Effects of short term lead exposure on gut microbiota and hepatic metabolism in adult zebrafish

Comparative Biochemistry and Physiology, Part C 209 (2018) 1–8

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Effects of short term lead exposure on gut microbiota and hepatic metabolism in adult zebrafish

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Jizhou Xia, Liang Lu, Cuiyuan Jin, Siyu Wang, Jicong Zhou, Yingchun Ni, Zhengwei Fu, ⁎ Yuanxiang Jin College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Lead Gut microbiota Hepatic metabolism Zebrafish

Lead (Pb) is one of the most prevalent toxic, nonessential heavy metals that has been associated with a wide range of toxic effects in humans and environmental animals. Here, effects of short time exposure to 10 and 30 μg/L Pb on gut microbiota and hepatic metabolism were analyzed in adult male zebrafish. We observed that both 10 and 30 μg/L Pb increased the volume of mucus in the gut. At phylum level, the abundance of αProteobacteria decreased significantly and the abundance of Firmicutes increased significantly in the gut when treated with 30 μg/L Pb for 7 days. In addition, the 16S rRNA gene sequencing for V3-V4 region revealed a significant change in the richness and diversity of gut microbiota in 30 μg/L Pb exposed group. A more depth analysis, at the genus level, discovered that 52 gut microbes identified by operational taxonomic unit analysis were changed significantly in 30 μg/L Pb treated group. Based on GC/MS metabolomics analysis, a total of 41 metabolites were significantly altered in 30 μg/L Pb treatment group. These changed metabolites were mainly associated with the pathways of glucose and lipid metabolism, amino acid metabolism, nucleotide metabolism. In addition, we also confirmed that the transcription of some genes related to glycolysis and lipid metabolism, including Gk, Aco, Acc1, Fas, Apo and Dgat, decreased significantly in the liver of zebrafish when exposed to 30 μg/L Pb for 7 days. Our results observed that Pb could cause gut microbiota dysbiosis and hepatic metabolic disorder in zebrafish.

1. Introduction Heavy metals induced the toxicity are increasing in recent years (Winneke, 2011; Nadella et al., 2013; Jin et al., 2016b). Lead (pb), one of the essential trace elements, is widely used in industrial activities, and it entered into the environment through different pathways. According to previous studies, Pb is a commonly detected heavy metal and is present in diverse environmental matrices, especially in the e-waste area at high concentrations (Wong et al., 2007; Leung et al., 2008). Undoubtedly, Pb can reach aquatic systems via the effluents of industrial, urban and mining sources (Senger et al., 2006). In particular, in water samples from e-waste recycling sites in Guiyu of China, Pb concentration even reached as high as 400 μg/L (Wang and Guo, 2006). More importantly, Pb has been associated with a wide range of toxic effects in different experimental animals. According to previous reports, in addition to its major neurotoxicity (Zhang et al., 2011; Lee and Freeman, 2014; Wang et al., 2016), the toxicity of Pb was also associated with hepatotoxicity (Hasanein et al., 2016), oxidative stress (Liu et al., 2015), endocrine disruption (He et al., 2017), and cardiovascular

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toxicity (Gump et al., 2011) as well as immune toxicity (Kasten-Jolly and Lawrence, 2017). Thus, high concentrations of Pb in aquatic system would cause the serious toxicity to aquatic organisms directly. However, till date, studies describing the mechanisms of Pb-induced gut microbiota dysbiosis and metabolism disorder in zebrafish are still remained unclear. As a frequently used experimental model, zebrafish (Danio rerio) has emerged as an ideal experimental model to study the aquatic toxicity of environmental chemicals including heavy metals (Jin et al., 2010; Lee and Freeman, 2014; Jin et al., 2015; Liu et al., 2016; Chen et al., 2017; Z.Z. Liu et al., 2017). As for Pb, a previous study reported that oral exposure to 10 mg/L Pb supplied in drinking water for 13 weeks could induce the gut microbiota dysbiosis and metabolism disorder in mice (Gao et al., 2017). Because Pb was often detected in the aquatic system, however, no study was focused on the effects of Pb on the gut microbiota and metabolism in fish. In this study, we hypothesized that Pb could affect the composition of gut microbiota, influence the gut function and cause metabolism disorder in adult zebrafish. Because egg protein transcripts are

Corresponding author at: 18, Chaowang Road, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China. E-mail address: [email protected] (Y. Jin).

https://doi.org/10.1016/j.cbpc.2018.03.007 Received 7 February 2018; Received in revised form 16 March 2018; Accepted 20 March 2018 Available online 22 March 2018 1532-0456/ © 2018 Elsevier Inc. All rights reserved.

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2.4. RNA extraction and gene transcription analysis

extremely abundant in the livers of breeding females and might confound the metabolic analysis, male fish were selected in the present study (Wang et al., 2005; Jin et al., 2008). For this purpose, male adult zebrafish was exposed to various concentrations of Pb for 7 days and determined whether or not they could induce microbiota dysbiosis in the gut and metabolism in the liver. We thought that the results acquired here will provide some new information regarding Pb-induced aquatic toxicity.

The livers from three zebrafish were dissected as one sample for total RNA extraction using TRIzol reagent (Takara, China). Six parallel total RNA samples were prepared for each Pb treated group. Then, a total of 500 ng RNA in each sample was used to synthase cDNA by a reverse transcription kit (Toyobo, Japan). The real-time quantitative polymerase chain reaction (RT-qPCR) was performed using the SYBR Green system (Toyobo, Japan) and Eppendorf MasterCycler® ep RealPlex2 system (Wesseling-Berzdorf, Germany). The sequences of the primers used in the present study were taken from previous studies (Freitas et al., 2010; Ballester et al., 2017; C.Y. Jin et al., 2017). The following PCR protocol was used: denaturation for 1 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The primer sequences are shown in Table S1. And, the transcription level of 18S rRNA gene was analyzed as a housekeeping gene. The quantification of relative abundances of gene transcription was performed as previously described (Livak and Schmittgen, 2001).

2. Materials and methods 2.1. Fish husbandry and exposure protocol Male adult wide type AB strain zebrafish (Danio rerio) were kept at standard laboratory conditions with temperature at 28 °C, a 14:10 dark/ light cycle according to the zebrafish breeding protocol (Westerfield, 1995). During the experiment, the fish were feed twice daily, and the water was changed every day. The selected male adult fish were exposed to 10 and 30 μg/L Pb (PbAC) for 7 days, respectively. In each group, 6 male adult fish were reared in 2 L of each solution in a glass tank, and 3 separate tanks was carried out in each group. A total of 18 fish was used in each group. The livers excised from 3 fish were collected as 1 sample, resulting in 6 pooled samples for mRNA transcription analysis. Additionally, 2 guts selected randomly in each glass tank (and total 6 samples in each group) were used for DNA extraction, and each gut excised from each fish was collected as one sample. Additionally, 2 guts selected randomly in each tank were used for histopathological analysis. No died fish was observed during the Pb exposure. For the hepatic metabolomic analysis, a total of 72 male adult fish (in 12 separate glass tanks, and 6 fish in each tank) were exposed to 30 μg/L Pb for 7 days. The same number of fish were reared in water as a control. After Pb exposure, the livers excised from 12 fish, from 2 tanks, were collected as one sample, and 6 pooled samples were collected both in control and 30 μg/L Pb treated groups, respectively. In all experiments, fish were anesthetized on ice before dissection. The livers and guts were kept on dry ice during preparation and then were stored at −80 °C until they were further analyzed.

2.5. GC/MS-based hepatic metabolomic analysis and the differential metabolites selection The sample preparation and GC/MS were performed according to a previous study (C.Y. Jin et al., 2017). Briefly, 30 mg accurately weighed sample was transferred to a 1.5-mL Eppendorf tube. Two small steel balls were added to the tube. Then, 20 μL of 2-chloro-L-phenylalanine (0.3 mg/mL) dissolved in methanol as internal standard and 600 μL mixture of methanol and water (4/1, vol/vol) were added to each sample, samples were placed at −80 °C for 2 min. Then grinded at 60 Hz for 2 min. Then ultrasonicated at ambient temperature for 10 min after vortexed, then placed at −20 °C for 30 min. Samples were centrifuged at 13,000 rpm, 4 °C for 15 min. 400 μL of supernatant in a glass vial was dried in a freeze concentration centrifugal dryer. And 80 μL of 15 mg/mL methoxylamine hydrochloride in pyridine was subsequently added. The resultant mixture was vortexed vigorously for 2 min and incubated at 37 °C for 90 min. A total of 80 μL of BSTFA (with 1% TMCS) and 20 μL n -hexane was added into the mixture, which was vortexed vigorously for 2 min and then derivatized at 70 °C for 60 min. The samples were placed at ambient temperature for 30 min before GC–MS analysis. In this study, six parallel samples (12 fish were sacrificed for each sample) were prepared for each treatment/control group. The derivatized samples were analyzed on an Agilent 7890A gas chromatography system coupled to an Agilent 5975C MSD system (Agilent, CA). A HP-5MS fused-silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J & W Scientific, Folsom, CA) was utilized to separate the derivatives. Helium (> 99.999%) was used as the carrier gas at a constant flow rate of 6.0 mL/min through the column. The injector temperature was maintained at 280 °C. Injection volume was 1 μL by splitless mode. The initial oven temperature was 60 °C, ramped to 125 °C at a rate of 8 °C/min, to 190 °C at a rate of 10 °C/min, to 210 °C at a rate of 4 °C/min, to 310 °C at a rate of 20 °C/ min, and finally held at 310 °C for 8.5 min. The temperature of MS quadrupole, and ion source (electron impact) was set to 150, and 230 °C, respectively. The collision energy was 70 eV. Mass data was acquired in a full-scan mode (m/z 50–600), and the solvent delay time was set to 5 min. The acquired MS data from GC–MS were analyzed by ChromaTOF software (v 4.34, LECO, St Joseph, MI). And Metabolites were quantified by the Fiehn database, which is linked to the ChromaTOF software. Briefly, after alignment with Statistic Compare component, the CSV file was obtained with three dimension data sets including sample information, peaks' name, retention time, m/z and peak intensities. The internal standard was used for data quality control (reproducibility). After internal standards and any known pseudo positive peaks, such as peaks caused by noise, column bleed and BSTFA derivatization

2.2. Histopathological analysis After 7 days of exposure with 10 and 30 μg/L Pb, a portion of the middle gut was fixed in 10% formalin at 4 °C for 24 h. Then, the fixed gut tissues were dehydrated in gradient ethanol, hyalinized in xylene, and embedded in paraffin wax. Next, the paraffin blocks were sectioned at 5-μm thickness. The sections were collected on glass slides and stained with hematoxylin and eosin (H&E) or Alcian Blue-Periodic Acid Schiff (AB-PAS). And the pictures of the guts were examined by a microscope (Olympus). 2.3. DNA extraction, PCR amplification, and 16S rRNA gene sequencing The genomic DNA (gDNA) was extracted from each gut using a commercial magnetic bead DNA isolation kit provided by Hangzhou Foreal Nanotechnology (Hangzhou, China). All the extracted gDNA was quantified by ultraviolet spectroscopy and electrophoresis for further analysis. Then, the gDNA samples were amplified by specific primers (Forward primer: 5′-ACTCCTACG GGAGGCAGCAG-3′; Reverse primer: 5′-GGACTA CHVGGGTWTCTAAT-3′) targeting the V3 and V4 regions of the bacterial 16S rRNA gene. Furthermore, the composition of the gut microbiota was detected using dual-indexing amplification and sequencing on the Illumina MiSeq platform followed by QIIME (vision 1.9.0) bioinformatics analysis. In addition, partial of the gDNA in each sample was amplified by real-time qPCR with following protocol: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, 56 °C for 30 s, and 72 °C for 1 min, repeated for 40 cycles, followed by 72 °C for 10 min. 2

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adult zebrafish were shifted by 30 μg/L Pb exposure (Fig. 2C). And the Shannon diversity indexes also suggested that the bacterial diversity of zebrafish gut microbiota was affected by Pb exposure (Fig. 2D). The changes in gut microbiota composition, the at the phylum level, was also detected by RT-qPCR (Fig. 3). The relative abundances of αProteobacteria decreased in the gut significantly when treated with 30 μg/L Pb for 7 days. And the relative abundance of Firmicutes in the 30 μg/L Pb treated group was significant higher than that in the control group (Fig. 3).

procedure, were removed from the data set, and the peaks from the same metabolite were combined. Finally, the differential metabolites were selected on the basis of the combination of a statistically significant threshold of variable influence on projection (VIP) values obtained from the OPLS-DA model and p values from a two-tailed Student's t-test on the normalized peak areas, where metabolites with VIP values larger than 1.0 and p values < 0.05 were included, respectively. 2.6. Data analysis

3.3. Effects of Pb on hepatic metabolism of zebrafish analyzed by GC/MS Statistical significance between the control and Pb treated groups was carried out using one-way ANOVA. A probability (p) value set at 0.05 was deemed to be significant. Data analyses were performed using SPSS (Statistical Package for the Social Sciences) 13.0 software (SPSS Inc., USA).

After exposure to 30 μg/L Pb for 7 days, the changed hepatic metabolites was analyzed by GC/MS based analysis. It was clearly observed that there was clear separation of samples from the two groups in the score plot of the OPLS-DA models, indicating that short Pb exposure induced significant changes in metabolomics in the liver of adult zebrafish (Fig. 4A). All the changed metabolites were selected by comparing the metabolites in the Pb-treated group with the control using the multivariate statistical analysis and the Wilcoxon-MannWhitney test. A total of 41 significantly changed metabolites, 22 ascending and 19 descending, were observed in the Pb-treated group (Table S1, Fig. 4B). Through the analysis of the pathways of the changed metabolic products, we found that the pathways of glucose and lipid metabolism (27 metabolites), amino acid metabolism (5 metabolites) and nucleotide metabolism (3 metabolites) showed a significant change (Fig. 4B). In addition, the main change metabolites involved in several pathways related to glucose and lipid metabolism in Fig. 4C.

3. Result 3.1. Gut histological analysis As shown in Fig. 1A, the H&E staining showed that there was no significant histological effects on the guts of zebrafish exposed to 30 μg/ L of Pb for 7 days (Fig. 1A). The results of AB-PAS staining of the gut demonstrated significant increases in the secretion of mucus (as indicated by arrows) in the guts of adult zebrafish when exposed to 10 and 30 μg/L Pb for 7 days (Fig. 1B, Fig. S1). 3.2. Effects of IMZ on the composition of gut microbiota in adult zebrafish

3.4. Effect of IMZ exposure on the transcription of genes related to glucose and lipid metabolism

At the phylum level, the levels of Proteobacteria decreased and the level of Firmicutes increased in the 30 μg/L pb treated group detected by high-throughput sequencing of 16S rRNA (Fig. 2A). In addition, the levels of Bacteroidetes increased while the level of Fusobacteria decreased after exposure with 30 μg/L pb for 7 days (Fig. 2A). At the genus level, the composition of microbiota in the gut of zebrafish was changed after short time Pb exposure (Fig. 2B). It was observed that about 30 kinds of microorganisms in the gut changed after short time Pb exposure (Fig. 2B). In addition, based on principal analysis, it was observed that the composition of the gut microbiota in

As shown in Fig. 5, the main genes related to glucose and lipid metabolism were affected by short term Pb exposure. The mRNA levels of GK in the liver of both 10 and 30 μg/L Pb treated group were significant lower than that in the control group (Fig. 5A). The mRNA levels of Pk and Pepckc were also had the tendency to decrease (Fig. 5A). In addition, we observed that the mRNA levels of Aco and Acc1 in the liver decreased significantly in the group when exposed to 10 and 30 μg/L Pb

Fig. 1. Representative middle gut sections from adult zebrafish after exposure to 0, 10 and 30 μg/L Pb for 7 days. (A) H&E staining; (B) AB-PAS staining. The arrows indicate the mucus compartment in the gut.

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Fig. 2. Effects of short time Pb exposure on gut microbiota in the gut, as detected by 16 s RNA gene sequencing. Adult male zebrafish was exposed to 30 μg/L Pb for 7 days. (A) Composition profiles of gut microbiota at the phylum level in the control and 30 μg/L Pb treated group; (B) Heat map of specimens showing the relative abundance of the main identified bacteria at the genus level of the control and 30 μg/L Pb treated group; (C) UniFrac principal component analysis (PCoA) estimates of gut microbiota of the control and 30 μg/L Pb treated group; (D) Shannon indexes of gut microbiota of the control and 30 μg/L Pb treated group. Fig. 3. Effects of short time Pb exposure on the structure of gut microbiota at the phylum level in the gut, as detected by quantitative real-time PCR. Adult male zebrafish was exposed to 10 and 30 μg/L Pb for 7 days. Data are expressed as the mean ± SEM of 6 individuals. The asterisk represents a statistically significant difference when compared with the control. *p < 0.05.

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Fig. 4. The OPLS-DA analysis and different metabolites involved in the potential perturbed pathways following exposure to 30 μg/L Pb for 7 days. (A) OPLS-DA analysis of the control and Pb treated samples; (B) The heat map of the main changed metabolites after Pb exposure (the average data originated from the six separate samples); (C) Pb induced the changed metabolites involved in glucose and lipid metabolism. The red arrows indicate significantly increased metabolites in Pb treated group, and the green arrows indicate significantly decreased metabolites in Pb treated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

specifically affected when the mice were exposed to various doses of Cd (5, 20 and 100 mg/L) and Pb (100 and 500 mg/L) chloride salts provided in drinking water for 4, 8 and 12 weeks (Breton et al., 2013). In this study, it was observed that the section of mucin increased after Pb exposure, indicating that the function of goblet cell and the physical barrier of gut was disturbed by Pb. In the gut, the microbiota plays very important roles in animal health including synthesis and assimilation of vitamins and other nutrients, production of short-chain fatty acids that provide energy for colonic epithelium, and adipose absorption and regulation of host glucose and energy metabolism with secondary bile acids (Preidis et al., 2015). Recently, a number of previous studies proved that some different kinds of environmental chemicals including heavy metals could induce gut microbiota dysbiosis in different experimental models (Lu et al., 2014; Zhang et al., 2015; Y.X. Jin et al., 2017; Chi et al., 2017; Y.X. Jin et al., 2018). As for Pb, three of previous studies observed that high doses of Pb exposure could cause gut microbiota dysbiosis in rat and mouse (Wu et al., 2016; Gao et al., 2017; Zhai et al., 2017), indicating that the gut microbiota was also the toxic target of Pb. According to our limited knowledge, whether low concentration of Pb exposure could affect the composition of gut microbiota and metabolism in fish still remained unclear. In this study, we observed that short time exposure with Pb would cause the change of the composition of microbiota in the gut of male adult zebrafish. Although the functions of most identified bacteria were unknown in zebrafish, several bacteria, namely, Bacteroides, Flavobacterium,

for 7 days (Fig. 5B). The mRNA levels of Fas, Apo and Dgat in the liver of 30 μg/L Pb treated group was significant lower than that in the control group (Fig.5B). In addition, we also observed that the mRNA levels of LDLR and HMGCR were also decreased in zebrafish when exposed to 30 μg/L Pb (Fig.5B). 4. Discussion The health concerns regarding to environmental chemicals including Pb are increasing in recent years (Henríquez-Hernández et al., 2017; Kasten-Jolly and Lawrence, 2017; Xia et al., 2018). Recently, a study reported that oral Pb exposure could change the composition of gut microbiota in mice (Gao et al., 2017), however, the effects of Pb exposure on gut microbiota and metabolism still remained unclear. In this study, we firstly observed that short period exposure to 10 and 30 μg/L Pb exposure would not only cause histological damage of gut but also induce gut microbiota dysbiosis and resulted in hepatic metabolism disorder in male adult zebrafish. The structure of gut was the very important barrier to keep exogenous microbe and or its metabolites out of the body (Newberry and Lorenz, 2005; Jin et al., 2016a; C.Y. Jin et al., 2018). Previous studies reported that heavy metals exposure could influence the gut function in animals. For example, the gene expression of representative intestinal markers revealed that the transport-, oxidative- and inflammatory status of the gut epithelium of the duodenum, ileum and colon were 5

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Fig. 5. Effect of short time Pb exposure on the transcription of glycolysis and lipid metabolism related genes in the liver of male adult zebrafish after exposure to 10 and 30 μg/L Pb for 7 days. Values were normalized against β-actin as a housekeeping gene, and represent mean mRNA expression value ± S.E.M (n = 6) relative to those of the controls. The asterisk represents a statistically significant difference when compared with the control. *p < 0.05; and **p < 0.01.

from the gut microbiota can also influence the liver function. As the most important tissue for detoxification, the liver was also easily be influenced by environmental chemicals (Liu et al., 2015). Recently, our study suggested that exposure with the fungicide of imazalil induced the metabolism disorder in the liver of adult male zebrafish (C.Y. Jin et al., 2017). Here, based on the GS/MS technology, a total of 41 metabolites in the liver were significant affected by short term Pb exposure, and these metabolites were tightly related to the pathways mainly including glucose and lipid metabolism, amino acid metabolism, nucleotide metabolism in the liver of adult zebrafish. Among these metabolites, the contents of some amino acids changed significantly after short time Pb exposure. Amino acids play very important roles between the gut microbiota and host metabolism (Sridharan et al., 2014). Gut bacteria can alter the bioavailability of amino acids by utilization of several amino acids originating from both alimentary and endogenous proteins (Neis et al., 2015). Moreover, the glucose and lipid

Ralstonia, Alloprevotella, Roseburia, Alloprevotella, Ruminococcus were closely related to metabolism, disease and inflammation (Pitcher and Cummings, 1996; Roediger et al., 1997; Lorenzen and Olesen, 1997; Xu et al., 2003; Ze et al., 2012; Keshavarzian et al., 2015). For example, the bacterial of Alloprevotella was associated with decreased lifetime cardiovascular disease in human (Kelly et al., 2016). Gauffin Cano et al. (2012) reported that oral administration of bacteroides uniformis CECT 7771 reduced body weight gain, liver steatosis and liver cholesterol and triglyceride concentrations and increased small adipocyte numbers in HFD-fed mice. In addition, it could also increase TNFα production and improve immune defense in mice. Short time Pb exposure could change the composition of microbiota indicated that gut microbiota played very important role in Pb induced toxicity in zebrafish. Generally, the gut microbiota dysbiosis is tightly related to metabolism disorder (Y.X. Jin et al., 2017; Wang et al., 2017; Heiss and Olofsson, 2017; Wu et al., 2018; Lu et al., 2018). The metabolites of

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is very important for energy metabolism. In this study, a total of 27 metabolites was influenced by short time Pb exposure which were tightly related to glucose and lipid metabolism. For example, in the changed metabolites, gluconic acid and pyruvic acids are important for glycolysis and synthesis of acety-CoA, which is important for TCA cycle and the synthesis of free fatty acid (Daniel et al., 2010; Li et al., 2017). In addition, the main genes related to glucose and lipid metabolism in the liver were significantly affected by Pb treatment (Fig. 5), indicated that the hepatic energy metabolism was influenced at transcriptional level. Although we did know whether the short time Pb exposure could induce hepatotoxicity in adult zebrafish, according to previous studies, Pb could induce hepatotoxicity in mouse or rat (Mabrouk et al., 2016; Moneim, 2016). These results indicated that the liver is also one of the main target tissues for Pb and could result in hepatic metabolism disorder directly in zebrafish. On the other hand, Pb induced gut microbiota dysbiosis may also an indirect reason for metabolism disorder in zebrafish. In conclusion, the results obtained from this study indicated that short term Pb exposure not only induced gut microbiota dysbiosis but also disturbed hepatic metabolism in adult zebrafish. The results also indicated that the gut microbiota was also the toxic target for environmental heavy metals such as Pb. We believed that the results obtained here provide a new insights into the toxicity of Pb and potential health risks in aquatic organisms. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpc.2018.03.007.

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