Structural and functional alterations of gut microbiome in mice induced by chronic cadmium exposure

Chemosphere 246 (2020) 125747

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Structural and functional alterations of gut microbiome in mice induced by chronic cadmium exposure Xiwei He a, Zhaodong Qi a, Hui Hou a, Ling Qian b, Jie Gao a, Xu-Xiang Zhang a, * a b

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, 210023, China Sino-Japan Friendship Center for Environmental Protection, Beijing, 100029, China

h i g h l i g h t s
Chronic
Chronic
Chronic
Chronic

Cd Cd Cd Cd

exposure exposure exposure exposure

induced gut barrier damage in mice. decreased gut microbial richness. inhibited SCFA-producing bacteria in mice gut. perturbed metabolic functions of gut microbiome.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 September 2019 Received in revised form 22 December 2019 Accepted 23 December 2019 Available online 24 December 2019

Mammalian gut microbiome is readily affected by acute or subchronic cadmium (Cd) intoxication, but it susceptibility following chronic Cd exposure at environmentally-relevant levels remains unknown. This study comprehensively assessed the effects of Cd exposure at doses of 10 and 50 ppm in drinking water for 20 weeks on gut microbiome in mice. Results showed that the Cd exposure induced alterations in gut morphology with potentially increased gut permeability and inflammation. These changes were accompanied by marked perturbation of gut microbiota characterized by significantly decreased gut microbial richness and lowered abundance of short chain fatty acid (SCFA)-producing bacteria, resulting in reduced SCFAs production in the gut. Moreover, the Cd exposure caused substantial metabolic functional changes of the gut microbiome, with significant inhibitions on gene pathways associated with metabolism of amino acid, carbohydrate, and energy, as well as promotions on metabolic pathways such as glutathione metabolism and aminobenzoate degradation. Our findings provide new insights into the hazards assessment of environmental Cd exposure towards gut microbiome. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: A. Gies Keywords: Cadmium Gut microbiome Gut permeability Short chain fatty acid Metabolic function

1. Introduction The gastrointestinal tract of vertebrates harbors an extremely complex and dense microbiota that interact closely with the host in nutrient metabolism, xenobiotic and drug metabolism, maintenance of structural integrity of the gut mucosal barrier, immunomodulation, and protection against pathogens (Jandhyala et al., 2015; Thursby and Juge, 2017; Rowland et al., 2018). Disturbance of the gut microbiota is associated with increased risk of developing metabolic disorders, inflammatory bowel disease, and allergic diseases (Clemente et al., 2012). Intrinsic and external factors affecting gut microbiota include genetic background, age, and diet

* Corresponding author. 163 Xianlin Road, Nanjing, 210023, China. E-mail address: [email protected] (X.-X. Zhang). https://doi.org/10.1016/j.chemosphere.2019.125747 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

(Yatsunenko et al., 2012; David et al., 2014). Recently, exposure to environmental pollutants such as heavy metals has also been postulated to shape gut microbiota (Jin et al., 2017; Tinkov et al., 2018). Cadmium (Cd) is one of the most prevalent toxic metal pollutants widely distributed in agricultural soil and drinking source water (Smolders et al., 2003; Bertin and Averbeck, 2006). Chronic Cd exposure has become increasingly prevalent with the access to contaminated food and water. This situation is particularly worrying in China, as over 16% of the farmland in China has been seriously contaminated by Cd, which is endangering the health of more than 200 million people (Ran and Li, 2011). Gut microbiota is susceptible to Cd, and Cd exposure via digestive tract can reduce the abundance of certain gut microbes such as Lachnospiraceae and Streptococcaceae while promote the colonization of Coriobacteriaceae and Lactobacillaceae (Zhang et al., 2015; Li et al., 2019). But

2

X. He et al. / Chemosphere 246 (2020) 125747

the impact of Cd on gut microbiota seems time-dependent, since Liu et al. (2014) have indicated that the number of Bifidobacteria decreased starting from the first week of Cd exposure, whereas Lactobacilli population showed the significant dose-dependent reduction after 3 weeks of exposure. The majority of existing studies investigating gut microbiota have principally focused on short-term or subchronic (no more than 8 weeks) Cd exposures (Liu et al., 2014; Go et al., 2015; Kim et al., 2015; Zhang et al., 2015; Zhai et al., 2016; Li et al., 2019). This has left much uncertainty about the potential effects of chronic or long-term Cd exposure, which often occurs in actual environmental exposure scenarios, on gut microbiome. In this study, we conducted a 20-week exposure mouse model to comprehensively assess the effects of chronic Cd exposure at environmentally-relevant doses on gut microbiome in mice. The gut tissue damages and gut microbiota disturbance in terms of alpha-, beta-diversity, and taxonomic composition were examined by using histological analysis and next-generation sequencing. Functional alterations of gut microbiota were also investigated based on the prediction of bacterial metagenome content. The results of this study may extend our knowledge regarding the roles of gut microbiome in the induction of health hazards by environmental Cd contamination.

2. Materials and methods 2.1. Animal exposure Toxicity tests were conducted on male C57BL/6 mice (5 weeks old) obtained from the Experimental Animal Center of the Academy of Military Medical Science of China. The mice were housed in stainless steel cages. After acclimated for two weeks under the ambient conditions of temperature at 25 ± 2o C, relative humidity at 50 ± 5%, and light/dark cycle at 12/12 h, 30 mice were randomly assigned to one control and two treatment groups (10 mice per group, 5 mice per cage) and treated for 20 weeks. For the low-dose group (L), a dose of 10 mg/L CdCl2 (in pure water) was used to mimic environmental Cd exposure, as Cd concentrations at ppm levels have been reported in drinking water (2.5 mg/L) (Wang et al., 2015) and rice grains (7.0 mg/kg) (Chen et al., 2013). This dose can also induce hepatic Cd deposition in mice close to the real hepatic Cd level in middle-aged human population after 20 weeks of exposure (Go et al., 2015). Accordingly, a higher dose of 50 mg/L CdCl2 (in pure water) was included in this study to mimic the Cd exposure in heavily polluted areas. Mice in the control group (Ctrl) were given pure water. During the exposure time, mice were fed with a normal diet containing less than 0.2 mg/kg Cd according to the Hygienic Standard for Formula Feeds (GB 14924.2-2001, 2001), and the food and water were provided ad libitum. After 20 weeks of exposure, mice were sacrificed, and intestine and feces samples were then collected and immediately put into ice-cold tubes for further use. The animal experiment was approved by the Institutional Animal Care and Use Committee of Nanjing University.

2.2. Histopathological analyses Four mice were randomly chosen from each group for histopathological analysis of the intestine. Part of the small bowel was fixed in 10% formalin solution for 4 h at room temperature and then overnight at 4  C with fresh fixative, embedded in paraffin and sectioned into 5 mm sections. The sections were stained with hematoxylin and eosin (H&E) and then subjected to microscope analysis.

2.3. Gene expression analyses Quantitative reverse-transcription PCRs (qRT-PCRs) were conducted to determine mRNA levels of the genes involved in inflammation (TNF-a), intestinal permeability (occludin and claudin-1), and SCFA receptors (GPR43 and GPR109A) (Details of the genes are shown in Table S1) in the small bowel samples of mice (eight mice in each group, randomly chosen). Total RNA from each sample was isolated using the TaKaRa MiniBEST Universal RNA Extraction Kit (Takara Bio Inc., Otsu, Shiga, Japan), and the first strand cDNA was synthesized using the PrimeScrip™ RT Reagent Kit (TaKaRa) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using the SYBR Premix EX Taq Super Mix (TaKaRa) in an ABI 7500 (Applied Biosystems, Foster City, CA, US) with b-actin gene as the housekeeping gene. The relative quantification of target mRNA and mtDNA was conducted using the 2-△△Ct method (Livak and Schmittgen, 2001). 2.4. Determination of short chain fatty acids (SCFAs) in mice feces Fecal pellets from four mice randomly chosen from each group were weighed and homogenized in 500 mL deionized water. After stored at 4  C for 12 h, pH of the suspension was adjusted to 2e3 by adding 5 M HCl at room temperature for 10 min with intermittent shaking. The supernatant was harvested after centrifuging the suspension at 12,000 rpm for 10 min. An internal standard, 2ethylbutyric acid, was added into the supernatant at a final concentration of 1 mM. Agilent 7890 (Agilent, USA) equipped with a flame ionization detector (FID) and a fused-silica capillary column (30 m  520mm  0.5 mm) with a free fatty acid phase (DB-FFAP 125e3237, J&W Scientific, Agilent Technologies Inc.) was used for the chromatographic analysis of SCFAs. Helium was supplied as the carrier gas at a flow rate of 14.4 mL/min. The initial oven temper ature was 100  C, maintained for 0.5 min, raised to 180  C at 8 C/   min and held for 60 s, then increased to 200 C at 20 C/min and held for 5 min. The temperature of the FID and the injection port was 240 and 200  C, respectively. The flow rates of hydrogen, air, and nitrogen were 30, 300 and 20 Ml/min, respectively. The injected sample volume was 1 mL, and the run time for each analysis was 17.5 min (Guohua et al., 2010). 2.5. 16 S rRNA gene sequencing of intestinal microbiota and data analyses Total DNA from fecal samples of mice (eight mice per group, randomly chosen) were extracted with FastDNA Soil Kit (MP Biomedicals) according to the manufacturer’s instructions. The concentration and quality of acquired DNA were measured by microspectrophotometry (Nano-Drop, 2000; US) and stored at 80  C for further analysis. The 16 S rRNA gene of bacteria was amplified using universal primers 16s-F (50 -AGAGTTTGATYMTGGCTCAG-30 ) and 16s-R (50 -TGCTGCCTCCCGTAGGAGT-30 ) targeting the V1eV2 region. Individual samples were barcoded and then pooled to construct the sequencing library before sequenced using an Illumina Miseq (Illumina, San Diego, CA). The raw sequencing data were processed with the Mothur software (www. mothur.org/) to remove low-quality and chimeric reads. After quality filtration, all datasets were rarefied to 16,557 sequences to achieve same sequencing depth. The Quantitative Insights into Microbial Ecology (QIIME, version 1.9.1) release was used to cluster high quality reads into operational taxonomic units (OTUs) at 97% identity level. OTU search was performed using the parallel_pick_open_reference_otus workflow script and the Greengenes 13_8 Database. The OTU rarefaction curve and rank abundance

X. He et al. / Chemosphere 246 (2020) 125747

curves were plotted in QIIME. The a-diversity indices (Chao, ACE, Simpson, and Shannon) and Good’s coverage were calculated by QIIME. Principal coordinate analysis (PCoA) was conducted and unweighted pair group method with arithmetic mean (UPGMA) clustering was performed to determine the b-diversity of the gut microbiota based on the OTUs obtained from each sample with PAST (version 3.16) (Hammer, 2001). Representative sequence of each OUT was assigned to taxonomy with Ribosomal Database Project (RDP) Classifier (http://rdp.cme.msu.edu/) using a confidence threshold of 80%. The sequencing data have been deposited in the NCBI sequencing reads archive database under the accession number SUB6236963. Bacterial metagenome content was predicted from the OTU data, and functional inferences were made from the Kyoto Encyclopedia of Gene and Genomes (KEGG) catalog using the Tax4Fun program (Aßhauer et al., 2015). UPGMA clustering of samples using the KEGG metabolic orthologies was performed with PAST. The zscores of KEGG orthologies were calculated with the following formula: z-score ¼ (treatment abundance e control mean)/standard deviation of control (Jordan et al., 2011). A heat map was generated to visualize the functional difference among the three groups using R software (version 3.6.1). 2.6. Statistical analyses All experimental data obtained in this study are expressed as the mean ± SEM, and Kruskal-Wallis test followed by multiple comparison was performed for comparisons between the treatment

3

groups and the control group using SPSS 16.0 software (SPSS Inc., Chicago, IL). Statistical difference of bacterial community structure was evaluated using One-way PERMANOVA test by PAST. The reported p-value is the Bonferroni-adjusted p-value corrected for multiple comparisons. 3. Results 3.1. Gut tissue damages induced by chronic Cd exposure No mortality or behavioral changes was observed for each mouse during the whole Cd exposure period (20 weeks). The control and Cd-treated mice had no significant difference in food intake, but the body weights of the Cd-treated showed a dosedependent decrease when compared to the control mice (Fig. S1). Under microscopy, the small bowel (ileum section) in the control group showed normal intestinal morphology with intact intestinal wall and regular-shaped villus. However, damaged intestinal morphology characterized by shedding intestinal villus was observed in the Cd (L) group (Fig. 1A). In comparison, the intestinal damage was more severe in the Cd (H) group, as the intestinal villus were markedly frayed and the intestinal walls were thinner (Fig. 1A). Additionally, compared with the control, mRNA levels of intestinal tight junction gene occludin and claudin-1 in the small bowel were decreased in both the Cd-treated groups (Fig. 1B), which was accompanied by dose-dependent decrease on mRNAs of G-protein-coupled receptor gene GPR43 and GPR109A (Fig. 1B), whereas intestinal mRNA level of cytokines TNF-a was significantly

Fig. 1. Gut damages induced by chronic Cd exposure. (A) H&E staining of the small bowel tissue sections. The white arrow indicates frayed intestinal villus and the black arrow indicates thinned intestinal wall, and the corresponding tissue damages are scored with the length of intestinal villus and the thickness of intestinal wall, respectively. (B) Gut mRNA levels of tight junction genes (occludin and claudin-1), G-protein-coupled receptor genes (GPR43 and GPR109A), and inflammatory cytokines (TNF-a) determined by quantitative real time RT-PCR. *p < 0.05, yp < 0.1 compared to the control.

4

X. He et al. / Chemosphere 246 (2020) 125747

increased in the Cd (H) group (Fig. 1B). 3.2. Alterations of gut microbial diversity induced by chronic Cd exposure High-throughput sequencing of 16 S rRNA gene amplicons revealed obvious changes of gut microbiota in mice induced by the chronic Cd exposure. A total of 397,368 sequences were obtained from all samples. Rarefaction curves of the sequences showed that high sampling coverage was gained in all samples (Fig. S2A). The Good’s coverage was nearly 95% for all sequences in the three groups (Fig. S2B). Both community richness indices Chao and ACE of the gut microbiota in mice showed a dose-dependent decrease in response of the chronic Cd exposure, and were markedly lower in Cd (H) group when compared to the control (Fig. 2A and B). In contrast, no significant difference was observed for the community diversity indices Simpson and Shannon between the control and Cd-treated groups (Fig. 2C and D). Rank-Abundance curves from the OTUs showed a long tail (Fig. S3), indicating that the majority of OTUs were present at low abundance in the gut microbiota of all samples. One-way PERMANOVA analysis of the OTUs with abundance greater than 1% of the total gut microbes in mice revealed that the chronic Cd exposure significantly altered the gut microbiome patterns (Control vs. Cd (L), p ¼ 0.0009; Control vs. Cd (H), p ¼ 0.0015), as revealed by the PCoA plot showing that the control and Cdtreated mice were well separated (Fig. 3A). Similarly, hierarchical clustering tree showed that most of the Cd (L) and Cd (H) mice were clustered into the same group, whereas most of the control mice were clustered into a different group (Fig. 3B). 3.3. Alterations of gut microbial abundance induced by chronic Cd exposure A total of 9 phyla were identified from the taxonomic assignments of gut microbiota (Fig. S4), among which Bacteroidetes and Firmicutes were the top two most abundant phyla in all samples,

followed by Proteobacteria. Other phyla were also present but with much lower abundance (Fig. S4). Kruskal-Wallis test showed no significant difference in the abundance of the detectable phyla between the control and Cd-treated groups except for Actinobacteria, which was enriched after the Cd exposure but still had relatively low abundance (mean 1.026%) (Fig. S5). The Cd exposure induced no evident changes in Bacteroidetes/Firmicutes ratio (Fig. S6). However, compared to the control, the Cd-treated groups had significantly (p < 0.05) lower abundance of Clostridia (class) and Clostridiales (order), while higher abundance of Erysipelotrichia (class) and Erysipelotrichales (order) (Fig. 4). At family level, Bacteroidales_S24-7_group, Erysipelotrichaceae, and Bifidobacteriaceae were enriched in the Cd-treated groups, while Bacteroidaceae and unclassified Clostridiales were more prevalent in the control group (Fig. 4). Notably, relative abundance of short chain fatty acid (SCFA)-producing bacteria such as Lachnospiraceae and Ruminococcaceae were dose-dependently decreased in the Cdtreated groups compared to the control (Fig. 4). The chronic Cd exposure significantly (p < 0.05) altered the relative abundance of 14 genera (Fig. 4). Among the predominant genera (>2%), unclassified Bacteroidales_S24-7_group, Lactobacillus, and Turicibacter were enriched in the Cd-treated mice, while the abundance of Bacteroides, unclassified Lachnospiraceae, and Ruminococcaceae_UCG-014 showed a dose-dependently decreasing trend (Fig. 4). Interestingly, the predominant genera inhibited by Cd were all SCFA-producing bacteria. This phenomenon was further accompanied by the growth inhibition of other SCFA-producing genera with lower abundance, including Unclassified Coriobacteriaceae and Ruminiclostridium (Fig. 4). In accordance with the observations for the bacteria, the concentrations of major fecal SCFAs, i.e. acetate, propionate, and butyrate, were reduced in a dosedependent manner after the Cd exposure (Fig. 5). 3.4. Metabolic functional changes of gut microbiota induced by chronic Cd exposure Tax4Fun computation revealed the alterations in the functional profiles of gut microbiota in mice by the Cd exposure. Comparison of the predicted KEGG orthologies demonstrated clear clustering of the control and Cd-treated mice (Fig. S7), which is similar to the results for OTUs. Kruskal-Wallis test identified a total of 54 KEGG metabolic functions that were differentially represented among the three groups (p < 0.05, Fig. 6). The altered metabolic pathways included amino acid metabolism, biosynthesis of other secondary metabolites, carbohydrate metabolism, energy metabolism, glycan biosynthesis and metabolism, lipid metabolism, metabolism of cofactors and vitamin, metabolism of other amino acids, metabolism of terpenoids and polyketides, and xenobiotics biodegradation and metabolism (Fig. 6). Notably, of the differentially represented metabolic functions, more than 2/3 were significantly down-regulated in either Cd (L) or Cd (H) group when compared to the control, including basic metabolic functions such as valine, leucine and isoleucine biosynthesis and degradation, and those related to core metabolic pathways, such as citrate cycle and oxidative phosphorylation. Conversely, metabolic functions associated with glutathione metabolism and aminobenzoate degradation were up-regulated in both the Cd-treated groups when compared to the control (Fig. 6). 4. Discussion

Fig. 2. Comparisons of a-diversity of the gut microbial community between the control and Cd-treated groups. (A) Chao index for richness; (B) Ace index for richness; (C) Simpson index for diversity; (D) Shannon index for diversity. *p < 0.05, yp < 0.1 compared to the control.

Oral ingestion of Cd-containing food or water is the primary route of environmental Cd exposure. The intestine is the first barrier against Cd after oral intake and is directly exposed to Cd. The small intestine is the primary organ for nutrient metabolism and

X. He et al. / Chemosphere 246 (2020) 125747

5

Fig. 3. The b-diversity of the gut microbial community. (A) The gut microbiome patterns of the control and Cd-treated mice differentiated by principal coordinate analysis. (B) Hierarchical clustering analysis of gut microbiome patterns by UPGMA. For each group, mice labeled 1e4 or 5e8 were housed in the same cage during the experiment.

energy harvest, and is susceptible to external stimulus. The present study showed malformation in the morphology of small intestine from Cd-treated mice, as evidenced by the observation of shedding and frayed intestinal villus as well as thinned intestinal walls in mice from Cd (L) and Cd (H) groups, indicating that the chronic Cd exposure could damage the intestinal barrier in mice. Cytotoxicity of Cd towards intestinal cells may at least partially account for the observed gut tissue damages induced by the chronic Cd exposure (Rusanov et al., 2015; Luo et al., 2019). The damages of intestinal barrier may impair the normal digestion and absorption functions of the gut and thereby cause the drop of body weight. One the other hand, disrupted intestinal barrier may lead to the alteration of gut permeability, as the Cd exposure was found to significantly reduce mRNA expression of the tight junction gene occludin and claudin-1 in mice gut, which were associated with increased gut permeability (Chen et al., 2017; Bashir et al., 2018). A previous study showed that 8 weeks of Cd exposure in drinking water at 100 ppm also reduced mRNA expression of tight junction genes in mice gut (Zhai et al., 2016). Here, we investigated the effects of a much lower dose (10 ppm) of Cd with prolonged exposure time, revealed the potential health risk in actual environmental exposures scenarios. Disrupted intestinal barrier and increased gut permeability facilitates the translocation of pathogenic microorganism and toxic

luminal substances from the gut to the circulation, which have been associated with the development of non-alcoholic fatty liver disease (NAFLD) (Bashiardes et al., 2016). This is in accordance with previous studies showing that chronic Cd exposure could induce NAFLD in mice (Go et al., 2015; He et al., 2019). Besides the intestinal histological changes induced, intestinal cytokines TNF-a was significantly increased after the chronic Cd exposure, suggesting an inflammatory response in mice gut to the Cd exposure. This is in agreement with previous studies showing intestinal inflammatory and immune response in mice with subchronic exposure at similar Cd doses (Liu et al., 2014; Ninkov et al., 2015). The shield-like function of small intestinal barrier is closely related to and can be modified by the gut microbiota (Jakobsson et al., 2015). Intestinal barrier dysfunction characterized by increased gut permeability has been associated with concomitant alterations in the gut microbiota (Camilleri et al., 2012). It has been reported that disruption of small intestinal barrier and activation of intestinal inflammatory response are often accompanied with gut microbial dysbiosis (Zheng et al., 2018; Lee et al., 2019). The present study showed that the Cd exposure decreased Chao and Ace indices of gut microbiota, indicating a significant reduction of microbial community richness. Additionally, the b-diversity analysis showed clear separation between the control and Cd-treated mice,

6

X. He et al. / Chemosphere 246 (2020) 125747

Fig. 4. Relative abundance of significantly changed gut bacteria taxa classified at class, order, family, and genus levels. *p < 0.05, **p < 0.01 compared to the control.

Fig. 5. Fecal concentrations of short chain fatty acid acetate, propionate, and butyrate from mice in the control and Cd-treated groups. *p < 0.05 compared to the control.

suggesting a marked alteration of the microbial community structure induced by Cd exposure. The changes in microbial community richness as well as community structure indicate a condition of gut microbial dysbiosis, which may contribute to the observed intestinal barrier damages in Cd-treated mice. Consistent with previous studies (Zhai et al., 2017; Zhang et al., 2015; Li et al., 2019), this study indicated that Bacteroidetes and Firmicutes were the dominant phyla in mice gut with or without Cd exposure. However, no significant difference in the relative abundance of these two phyla was observed between the control and Cd-treated mice. This differs from the findings obtained from previously reported subchronic Cd exposures at similar doses which showed significantly decreased Firmicutes to Bacteroidetes ratio (Liu et al., 2014; Kim et al., 2015). At lower taxonomy level, we found that the gut bacteria families Bifidobacteriaceae and Bacteroidales__S24-7_group were significantly increased after exposed to 10 mg/L Cd for 20 weeks; however, no significant changes were found for the bacteria after 8-week Cd exposure at the same dose (Zhang et al., 2015). Additionally, the present study

revealed that the 20-week exposure to 50 mg/L Cd induced evident alterations of the genera Bacteroides, Allobaculum, and Turicibacter, but Li et al. (2019) revealed no significant changes in mice gut when exposed to Cd at the same dose for 2 weeks. The divergences of the results obtained from this study and previous reports suggest that exposure time is an important factor influencing the toxicity of Cd on gut microbiota. Although the exact mechanism remains unclear, we speculate that the long-term Cd exposure may not share the same toxicological mechanisms with the short-term exposure towards prokaryotic cells, as this has been observed for eukaryocytes (Liu et al., 2009). The discrepancy of gut microbial variations between short-term and long-term Cd exposure calls for more longterm exposure investigations given the scarce studies of relevance, so as to gain a better understanding of Cd toxicity to gut microbiome. It is worth noting that, despite the discrepancy mentioned above, the present study and previous reports consistently indicated that the SCFA-producing bacteria, especially Lachnospiraceae and Ruminococcaceae, had lower abundance in response to chronic or sunchronic Cd exposure (Zhang et al., 2015; Li et al., 2019). The inhibition of these bacteria can lead to decreased production of SCFAs in gut, which is confirmed by the observation of lowered fecal SCFAs concentrations in the Cd-treated mice. This agrees with Liu et al. (2014) indicating that the reduction of SCFAs concentrations in fecal samples after challenging mice with 20 and 100 mg/kg Cd for 3 weeks. SCFAs are known for their anti-inflammatory effects in the gut mediated through G-protein-coupled receptors including GPR43 and GPR109a (Tan et al., 2014). Here, we found that mRNA expressions of GPR43 and GPR109a were markedly down-regulated in mice gut after the chronic Cd exposure, indicating that the SCFAassociated pathways may be involved in the Cd-induced gut inflammation. Additionally, SCFAs are reported to participate in systematic energy metabolism (Canfora et al., 2015), so the decreased gut-to-body supply of SCFAs may potentially contribute

X. He et al. / Chemosphere 246 (2020) 125747

7

Fig. 6. Heat map for all significantly differentially represented (all p < 0.05) metabolic functional pathways in the Cd-treated groups calculated by z-scores. Gene pathways were grouped by major functional categories. þ indicates that the pathway is up-regulated in at least one Cd-treated group when compared to the control; - indicates that the pathway is down-regulated in at least one Cd-treated group when compared to the control.

to the progression of energy metabolism disorder induced by Cd (Go et al., 2015; Zhang et al., 2015). In this study, we further explored the functional alterations of gut microbiome induced by the chronic Cd exposure, because many of the functions of a normal gut microbiome can be carried out by a number of microbial groups (Huttenhower et al., 2012), and simply knowing how treatments affect taxonomic abundance cannot provide us with a complete understanding of how they impact the gut microbiome. Here we found that a large number of gene pathways associated with a variety of substances and energy metabolism were significantly differentially represented in the Cdtreated mice compared to the control, suggesting that the chronic Cd exposure markedly influenced the metabolic functions of the gut microbiome. Interestingly, we found that most of the altered pathways associated with amino acid, carbohydrate, and energy metabolism were significantly down-regulated after Cd exposure. Similar results were observed in the patients with type-2 diabetes mellitus (T2DM) (Sanchez-Alcoholado et al., 2017). Cd exposure has been implicated in the development of T2DM (Tinkov et al., 2017; Liu et al., 2018), indicating that a link between the functional changes of gut microbiome and the Cd-induced T2DM. Notably, metabolic functions associated with glutathione metabolism were up-regulated in the Cd-treated mice when compared to the control. Glutathione is an important agent of the endogenous antioxidant defense system, and loss of the oxidant/antioxidant balance has been implicated in Cd toxicity (Cuypers et al., 2010). Aminobenzoate degradation was also found to be up-regulated in the Cdtreated mice. Although its exact mechanism remains unclear, this pathway is tied to cross-regulation between aerobic and anaerobic pathways of catabolism in bacteria (Valderrama et al., 2012). Together, these findings demonstrated that the chronic Cd

exposure altered the gut microbiome both in structure and function.

5. Conclusions This study demonstrated that the chronic Cd exposure at environmentally-relevant levels could induce alterations in gut morphology with potentially increased gut permeability and inflammation. These changes are accompanied with marked perturbation of gut microbiota characterized by significantly decreased gut microbial richness and altered microbial abundance at taxonomical levels from phylum to genus. Specifically, gut SCFAproducing bacteria underwent significant inhibition following the chronic Cd exposure, resulting in the reduced SCFAs production in the gut. The gut microbiota disturbance was associated with substantial metabolic functional changes, characterized by significant inhibitions on gene pathways associated with metabolism of amino acid, carbohydrate, and energy, as well as promotions on metabolic pathways such as glutathione metabolism and aminobenzoate degradation. Taken together, these data indicate that chronic Cd exposure is structurally and functionally detrimental to gut barrier and gut microbiome. The perturbations of the gut microbiome may serve as a new pathway, via which environmental Cd exposure leads to or exacerbates human disease.

Financial disclosures The authors declare they have no actual or potential competing financial interests.

8

X. He et al. / Chemosphere 246 (2020) 125747

CRediT authorship contribution statement Xiwei He: Methodology, Data curation, Writing - original draft. Zhaodong Qi: Formal analysis, Validation. Hui Hou: Methodology. Ling Qian: Methodology, Data curation. Jie Gao: Software. XuXiang Zhang: Conceptualization, Funding acquisition, Project administration, Writing - review & editing. Acknowledgements This study was financially supported by the National Key Research and Development Program of China (2018YFF0214105), the Key R&D Program of Jiangsu Province, China (BE2018632) and the Fundamental Research Funds for the Central Universities, China (14380116). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125747. References Aßhauer, K.P., Wemheuer, B., Daniel, R., Meinicke, P., 2015. Tax4Fun: predicting functional profiles from metagenomic 16S rRNA data. Bioinformatics 31, 2882e2884. Bashiardes, S., Shapiro, H., Rozin, S., Shibolet, O., Elinav, E., 2016. Non-alcoholic fatty liver and the gut microbiota. Mol. Metab. 5, 782e794. Bashir, M., Meddings, J., Alshaikh, A., Jung, D., Le, K., Amin, R., Ratakonda, S., Sharma, S., Granja, I., Satti, M., Asplin, J., Hassan, H., 2018. Enhanced gastrointestinal passive paracellular permeability contributes to the obesity-associated hyperoxaluria. Am. J. Physiol. Gastrointest. Liver Physiol. 316, G1eG14. Bertin, G., Averbeck, D., 2006. Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review). Biochimie 88, 1549e1559. Camilleri, M., Lasch, K., Zhou, W., 2012. The confluence of increased permeability, inflammation, and pain in irritable bowel syndrome. Am. J. Physiol. Gastrointest. Liver Physiol. 303, G775eG785. Canfora, E.E., Jocken, J.W., Blaak, E.E., 2015. Short-chain fatty acids in control of body weight and insulin sensitivity. Nat. Rev. Endocrinol. 11, 577e591. Chen, A.K., Gong, Y.L., Zhou, X.J., Liu, X.H., 2013. Accumulation of Cd, as and their interaction with mineral elements in different rice (Oryza sativa L.) cultivars grown in Gejiu mine, Yunnan Province. Ecol. Sci. 32, 769e774. Chen, W.Y., Wang, M., Zhang, J., Barve, S.S., McClain, C.J., Joshi-Barve, S., 2017. Acrolein disrupts tight junction proteins and causes ER stress-mediated epithelial cell death leading to intestinal barrier dysfunction and permeability. Am. J. Pathol. 187, 2686e2697. Clemente, J.C., Ursell, L.K., Parfrey, L.W., Knight, R., 2012. The impact of the gut microbiota on human health: an integrative view. Cell 148, 1258e1270. Cuypers, A., Plusquin, M., Remans, T., Jozefczak, M., Keunen, E., Gielen, H., Opdenakker, K., Nair, A.R., Munters, E., Artois, T.J., 2010. Cadmium stress: an oxidative challenge. Biometals 23, 927e940. David, L.A., Maurice, C.F., Carmody, R.N., Gootenberg, D.B., Button, J.E., Wolfe, B.E., Ling, A.V., Devlin, A.S., Varma, Y., Fischbach, M.A., Biddinger, S.B., Dutton, R.J., Turnbaugh, P.J., 2014. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559e563. GB 14924.2-2001, 2001. Laboratory Animals-Hygienic Standard for Formula Feeds. General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. Go, Y.-M., Sutliff, R.L., Chandler, J.D., Khalidur, R., Kang, B.-Y., Anania, F.A., Orr, M., Hao, L., Fowler, B.A., Jones, D.P., 2015. Low-dose cadmium causes metabolic and genetic dysregulation associated with fatty liver disease in mice. Toxicol. Sci. 147, 524e534. Guohua, Z., Margareta, N., Jan Ake, J.N., 2010. Rapid determination of short-chain fatty acids in colonic contents and faces of humans and rats by acidified water-extraction and direct-injection gas chromatography. Biomed. Chromatogr. 20, 674e682. Hammer, O., 2001. PAST: paleontological statistics software package for education and data analysis. Palaeontol. Electron. 4, 1e9. He, X., Gao, J., Hou, H., Qi, Z., Chen, H., Zhang, X.-X., 2019. Inhibition of mitochondrial fatty acid oxidation contributes to development of nonalcoholic fatty liver disease induced by environmental cadmium exposure. Environ. Sci. Technol. https://doi.org/10.1021/acs.est.9b05131. Huttenhower, C., Gevers, D., Knight, R., Abubucker, S., Badger, J.H., Chinwalla, A.T., Creasy, H.H., Earl, A.M., FitzGerald, M.G., Fulton, R.S., Giglio, M.G., HallsworthPepin, K., Lobos, E.A., Madupu, R., Magrini, V., Martin, J.C., Mitreva, M., Muzny, D.M., Sodergren, E.J., Versalovic, J., Wollam, A.M., Worley, K.C., Wortman, J.R., Young, S.K., Zeng, Q., Aagaard, K.M., Abolude, O.O., Allen-

Vercoe, E., Alm, E.J., Alvarado, L., Andersen, G.L., Anderson, S., Appelbaum, E., Arachchi, H.M., Armitage, G., Arze, C.A., Ayvaz, T., Baker, C.C., Begg, L., Belachew, T., Bhonagiri, V., Bihan, M., Blaser, M.J., Bloom, T., Bonazzi, V., Brooks, J.P., Buck, G.A., Buhay, C.J., Busam, D.A., Campbell, J.L., Canon, S.R., Cantarel, B.L., Chain, P.S.G., Chen, I.M.A., Chen, L., Chhibba, S., Chu, K., Ciulla, D.M., Clemente, J.C., Clifton, S.W., Conlan, S., Crabtree, J., Cutting, M.A., Davidovics, N.J., Davis, C.C., DeSantis, T.Z., Deal, C., Delehaunty, K.D., Dewhirst, F.E., Deych, E., Ding, Y., Dooling, D.J., Dugan, S.P., Dunne, W.M., Durkin, A.S., Edgar, R.C., Erlich, R.L., Farmer, C.N., Farrell, R.M., Faust, K., Feldgarden, M., Felix, V.M., Fisher, S., Fodor, A.A., Forney, L.J., Foster, L., Di Francesco, V., Friedman, J., Friedrich, D.C., Fronick, C.C., Fulton, L.L., Gao, H., Garcia, N., Giannoukos, G., Giblin, C., Giovanni, M.Y., Goldberg, J.M., Goll, J., Gonzalez, A., Griggs, A., Gujja, S., Haake, S.K., Haas, B.J., Hamilton, H.A., Harris, E.L., Hepburn, T.A., Herter, B., Hoffmann, D.E., Holder, M.E., Howarth, C., Huang, K.H., Huse, S.M., Izard, J., Jansson, J.K., Jiang, H., Jordan, C., Joshi, V., Katancik, J.A., Keitel, W.A., Kelley, S.T., Kells, C., King, N.B., Knights, D., Kong, H.H., Koren, O., Koren, S., Kota, K.C., Kovar, C.L., Kyrpides, N.C., La Rosa, P.S., Lee, S.L., Lemon, K.P., Lennon, N., Lewis, C.M., Lewis, L., Ley, R.E., Li, K., Liolios, K., Liu, B., Liu, Y., Lo, C.-C., Lozupone, C.A., Lunsford, R.D., Madden, T., Mahurkar, A.A., Mannon, P.J., Mardis, E.R., Markowitz, V.M., Mavromatis, K., McCorrison, J.M., McDonald, D., McEwen, J., McGuire, A.L., McInnes, P., Mehta, T., Mihindukulasuriya, K.A., Miller, J.R., Minx, P.J., Newsham, I., Nusbaum, C., O’Laughlin, M., Orvis, J., Pagani, I., Palaniappan, K., Patel, S.M., Pearson, M., Peterson, J., Podar, M., Pohl, C., Pollard, K.S., Pop, M., Priest, M.E., Proctor, L.M., Qin, X., Raes, J., Ravel, J., Reid, J.G., Rho, M., Rhodes, R., Riehle, K.P., Rivera, M.C., Rodriguez-Mueller, B., Rogers, Y.-H., Ross, M.C., Russ, C., Sanka, R.K., Sankar, P., Sathirapongsasuti, J.F., Schloss, J.A., Schloss, P.D., Schmidt, T.M., Scholz, M., Schriml, L., Schubert, A.M., Segata, N., Segre, J.A., Shannon, W.D., Sharp, R.R., Sharpton, T.J., Shenoy, N., Sheth, N.U., Simone, G.A., Singh, I., Smillie, C.S., Sobel, J.D., Sommer, D.D., Spicer, P., Sutton, G.G., Sykes, S.M., Tabbaa, D.G., Thiagarajan, M., Tomlinson, C.M., Torralba, M., Treangen, T.J., Truty, R.M., Vishnivetskaya, T.A., Walker, J., Wang, L., Wang, Z., Ward, D.V., Warren, W., Watson, M.A., Wellington, C., Wetterstrand, K.A., White, J.R., Wilczek-Boney, K., Wu, Y., Wylie, K.M., Wylie, T., Yandava, C., Ye, L., Ye, Y., Yooseph, S., Youmans, B.P., Zhang, L., Zhou, Y., Zhu, Y., Zoloth, L., Zucker, J.D., Birren, B.W., Gibbs, R.A., Highlander, S.K., Methe, B.A., Nelson, K.E., Petrosino, J.F., Weinstock, G.M., Wilson, R.K., White, O., Human Microbiome Project, C., 2012. Structure, function and diversity of the healthy human microbiome. Nature 486, 207e214. Jandhyala, S.M., Talukdar, R., Subramanyam, C., Vuyyuru, H., Sasikala, M., Reddy, D.N., 2015. Role of the normal gut microbiota. World J. Gastroenterol. 21, 8787e8803. Jakobsson, H.E., Rodriguez-Pineiro, A.M., Schutte, A., Ermund, A., Boysen, P., Bemark, M., Sommer, F., Backhed, F., Hansson, G.C., Johansson, M.E.V., 2015. The composition of the gut microbiota shapes the colon mucus barrier. EMBO Rep. 16, 164e177. Jin, Y., Wu, S., Zeng, Z., Fu, Z., 2017. Effects of environmental pollutants on gut microbiota. Environ. Pollut. 222, 1e9. Jordan, J., Zare, A., Jackson, L.J., Habibi, H.R., Weljie, A.M., 2011. Environmental contaminant mixtures at ambient concentrations invoke a metabolic stress response in goldfish not predicted from exposure to individual compounds alone. J. Proteome Res. 11, 1133e1143. Kim, E., Xu, X., Steiner, H., Ahmer, B., Cormetboyaka, E., Boyaka, P., 2015. Chronic ingestion of low doses of cadmium alters the gut microbiome and immune homeostasis to enhance allergic sensitization (MUC9P.743). J. Immunol. 194, 205e207. Lee, S., Kirkland, R., Grunewald, Z.I., Sun, Q., Wicker, L., de La Serre, C.B., 2019. Beneficial effects of non-encapsulated or encapsulated probiotic supplementation on microbiota composition, intestinal barrier functions, inflammatory profiles, and glucose tolerance in high fat fed rats. Nutrients 11. Li, X., Brejnrod, A.D., Ernst, M., Rykaer, M., Herschend, J., Olsen, N.M.C., Dorrestein, P.C., Rensing, C., Sorensen, S.J., 2019. Heavy metal exposure causes changes in the metabolic health-associated gut microbiome and metabolites. Environ. Int. 126, 454e467. Liu, J., Qu, W., Kadiiska, M.B., 2009. Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol. Appl. Pharmacol. 238, 209e214. Liu, W., Zhang, B., Huang, Z., Pan, X., Chen, X., Hu, C., Liu, H., Jiang, Y., Sun, X., Peng, Y., 2018. Cadmium body burden and gestational diabetes mellitus: a prospective study. Environ. Health Perspect. 126, 027006. Liu, Y., Li, Y., Liu, K., Shen, J., 2014. Exposing to cadmium stress cause profound toxic effect on microbiota of the mice intestinal tract. PLoS One 9, e85323. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402e408. Luo, S., Terciolo, C., Bracarense, A., Payros, D., Pinton, P., Oswald, I.P., 2019. In vitro and in vivo effects of a mycotoxin, deoxynivalenol, and a trace metal, cadmium, alone or in a mixture on the intestinal barrier. Environ. Int. 132, 105082. Ninkov, M., Aleksandrov, A.P., Demenesku, J., Mirkov, I., Mileusnic, D., Petrovic, A., Grigorov, I., Zolotarevski, L., Tolinacki, M., Kataranovski, D., 2015. Toxicity of oral cadmium intake: impact on gut immunity. Toxicol. Lett. 237, 89e99. Ran, L., Li, H.H., 2011. State of art of cadmium pollution and hazard in soil. J. Chongqing Univ. Arts. Sci. 30, 69e73. Rowland, I., Gibson, G., Heinken, A., Scott, K., Swann, J., Thiele, I., Tuohy, K., 2018. Gut microbiota functions: metabolism of nutrients and other food components. Eur. J. Nutr. 57, 1e24.

X. He et al. / Chemosphere 246 (2020) 125747 Rusanov, A.L., Smirnova, A.V., Poromov, A.A., Fomicheva, K.A., Luzgina, N.G., Majouga, A.G., 2015. Effects of cadmium chloride on the functional state of human intestinal cells. Toxicol. In Vitro 29, 1006e1011. Sanchez-Alcoholado, L., Castellano-Castillo, D., Jord
an-Martínez, L., Moreno~ oz-Garcia, A.J., Queipo-Ortun ~ o, M.I., Indias, I., Cardila-Cruz, P., Elena, D., Mun Jimenez-Navarro, M., 2017. Role of gut microbiota on cardio-metabolic parameters and immunity in coronary artery disease patients with and without type-2 diabetes mellitus. Front. Microbiol. 8, 1936. Smolders, A.J.P., Lock, R.A.C., Van der Velde, G., Hoyos, R.I.M., Roelofs, J.G.M., 2003. Effects of mining activities on heavy metal concentrations in water, sediment, and macroinvertebrates in different reaches of the Pilcomayo River, South America. Arch. Environ. Contam. Toxicol. 44, 314e323. Tan, J., McKenzie, C., Potamitis, M., Thorburn, A.N., Mackay, C.R., Macia, L., 2014. The role of short-chain fatty acids in health and disease. Adv. Immunol. 121, 91e119. Thursby, E., Juge, N., 2017. Introduction to the human gut microbiota. Biochem. J. 474, 1823e1836. Tinkov, A.A., Filippini, T., Ajsuvakova, O.P., Aaseth, J., Gluhcheva, Y.G., Ivanova, J.M., Bjørklund, G., Skalnaya, M.G., Gatiatulina, E.R., Popova, E.V., 2017. The role of cadmium in obesity and diabetes. Sci. Total Environ. 601e602, 741e755. Tinkov, A.A., Gritsenko, V.A., Skalnaya, M.G., Cherkasov, S.V., Aaseth, J., Skalny, A.V., 2018. Gut as a target for cadmium toxicity. Environ. Pollut. 235, 429e434.
Luis, G., Manuel, C., Eduardo, D., 2012. Valderrama, J.A., Gonzalo, D.R., Blas, B., Jose Bacterial degradation of benzoate: cross-regulation between aerobic and

9

anaerobic pathways. J. Biol. Chem. 287, 10494e10508. Wang, Q., Feng, Y.M., Wang, S.M., Du, Y.Q., Yin, J.Z., Yang, Y.L., 2015. Assessment of the cadmium exposure in the blood, diet, and water of the Pumi People in Yunnan, China. Biol. Trace Elem. Res. 168, 349e355. Yatsunenko, T., Rey, F.E., Manary, M.J., Trehan, I., Dominguez-Bello, M.G., Contreras, M., Magris, M., Hidalgo, G., Baldassano, R.N., Anokhin, A.P., Heath, A.C., Warner, B., Reeder, J., Kuczynski, J., Caporaso, J.G., Lozupone, C.A., Lauber, C., Clemente, J.C., Knights, D., Knight, R., Gordon, J.I., 2012. Human gut microbiome viewed across age and geography. Nature 486, 222e227. Zhai, Q., Li, T., Yu, L., Xiao, Y., Feng, S., Wu, J., Zhao, J., Zhang, H., Chen, W., 2017. Effects of subchronic oral toxic metal exposure on the intestinal microbiota of mice. Sci. Bull. 62, 831e840. Zhai, Q., Tian, F., Zhao, J., Zhang, H., Narbad, A., Chen, W., 2016. Oral administration of probiotics inhibits absorption of the heavy metal cadmium by protecting the intestinal barrier. Appl. Environ. Microbiol. 82, 4429e4440. Zhang, S., Jin, Y., Zeng, Z., Liu, Z., Fu, Z., 2015. Subchronic Exposure of mice to cadmium perturbs their hepatic energy metabolism and gut microbiome. Chem. Res. Toxicol. 28, 2000e2009. Zheng, H., You, Y., Hua, M., Wu, P., Liu, Y., Chen, Z., Zhang, L., Wei, H., Li, Y., Luo, M., Zeng, Y., Liu, Y., Luo, D.X., Zhang, J., Feng, M., Hu, R., Pandol, S.J., Han, Y.P., 2018. Chlorophyllin modulates gut microbiota and inhibits intestinal inflammation to ameliorate hepatic fibrosis in mice. Front. Physiol. 9, 1671.