
CG
Epoxiconazole treatment LDG
Results
HDG
HDG
ALT1, U/l 31.8±13.2a 34.6±10.2a 27.9±6.2a AST1, U/l 119.8±59.1a 131.9±38.8a 90.4±18.3a TP2, g/l 70.8±4.9a 71.0±5.3a 67.4±4.0a 2 a a ALB , g/l 41.8±2.6 42.0±2.6 38.8±2.0a TBIL1, μmol/l 0.79±0.43a 0.70±0.55b 0.06±0.12c 2 a a ALP , U/l 28.6±7.8 30.0±7.2 29.6±6.0a 2 a a, b GLU , mmol/l 8.68±0.95 8.48±1.29 10.29±1.24c BUN2, mmol/l 6.73±0.79a 7.00±1.39a 6.51±0.82a 2 a a CREA , μmol/l 40.4±5.58 44.1±5.80 47.67±5.96a CHOL2, mg/dl 3.49±0.82a 3.32±0.79a 3.58±0.58a CHE2, U/l 1,369.0±231.4a, b 1,389.2±252.6a 1,090.0±256.8b Means ± SD (n = 10 rats/group). Values in the same row with different letters are significantly different at p < 0.05. TP = Total protein; ALB = albumin; ALP = alkaline phosphatase; GLU = glucose; BUN = blood urea nitrogen; CREA = creatinine; CHOL = cholesterol. 1 Data present nonnormal distribution (Kruskal-Wallis test). 2 Data present normal distribution (ANOVA).
Histological Evaluation of the Liver No microscopic abnormality was found in the liver tissue of rats after low- or high-dose epoxiconazole administration (fig. 1).
Effect of Epoxiconazole on Gut Microbiota A total of 529 OTUs were obtained from three pooled samples (every pooled sample contains feces from 6 rats) through Illumina MiSeq sequencing analysis: 8,612, 15,236 and 13,362 sequencing reads were obtained in CG, LDG and HDG, respectively. Each library contains different phylogenetic OTUs ranging from 294 to 427.
Epoxiconazole Exposure Alters Gut Microbiota
Chemotherapy 2014;60:135–142 DOI: 10.1159/000371837
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to the manufacturer’s instructions and quantified using QuantiFluorTM-ST (Promega, Madison, Wisc., USA). Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to standard protocols. Raw data were deposited ino the NCBI SRA (Sequence Read Archive) database. Processing of Sequencing Data. Raw FastQ files were demultiplexed and quality filtered using QIIME using the following criteria: (i) The 250-bp reads were truncated at any site receiving a quality score 5% in each sample, 138
Chemotherapy 2014;60:135–142 DOI: 10.1159/000371837
LDG
HDG
but they jointly hold 90.53% of the total reads. Among them, Firmicutes are the most abundant (fig. 2), comprising approximately 77.73% in CG, decreasing to 40.57% in LDG and 28.36% in HDG after exposure to epoxiconazole. In contrast, Bacteroidetes, the second abundant, increased from 20.66% in CG to 51.82% in LDG and 52.45% in HDG. Proteobacteria showed a similar trend of change as Bacteroidetes. Tenericutes and the average radius of an Xu/Liu/Huan/Qu/Liu/Gu/Wang/Jiang
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Fig. 2. Bacterial composition of the differ-
Relative abundance (%)
100
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pictures) and 5 μm (larger pictures).
60 40 20 0
CG
unassigned group account for 0.97 and 0.44%, fluctuating among groups. The other lineages represent much smaller fractions (∼0.1%) of the bacterial communities. At family level, there are 13 lineages among the total families which exist in all samples (fig. 3), but these subgroups account for 95.4, 91.34 and 94.22% of the total reads in the three groups, respectively. The top 5 families, by descending read abundance, were unassigned (27.91%), Bacillaceae (12.39%), Ruminococcaceae (10.76%), Lactobacillaceae (8.08%) and Erysipelotrichaceae (6.12%). Epoxiconazole exposure showed higher Bacteroidaceae and Enterobacteriaceae richness in the HDG than in the CG as well as LDG. In contrast, the second predominant family of Bacillaceae showed a descending trend in LDG and HDG, as well as Lactobacillaceae, Peptostreptococcaceae and Erysipelotrichaceae. The Ruminococcaceae profile appeared stable. However, the profile of Prevotellaceae demonstrates a variable order among groups. The communities shared among the different groups are further determined via a Venn diagram (fig. 4). A total of 209 OTUs are shared by the three groups, accounting for 39.51% of the total 529 bacterial taxa across all samples. Throughout the whole profiles, the unique bacterial communities in CG, LDG and HDG occupy 30, 51 and 86 bacterial taxa, respectively. The top 10 abundant OTUs shared by LDG and HDG after exposure to epoxiconazole are listed in table 3. They belong to different phyla, including Bacteriodetes, Proteobacteria and Firmicutes. The two greatest bacterial taxa changed were Lachnospiraceae and Enterobacteriaceae.
LDG
Color version available online
80
Staphylococcaceae Lachnospiraceae Moraxellaceae Enterobacteriaceae Others
HDG
CG
HDG 21
30
51
209
34
98
86
LDG
Fig. 4. Venn diagram representing shared and unique OTUs of the gut microbiome. Numbers in the diagram represent the number of OTUs in the different groups. There are 529 OTUs in all three groups. Pooled feces of 6 rats were analyzed in every group.
In view of the recent evidence, the gut microbiome may play a role in the modulation of health risks when exposed to environmental chemicals or heavy metals [22]. In this
study, we aimed to detect changes in the gut microbiome in rats after long-term oral exposure to epoxiconazole, an important agricultural fungicide, as well as its effect on key organ functions. Although 90-day exposure to epoxiconazole did not lead to apparent pathological hepatic lesions following HE staining, increases in liver weight and decreases in TBIL and CHE levels, which reflect impaired liver function, already occurred. Furthermore, epoxiconazole exposure affected the abundance and composition of gut microbiota. However, 209 OTUs were shared by the three groups. It is noteworthy that the kidney weight was increased in HDG compared to CG, but this mechanism requires elucidation by future trials.
Epoxiconazole Exposure Alters Gut Microbiota
Chemotherapy 2014;60:135–142 DOI: 10.1159/000371837
Discussion
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of the gut microbiome at family level. Pooled feces of 6 rats were analyzed in every group.
No-Rank Lactobacillaceae Bacillaceae Bacteroidaceae Prevotellaceae Peptostreptococcaceae Ruminococcaceae Erysipelotrichaceae Enterococcaceae
Color version available online
Fig. 3. HE histogram of relative abundance
Relative abundance (%)
100
Table 3. The 10 bacterial taxa with the greatest increase in abundance following exposure of rats to epoxiconazole
Phylum
Class
Order
Family
Firmicutes Proteobacteria Bacteroidetes Firmicutes Bacteroidetes Firmicutes Bacteroidetes Bacteroidetes Bacteroidetes Firmicutes
Clostridia Gammaproteobacteria Bacteroidia Clostridia Bacteroidia Clostridia Bacteroidia Bacteroidia Bacteroidia Clostridia
Clostridiales Enterobacteriales Bacteroidales Clostridiales Bacteroidales Clostridiales Bacteroidales Bacteroidales Bacteroidales Clostridiales
Lachnospiraceae Enterobacteriaceae S24-7 Ruminococcaceae Bacteroidaceae Ruminococcaceae Bacteroidaceae Rikenellaceae Rikenellaceae Christensenellaceae
Species Proteus mirabilis Uncultured organism Human gut metagenome Bacteroides intestinalis Uncultured bacterium
Bacterium_YE57
LDG
HDG
9 7 28 17 9 6 7 15 10 7
47 49 19 25 31 32 21 6 11 12
Epoxiconazole is widely used for many products and product mixtures, and is a fungicide targeting a large number of pathogens in various crops worldwide. It has been found in groundwater and soil with medium/high persistence (http://www.efsa.europa.eu/en/efsajournal/ pub/138r.htm). Humans may be exposed via the drinking water and epoxiconazole-contaminated food. Therefore, we chose to evaluate the effect of epoxiconazole exposure on the abundance and diversity of the gut microbiome. A 90-day epoxiconazole diet significantly increased liver and kidney weight in HDG. This might be due to cell injury, tissue edema and histopathological alterations in the liver and kidney of male rats caused by 75 and 150 mg/kg of epoxiconazole via oral gavage for 4 weeks [23]. In epoxiconazole-treated male mice, increases in hepatic cell proliferation have also been reported [18]. Biochemical analysis showed decreased TBIL levels after epoxiconazole exposure. These defects occur before pathological changes can be visualized the in liver tissue. Furthermore, CHE levels were also decreased, possibly due to injury of the liver, where CHE is synthesized [24]. The effects of this fungicide on the gut microbiome in the intestinal tract and, in turn, on enterohepatic recycling may possibly explain the underlying mechanism. Interestingly, epoxiconazole exposure appears to be a source of environmental stress and consequently affects the composition of bacterial communities. The abundance and composition of the gut microbiome changed following epoxiconazole administration across the groups. These changes selectively affected bacterial phylotypes in phyla such as Firmicutes, Bacteroidetes and Proteobacteria. Epoxiconazole exposure led to phylum shifts. The gut microbiome plays an important role in mucosal immunity and interactions with intestinal and 140
Chemotherapy 2014;60:135–142 DOI: 10.1159/000371837
colonic epithelial cells, dendritic cells, and T and B cells [25]. The microbiotic composition has functional effects on T-effector/T-regulatory cell ratios, immune responsiveness and homeostasis [26]. Therefore, changes in gut bacteria may lead to defects in mucosal immunity. At family level, we found a 17-fold decrease in the abundance of Lactobacillaceae in the gut microbiome of HDG, and, in contrast, a 12-fold increase in the abundance of Bacteroidaceae. Lactobacillaceae are beneficial for the human gut due to its ability to maintain the homeostasis of the intestinal environment [27]. The fact that high-dose epoxiconazole resulted in a lower abundance of Lactobacillaceae may possibly be associated with liver damage since Lactobacillaceae have shown to protect liver from damage [28]. Changes in Bacteroidaceae were reported to be associated with disease: lower abundance of Bacteroidaceae was associated with pouchitis [29], and Crohn’s disease has been found to be related with an increased abundance of Bacteroidaceae [30]. The Bacteroidaceae family has amylolytic and cellulolytic properties [31]. In our study, we found an increased abundance of Bacteroidaceae following epoxiconazole administration, which might reflect compensatory growth. Although the abundance and composition of the gut microbiome varied following epoxiconazole treatment, 209 OTUs were shared by all the three groups. This suggested that they are resistant to epoxiconazole exposure. In contrast, only 98 OTUs were noted in both epoxiconazole groups. Among the 10 bacterial taxa with the greatest increase in abundance of the gut microbiome, the two greatest changes were observed in Lachnospiraceae and Enterobacteriaceae. The main benefit of Lachnospiraceae family members in the human intestine is their participaXu/Liu/Huan/Qu/Liu/Gu/Wang/Jiang
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Values of LDG and HDG were calculated from the number of sequence reads.
tion in carbohydrate fermentation into butyrate (a kind of short-chain fatty acid) and gas [32]. Short-chain fatty acids are important sources of energy for colonic epithelial cells. It may enhance epithelial barrier integrity and modulate gastrointestinal immune responses [33]. In our study, the increased number of Lachnospiraceae might be the result of host self-modulation, since Lachnospiraceae was conducive to host health. Enterobacteriaceae function as facultative anaerobes producing lactate, succinate, ethanol, acetate and carbon dioxide [34]. Increased abundance of Enterobacteriaceae was associated with gut inflammation. Induction of experimental colitis in rodents was followed by an increase in this family and it might be a consequence of gut inflammation rather than a cause [35]. On the other hand, antibiotic treatment had a substantial effect on the gut microbiome, increasing Enterobacteriaceae abundance [36]. Since Lachnospiraceae and Enterobacteriaceae were significantly enriched following epoxiconazole administration, they might be used as indicators for monitoring the health risk of the host. To our knowledge, this is the first study demonstrated that chronic oral exposure to epoxiconazole significantly
altered the composition of the gut microbiome by increasing the levels of Proteobacteria and Bacteroidetes, and decreasing the levels of Firmicutes. The results suggest that the gut microbiome may be one of the primary targets of epoxiconazole-induced toxicity in subjects prior to the occurrence of hepatic and intestinal pathological changes. Changes in the gut microbiome may be used as early indicators for monitoring the health risk of the host. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant Nos. 81172694, 81270537, 81373041 and 81302453), the Research Project of the Chinese Ministry of Education (No. 213015A), the Outstanding Youth Fund of Jiangsu Province SBK2014010296), and the Qinglan project of Jiangsu Province (JX2161015124), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Disclosure Statement The authors have declared that no competing interests exist.
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Chemotherapy 2014;60:135–142 DOI: 10.1159/000371837
Changes in Gut Microbiota May Be Early Signs of Liver Toxicity Induced by Epoxiconazole in Rats Cheng Xu a, b Qian Liu a, b Fei Huan c Jianhua Qu a, b Wei Liu a, b Aihua Gu a, b Yubang Wang a, c Zhaoyan Jiang d
a
State Key Laboratory of Reproductive Medicine, Institute of Toxicology, b Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, and c Safety Assessment and Research Center for Drugs, Pesticides and Veterinary Drugs of the Jiangsu Province, Nanjing Medical University, Nanjing, d Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, China
Abstract Objective: The gut microbiome is essential for human health due to its effects on disease development, drug metabolism and the immune system. It may also play a role in the interaction with environmental toxicants. However, the effect of epoxiconazole, a fungicide active ingredient from the class of azoles developed to protect crops, on the abundance and composition of the gut microbiome has never been studied. We put forward the hypothesis that changes in gut microbiota may be early signs of toxicity induced by epoxiconazole. Methods: In this study, female rats were fed with epoxiconazole-adulterated diets (0, 4 and 100 mg/kg/day) for 90 days. The gut microbiome was determined by 16S rRNA gene sequencing. Body and organ weight, and blood biochemistry were also measured after 90 days of oral epoxiconazole exposure. Results: Interestingly, the abundance of gut Firmicutes decreased, and Bacteroidetes and ProteobacC. Xu, Q. Liu and F. Huan contributed equally to this study and should be regarded as joint first authors.
© 2015 S. Karger AG, Basel 0009–3157/15/0602–0135$39.50/0 E-Mail [email protected] www.karger.com/che
teria increased. At family level, Lachnospiraceae and Enterobacteriaceae were selectively enriched following epoxiconazole exposure. Our results indicate that epoxiconazole exposure may induce changes in the gut microbiome and potential liver toxicity. Conclusion: Changes in the gut microbiome may be used as early indicators for monitoring the health risk of the host. © 2015 S. Karger AG, Basel
Introduction
The microbiome is found all over the biosphere; it is even included in a portion of the alimentary system of the host. Approximately 100 trillion microbes exist as common microbiota in the gastrointestinal tract of hosts, with bacteria being the major participants of the human microbiome [1]. In the human gut microbiome, there are >300,000 gene-encoding genes [2], whereas in the host, the human genome encodes only 20,000–25,000 proteincoding genes. Recent investigations into the human gut microbiome found that nearly 98% of bacterial phyla Aihua Gu, Yubang Wang State Key Laboratory of Reproductive Medicine Institute of Toxicology, Nanjing Medical University Nanjing 210029 (China) E-Mail aihuagu @ njmu.edu.cn, wyb @ njmu.edu.cn Zhaoyan Jiang Department of Surgery, Shanghai Institute of Digestive Surgery Ruijin Hospital, Shanghai JiaoTong University School of Medicine Shanghai 200025 (China) E-Mail zhaoyanjiang @ gmail.com
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Key Words Epoxiconazole · Gut microbiota · Host health · Liver toxicity · Microarray analysis
Materials and Methods
Epoxiconazole-adulterated diets were prepared according to a previous report [20] with some modifications. The following adulterated feeds were prepared: epoxiconazole (0, 40 and 1,000 ppm) with phytoestrogen-free food (Shuangshi Animal Food Inc., Suzhou, China). The feeding concentrations were selected based on the feeding levels used in our preliminary 14-day acute toxicity study (data not shown). Based on the preliminary study, 4 and 100 mg/ kg body weight/day were chosen as low- and high-concentration diets, respectively. These doses, 4 and 100 mg/kg/day, were chosen as the lower dose (less than no observed adverse effect level) and higher dose (more than the lowest observed adverse effect level), respectively (http://enfo.agt.bme.hu/drupal/sites/default/ files/epoxiconazole_0.pdf). The rats were allowed to acclimate for 1 week before study commencement and were then given the above adulterated feeds for 90 days. The rats were randomly divided into three groups (10 rats/ group and 2 rats/metabolic cage): the control group (CG), the lowepoxiconazole group (LDG) and the high-epoxiconazole group (HDG) and fed adulterated feeds and water ad libitum. All animal procedures were approved by the local Animal Care and Use Committee of the Nanjing Medical University. Collection of Biological Samples, Determination of Serum Biochemical Parameters and Histological Analysis Twenty-four hours after completion of the final task session, the rats were euthanized by CO2 asphyxiation. Blood was collected from all rats by cardiac puncture using a 23-gauge needle into BD Vacutainer serum blood collection tubes and centrifuged immediately at 1,200 g for 20 min at 4 ° C. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, albumin, total bilirubin (TBIL), alkaline phosphatase, glucose, blood urea nitrogen, creatinine, cholesterol and cholinesterase (CHE) of all animals were measured by automatic biochemical analyzer in the Center for Hygienic Analysis and Detection, Nanjing Medical University. The initial and final body weights were recorded. The relevant primary organs were excised and weighed. The liver was prepared for histological studies: tissues were fixed in 4% (w/v) paraformaldehyde, embedded in paraffin, stained with hematoxylin-eosin (HE) and observed under a light microscope. Feces were collected from the bottom of the metabolic cage on day 90, transferred to 15-ml Corning tubes and stored at –80 ° C until analysis.
Genomic DNA Extraction and PCR Amplification DNA Extraction and PCR Amplification. Microbial DNA was extracted from fecal samples using the E.Z.N.A.® stool DNA kit (Omega Bio-Tek, Norcross, Ga., USA) according to the manufacturer’s protocols. The V4–V5 region of the bacterial 16S ribosomal RNA gene was amplified by PCR (95 ° C for 2 min, followed by 25 cycles at 95 ° C for 30 s, 55 ° C for 30 s and 72 ° C for 30 s, and a final extension at 72 ° C for 5 min) using primers 515F 5′-barcodeGTG CCA GCM GCC GCG G-3′ and 907R 5′-CCG TCA ATT CMT TTR AGT TT-3′ [21], where the barcode is an 8-base sequence unique to each sample. PCR reactions were performed in triplicate using 20 μl of mixture containing 4 μl of 5× FastPfu buffer, 2 μl of the 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl of FastPfu polymerase and 10 ng of template DNA. Illumina MiSeq Sequencing. Amplicons were extracted from 2% agarose gel and purified using the AxyPrep DNA gel extraction kit (Axygen Biosciences, Union City, Calif., USA) according
Animals and Epoxiconazole Treatment Epoxiconazole (97% purity; molecular formula: C17H13ClFN3O; CAS No. 133855-98-8) was obtained from Hebei Chinally International Trade Co., Ltd. (Hebei, China). Thirty female Sprague-Dawley rats (weight: 83.4 ± 4.38 g, age: 55–65 days) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) under license No. SCXK (HU) 2012-0002. They were housed in a barrier environment at controlled temperature (22.5 ± 2.5 ° C) and humidity (30–70%) on a 12-hour light/12-hour dark cycle.
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were Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria [3], with the dominant phyla in the human gut being Firmicutes and Bacteroidetes. Gut bacteria can provide several trophic factors for the host via the metabolism of substances such as bile salts [4] and short-chain fatty acids, which can be involved in the communication of gut-liver recycling and gut-brain axis [5]. Associations between the gut microbiome and diseases such as obesity [6], necrotizing enterocolitis [7], diabetes [8], irritable bowel syndrome [9], nonalcoholic fatty liver disease [10] and colon cancer [11] have been reported. Epoxiconazole, a broad-spectrum azole fungicide, is widely used as antifungal in agriculture [12], where it is effective in preventing leaf blotch (Septoria tritici) [13] and rust (Puccinia triticina) in wheat [14], which ensues the possibility of human exposure due to the consumption of azole fungicide-contaminated food. Epoxiconazole may antagonize the androgen receptor and inhibit testosterone formation in vitro [15]. It has fetotoxic effects in rats following exposure in utero or during pregnancy [16, 17], which is related with increased maternal plasma testosterone and reduced estradiol levels [17]. Epoxiconazole may also affect key enzymes such as cytochrome P450 17A1 or 17α-hydroxylase/17,20-lyase (CYP17), sequentially affecting the synthesis of steroid hormones [16]. A carcinogenic dose of epoxiconazole could reduce serum cholesterol levels and all-trans-retinoic acid levels, increase response to CYP and glutathione S-transferases genes, and up-regulate overexpression of transforming growth factor-α in mice [18]. Oral exposure to environmental chemicals may influence systemic toxicity by altering the gut microbiome [19]. However, little is known on the effects of pesticides on gut microbiota. Therefore, in this study, we aimed to evaluate whether exposure to epoxiconazole may impact on the abundance and composition of the gut microbiome, as well as their adverse effects on key organ functions.
Statistical Analyses Data are expressed as means ± SD. One-way analysis of variance (ANOVA) with post hoc Bonferroni’s multiple comparison test and nonparametric Kruskal-Wallis test was performed across the groups. Significance was defined as p < 0.05.
Table 1. Body and organ weights of the rats
Parameters, g
CG
Epoxiconazole treatment LDG
Initial body weight1 79.5±4.38a 79.2±3.43a Final body weight2 336.5±24.0a 331.7±23.1a Brain1 1.89±0.1a 1.94±0.1a 2 a Heart 1.01±0.1 1.00±0.1a Lung1 1.33±0.2a 1.21±0.2a 2 a Liver 11.0±1.6 10.5±1.3a, b 2 a Spleen 0.60±0.07 0.59±0.06a Kidney2 2.2±0.2a 2.2±0.1a, b 1 a Paranephros 0.06±0.01 0.07±0.01a Ovary2 0.11±0.02a 0.12±0.03a
Effect of Epoxiconazole on Organ Weights The body weight and relative vital organ weights of rats before and after epoxiconazole exposure are shown in table 1. No difference existed regarding the weight of the body and vital organs such as the brain, heart, lung, spleen, paranephros or ovary after exposure to epoxiconazole. However, the weight of the liver was significantly increased (p < 0.001) in HDG as well as the kidney (p < 0.05). Effect of Epoxiconazole on Serum Biochemical Parameters The effect of epoxiconazole on serum biochemical parameters is summarized in table 2. Epoxiconazole significantly decreased serum levels of TBIL and CHE in HDG compared with LDG and CG. Glucose levels were also increased in HDG. No changes were observed in the levels of AST, ALT, total protein, albumin, alkaline phosphatase, blood urea nitrogen, creatinine and cholesterol.
78.9±2.81a 325.3±29.2a 1.80±0.2a 0.98±0.1a 1.31±0.2a 12.9±1.2c 0.59±0.10a 2.4±0.2b 0.08±0.02a 0.14±0.05a
Means ± SD (n = 10 rats/group). Values in the same row with different letters are significantly different at p < 0.05. 1 Data present nonnormal distribution (Kruskal-Wallis test). 2 Data present normal distribution (ANOVA).
Table 2. Serum biochemical parameters
Parameters
CG
Epoxiconazole treatment LDG
Results
HDG
HDG
ALT1, U/l 31.8±13.2a 34.6±10.2a 27.9±6.2a AST1, U/l 119.8±59.1a 131.9±38.8a 90.4±18.3a TP2, g/l 70.8±4.9a 71.0±5.3a 67.4±4.0a 2 a a ALB , g/l 41.8±2.6 42.0±2.6 38.8±2.0a TBIL1, μmol/l 0.79±0.43a 0.70±0.55b 0.06±0.12c 2 a a ALP , U/l 28.6±7.8 30.0±7.2 29.6±6.0a 2 a a, b GLU , mmol/l 8.68±0.95 8.48±1.29 10.29±1.24c BUN2, mmol/l 6.73±0.79a 7.00±1.39a 6.51±0.82a 2 a a CREA , μmol/l 40.4±5.58 44.1±5.80 47.67±5.96a CHOL2, mg/dl 3.49±0.82a 3.32±0.79a 3.58±0.58a CHE2, U/l 1,369.0±231.4a, b 1,389.2±252.6a 1,090.0±256.8b Means ± SD (n = 10 rats/group). Values in the same row with different letters are significantly different at p < 0.05. TP = Total protein; ALB = albumin; ALP = alkaline phosphatase; GLU = glucose; BUN = blood urea nitrogen; CREA = creatinine; CHOL = cholesterol. 1 Data present nonnormal distribution (Kruskal-Wallis test). 2 Data present normal distribution (ANOVA).
Histological Evaluation of the Liver No microscopic abnormality was found in the liver tissue of rats after low- or high-dose epoxiconazole administration (fig. 1).
Effect of Epoxiconazole on Gut Microbiota A total of 529 OTUs were obtained from three pooled samples (every pooled sample contains feces from 6 rats) through Illumina MiSeq sequencing analysis: 8,612, 15,236 and 13,362 sequencing reads were obtained in CG, LDG and HDG, respectively. Each library contains different phylogenetic OTUs ranging from 294 to 427.
Epoxiconazole Exposure Alters Gut Microbiota
Chemotherapy 2014;60:135–142 DOI: 10.1159/000371837
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to the manufacturer’s instructions and quantified using QuantiFluorTM-ST (Promega, Madison, Wisc., USA). Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to standard protocols. Raw data were deposited ino the NCBI SRA (Sequence Read Archive) database. Processing of Sequencing Data. Raw FastQ files were demultiplexed and quality filtered using QIIME using the following criteria: (i) The 250-bp reads were truncated at any site receiving a quality score 5% in each sample, 138
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LDG
HDG
but they jointly hold 90.53% of the total reads. Among them, Firmicutes are the most abundant (fig. 2), comprising approximately 77.73% in CG, decreasing to 40.57% in LDG and 28.36% in HDG after exposure to epoxiconazole. In contrast, Bacteroidetes, the second abundant, increased from 20.66% in CG to 51.82% in LDG and 52.45% in HDG. Proteobacteria showed a similar trend of change as Bacteroidetes. Tenericutes and the average radius of an Xu/Liu/Huan/Qu/Liu/Gu/Wang/Jiang
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Fig. 2. Bacterial composition of the differ-
Relative abundance (%)
100
Color version available online
pictures) and 5 μm (larger pictures).
60 40 20 0
CG
unassigned group account for 0.97 and 0.44%, fluctuating among groups. The other lineages represent much smaller fractions (∼0.1%) of the bacterial communities. At family level, there are 13 lineages among the total families which exist in all samples (fig. 3), but these subgroups account for 95.4, 91.34 and 94.22% of the total reads in the three groups, respectively. The top 5 families, by descending read abundance, were unassigned (27.91%), Bacillaceae (12.39%), Ruminococcaceae (10.76%), Lactobacillaceae (8.08%) and Erysipelotrichaceae (6.12%). Epoxiconazole exposure showed higher Bacteroidaceae and Enterobacteriaceae richness in the HDG than in the CG as well as LDG. In contrast, the second predominant family of Bacillaceae showed a descending trend in LDG and HDG, as well as Lactobacillaceae, Peptostreptococcaceae and Erysipelotrichaceae. The Ruminococcaceae profile appeared stable. However, the profile of Prevotellaceae demonstrates a variable order among groups. The communities shared among the different groups are further determined via a Venn diagram (fig. 4). A total of 209 OTUs are shared by the three groups, accounting for 39.51% of the total 529 bacterial taxa across all samples. Throughout the whole profiles, the unique bacterial communities in CG, LDG and HDG occupy 30, 51 and 86 bacterial taxa, respectively. The top 10 abundant OTUs shared by LDG and HDG after exposure to epoxiconazole are listed in table 3. They belong to different phyla, including Bacteriodetes, Proteobacteria and Firmicutes. The two greatest bacterial taxa changed were Lachnospiraceae and Enterobacteriaceae.
LDG
Color version available online
80
Staphylococcaceae Lachnospiraceae Moraxellaceae Enterobacteriaceae Others
HDG
CG
HDG 21
30
51
209
34
98
86
LDG
Fig. 4. Venn diagram representing shared and unique OTUs of the gut microbiome. Numbers in the diagram represent the number of OTUs in the different groups. There are 529 OTUs in all three groups. Pooled feces of 6 rats were analyzed in every group.
In view of the recent evidence, the gut microbiome may play a role in the modulation of health risks when exposed to environmental chemicals or heavy metals [22]. In this
study, we aimed to detect changes in the gut microbiome in rats after long-term oral exposure to epoxiconazole, an important agricultural fungicide, as well as its effect on key organ functions. Although 90-day exposure to epoxiconazole did not lead to apparent pathological hepatic lesions following HE staining, increases in liver weight and decreases in TBIL and CHE levels, which reflect impaired liver function, already occurred. Furthermore, epoxiconazole exposure affected the abundance and composition of gut microbiota. However, 209 OTUs were shared by the three groups. It is noteworthy that the kidney weight was increased in HDG compared to CG, but this mechanism requires elucidation by future trials.
Epoxiconazole Exposure Alters Gut Microbiota
Chemotherapy 2014;60:135–142 DOI: 10.1159/000371837
Discussion
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of the gut microbiome at family level. Pooled feces of 6 rats were analyzed in every group.
No-Rank Lactobacillaceae Bacillaceae Bacteroidaceae Prevotellaceae Peptostreptococcaceae Ruminococcaceae Erysipelotrichaceae Enterococcaceae
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Fig. 3. HE histogram of relative abundance
Relative abundance (%)
100
Table 3. The 10 bacterial taxa with the greatest increase in abundance following exposure of rats to epoxiconazole
Phylum
Class
Order
Family
Firmicutes Proteobacteria Bacteroidetes Firmicutes Bacteroidetes Firmicutes Bacteroidetes Bacteroidetes Bacteroidetes Firmicutes
Clostridia Gammaproteobacteria Bacteroidia Clostridia Bacteroidia Clostridia Bacteroidia Bacteroidia Bacteroidia Clostridia
Clostridiales Enterobacteriales Bacteroidales Clostridiales Bacteroidales Clostridiales Bacteroidales Bacteroidales Bacteroidales Clostridiales
Lachnospiraceae Enterobacteriaceae S24-7 Ruminococcaceae Bacteroidaceae Ruminococcaceae Bacteroidaceae Rikenellaceae Rikenellaceae Christensenellaceae
Species Proteus mirabilis Uncultured organism Human gut metagenome Bacteroides intestinalis Uncultured bacterium
Bacterium_YE57
LDG
HDG
9 7 28 17 9 6 7 15 10 7
47 49 19 25 31 32 21 6 11 12
Epoxiconazole is widely used for many products and product mixtures, and is a fungicide targeting a large number of pathogens in various crops worldwide. It has been found in groundwater and soil with medium/high persistence (http://www.efsa.europa.eu/en/efsajournal/ pub/138r.htm). Humans may be exposed via the drinking water and epoxiconazole-contaminated food. Therefore, we chose to evaluate the effect of epoxiconazole exposure on the abundance and diversity of the gut microbiome. A 90-day epoxiconazole diet significantly increased liver and kidney weight in HDG. This might be due to cell injury, tissue edema and histopathological alterations in the liver and kidney of male rats caused by 75 and 150 mg/kg of epoxiconazole via oral gavage for 4 weeks [23]. In epoxiconazole-treated male mice, increases in hepatic cell proliferation have also been reported [18]. Biochemical analysis showed decreased TBIL levels after epoxiconazole exposure. These defects occur before pathological changes can be visualized the in liver tissue. Furthermore, CHE levels were also decreased, possibly due to injury of the liver, where CHE is synthesized [24]. The effects of this fungicide on the gut microbiome in the intestinal tract and, in turn, on enterohepatic recycling may possibly explain the underlying mechanism. Interestingly, epoxiconazole exposure appears to be a source of environmental stress and consequently affects the composition of bacterial communities. The abundance and composition of the gut microbiome changed following epoxiconazole administration across the groups. These changes selectively affected bacterial phylotypes in phyla such as Firmicutes, Bacteroidetes and Proteobacteria. Epoxiconazole exposure led to phylum shifts. The gut microbiome plays an important role in mucosal immunity and interactions with intestinal and 140
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colonic epithelial cells, dendritic cells, and T and B cells [25]. The microbiotic composition has functional effects on T-effector/T-regulatory cell ratios, immune responsiveness and homeostasis [26]. Therefore, changes in gut bacteria may lead to defects in mucosal immunity. At family level, we found a 17-fold decrease in the abundance of Lactobacillaceae in the gut microbiome of HDG, and, in contrast, a 12-fold increase in the abundance of Bacteroidaceae. Lactobacillaceae are beneficial for the human gut due to its ability to maintain the homeostasis of the intestinal environment [27]. The fact that high-dose epoxiconazole resulted in a lower abundance of Lactobacillaceae may possibly be associated with liver damage since Lactobacillaceae have shown to protect liver from damage [28]. Changes in Bacteroidaceae were reported to be associated with disease: lower abundance of Bacteroidaceae was associated with pouchitis [29], and Crohn’s disease has been found to be related with an increased abundance of Bacteroidaceae [30]. The Bacteroidaceae family has amylolytic and cellulolytic properties [31]. In our study, we found an increased abundance of Bacteroidaceae following epoxiconazole administration, which might reflect compensatory growth. Although the abundance and composition of the gut microbiome varied following epoxiconazole treatment, 209 OTUs were shared by all the three groups. This suggested that they are resistant to epoxiconazole exposure. In contrast, only 98 OTUs were noted in both epoxiconazole groups. Among the 10 bacterial taxa with the greatest increase in abundance of the gut microbiome, the two greatest changes were observed in Lachnospiraceae and Enterobacteriaceae. The main benefit of Lachnospiraceae family members in the human intestine is their participaXu/Liu/Huan/Qu/Liu/Gu/Wang/Jiang
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Values of LDG and HDG were calculated from the number of sequence reads.
tion in carbohydrate fermentation into butyrate (a kind of short-chain fatty acid) and gas [32]. Short-chain fatty acids are important sources of energy for colonic epithelial cells. It may enhance epithelial barrier integrity and modulate gastrointestinal immune responses [33]. In our study, the increased number of Lachnospiraceae might be the result of host self-modulation, since Lachnospiraceae was conducive to host health. Enterobacteriaceae function as facultative anaerobes producing lactate, succinate, ethanol, acetate and carbon dioxide [34]. Increased abundance of Enterobacteriaceae was associated with gut inflammation. Induction of experimental colitis in rodents was followed by an increase in this family and it might be a consequence of gut inflammation rather than a cause [35]. On the other hand, antibiotic treatment had a substantial effect on the gut microbiome, increasing Enterobacteriaceae abundance [36]. Since Lachnospiraceae and Enterobacteriaceae were significantly enriched following epoxiconazole administration, they might be used as indicators for monitoring the health risk of the host. To our knowledge, this is the first study demonstrated that chronic oral exposure to epoxiconazole significantly
altered the composition of the gut microbiome by increasing the levels of Proteobacteria and Bacteroidetes, and decreasing the levels of Firmicutes. The results suggest that the gut microbiome may be one of the primary targets of epoxiconazole-induced toxicity in subjects prior to the occurrence of hepatic and intestinal pathological changes. Changes in the gut microbiome may be used as early indicators for monitoring the health risk of the host. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant Nos. 81172694, 81270537, 81373041 and 81302453), the Research Project of the Chinese Ministry of Education (No. 213015A), the Outstanding Youth Fund of Jiangsu Province SBK2014010296), and the Qinglan project of Jiangsu Province (JX2161015124), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Disclosure Statement The authors have declared that no competing interests exist.
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