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Arsenic metabolism and toxicity influenced by ferric iron in simulated gastrointestinal tract and the roles of gut microbiota Haiyan Yu, Bing Wu, Xu-Xiang Zhang, Su Liu, Jing Yu, Shupei Cheng, Hong-qiang Ren, and Lin Ye Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01533 • Publication Date (Web): 09 Jun 2016 Downloaded from http://pubs.acs.org on June 10, 2016
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Environmental Science & Technology
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Arsenic metabolism and toxicity influenced by ferric iron in
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simulated gastrointestinal tract and the roles of gut microbiota
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Haiyan Yu, Bing Wu, Xu-Xiang Zhang, Su Liu, Jing Yu, Shupei Cheng, Hong-qiang
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Ren, Lin Ye
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State Key Laboratory of Pollution Control and Resource Reuse, School of the
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Environment, Nanjing University, Nanjing, 210023, P.R. China
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*Corresponding Author:
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School of the Environment, Nanjing University
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NO.163 Xianlin Road, Nanjing, P.R. China
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Tel. & Fax: 0086-25-89680720
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E-mail:
[email protected] (B Wu) or
[email protected] (L Ye)
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ABSTRACT
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Iron (Fe) is a common trace element in drinking water. However, little is known about
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how environmental concentrations of Fe affect the metabolism and toxicity of arsenic
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(As) in drinking water. In this study, influence of Fe at drinking water-related
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concentrations (0.1, 0.3 and 3 mg Fe (total)/L) on As metabolism and toxicity, and the
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roles of gut microbiota during this process were investigated by using in vitro
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Simulator of the Human Intestinal Microbial Ecosystem (SHIME). Results showed
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that Fe had ability to decrease bioaccessible As by co-flocculation in small intestine.
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0.1 and 0.3 mg/L Fe significantly increased As methylation in simulated transverse
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and descending colon. Gut microbiota played an important role in alteration of As
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species, and Fe could affect As metabolism by changing the gut microbiota.
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Bacteroides, Clostridium, Alistipes and Bilophila had As resistance and potential
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ability to methylate As. Cytotoxicity assays of effluents from simulated colons
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showed that the low levels of Fe decreased As toxicity on human hepatoma cell line
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HepG2, which might be due to the increase of methylated As. When assessing the
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health risk of As in drinking water, the residual Fe should be considered.
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Keywords: Arsenic; Iron; Toxicity; Metabolism; Gut microbial community;
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Simulator of the Human Intestinal Microbial Ecosystem
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TOC Art
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INTRODUCTION
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Arsenic (As) is a widely distributed metalloid on Earth. Epidemiological studies
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and clinical observations have indicated that As is associated with many kinds of
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human cancers and noncancerous diseases 1. Drinking water is the main source of As
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exposure
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around the world, such as Bangladesh, India, Mongolia, China, etc. 4, 5 Toxicity of As
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is largely determined by its species, which include inorganic arsenic (iAs),
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monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Generally, iAs is
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more toxic than organic As (MMA and DMA)，and the toxicity of AsIII is higher than
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AsV 6. Besides species, As toxicity can also be affected by some compounds and
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elements. For instance, vitamins can reduce As toxicity by increasing As methylation
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and antioxidative enzymes to antagonize oxidative stress which is thought to be main
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mechanism of As toxicity 7, 8. Among the factors which can affect As toxicity, iron (Fe)
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is a common element. In the past few years, several studies have been conducted to
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investigate the toxicity of co-existence of As and Fe. Chandrasekaran et al.,
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that a combination of As and Fe could cause synergistically hepatic damage in rats by
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gavage. Our previous studies showed that co-exposure of As and Fe could cause a
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synergetic effect based on HepG2 cell lines. However an antagonistic effect was
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found in the animal experiment, which indicates that Fe could change the metabolism
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and bioaccessibility of As in gastrointestinal tract and further alter its toxicity 10, 11. It
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is noticed that very high Fe concentrations (5~200 mg/L) have been applied in
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abovementioned researches. To our knowledge, few studies have been conducted to
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investigate how the normal environmental concentrations of Fe affect the
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bioaccessibility and toxicity of As, which is very important to characterize the actual
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risk of As in drinking water.
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. Safety of As in drinking water is a major concern in many countries
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In order to characterize the metabolism and bioacessibility of xenobiotics in
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gastrointestinal tract, many kinds of in vitro simulated system have been developed.
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Simulator of the Human Intestinal Microbial Ecosystem (SHIME) is one of these
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simulated systems which can dynamically simulate the digestive processes of stomach,
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small intestine as well as ascending, transverse, and descending colon of human
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gastrointestinal tract
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unique advantages in simulating the gut microbial community and functions in
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different regions of the human colon, therefore, it was widely used to study
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metabolism and bioaccessibility of xenobiotics and the underlying roles of gut
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microbiota
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and bioaccessibility in human gastrointestinal tract 3, 17, 18.
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. Compared with other in vitro simulations, SHIME offers
3, 15,16
. Recently, SHIME has also been applied to explore As metabolism
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The aim of this study is to explore the influence of Fe at environmental level on
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As metabolism, bioaccessibility and toxicity, and the roles of gut microbiota during
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this process in SHIME. High performance liquid chromatography and inductively
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coupled plasma mass spectrometry (HPLC-ICP-MS) was applied to analyze As
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species, and the atomic absorption spectrometry (AAS) was used to determine Fe
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concentrations in different parts of SHIME. High-throughput sequencing was
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conducted to characterize the microbial community. Cytotoxicity experiments in
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human hepatoma cell line HepG2 were conducted to evaluate toxicity of bioaccessible
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As in gastrointestinal tract. HepG2 cells are a suitable in vitro model system for the
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study of hepatotoxicity, which have been widely used to analyze the metabolism and
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toxicity of As 19, 20. This study improves our understanding on the As metabolism and
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provides insights into the health risk assessment of As and residual Fe in drinking
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water.
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MATERIALS AND METHODS 5
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SHIME
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The SHIME, consisting of five double-jacketed vessels maintained at 37 oC, was
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fed three times a day using a nutritional medium as described by Van de Wiele et al, 21.
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1L of the nutritional medium contained 1 g arabinogalactan, 2 g pectin, 1 g xylan, 4 g
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starch, 0.4 g glucose, 3 g yeast extract, 1 g peptone, 4 g mucin, and 0.5 g cystein. The
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five vessels simulate stomach, small intestine, ascending colon, transverse colon and
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descending colon, respectively. Pancreatic juices were pumped into small intestine 22.
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Fecal microbiota was obtained from a volunteer who did not receive antibiotic
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treatment in the 6 months before the study. The fecal sample was disposed and
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transferred into the last three vessels of simulator whose pH values were controlled in
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the ranges of 5.5–5.9, 6.0–6.4 and 6.6–6.9, respectively
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experiments, the SHIME was fed with nutritional medium for two weeks to stabilize
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the microbial community
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medium (CK), nutritional medium + 100 µg/L As, nutritional medium + 600 µg/L As,
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nutritional medium + 600 µg/L As + 0.1 mg/L Fe, nutritional medium + 600 µg/L
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As+0.3 mg/L Fe, and nutritional medium + 600 µg/L As+3mg/L Fe. The exposure
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concentrations of Fe were selected according to actual concentration of Fe in drinking
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water and National Standards for Drinking Water Quality of China (GB5749-2006). In
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normal drinking water, the concentration of Fe is about 0.1mg/L. The limit of Fe
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concentration in drinking water shown in GB5749-2006 is 0.3mg/L. The 3mg/L as the
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high control concentration of Fe was also applied in this study. Since As has been
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detected to be > 100 µg/L in drinking water in some regions, such as China, Argentina
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and Chile
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was iAsIII, which is present in a significant amount under reducing conditions in
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groundwater
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. Prior to exposure
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. The SHIME was sequentially exposed to nutritional
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, 100 µg/L and 600 µg/L As were chosen in this study. The exposed As
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. The As2O3 solution and FeCl3 powder was purchased from O2si 6
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(USA) and Adamas reagent (China), respectively. In purchased As2O3 solution, As2O3
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was dissolved in 2% nitric acid solution. The exposure duration was set to 7 days for
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each condition according to the previous studies 26, 27. At the end of each condition 30
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mL sample was taken from each vessel of SHIME. Then the samples were centrifuged
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at 10400×g for 10 min. The separated supernatants and pellets were flash frozen with
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liquid nitrogen, and stored at -80 oC for further analyses 18.
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Determination of As species and Fe
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Concentrations of As species in supernatants were measured. The supernatants
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were diluted with ultrapure water, and then filtered through 0.22 µm polyether sulfone
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(PES) membranes with 5-mL syringes for further analyses. Four As species, including
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iAsIII, iAsV, MMA and DMA, were measured by HPLC-ICP-MS (PerkinElmer), and
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three repetitions for one sample were performed. Separation of different As species
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was performed on a PRP-X100 HPLC column (Hamilton, UK). The chromatographic
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mobile phase was a solution of 8 mM NH4NO3 and 8 mM NH4H2PO4, and the pH was
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adjusted to 7.2 before use. Flow rate of mobile phase was 1.2 mL/min, and sample
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injection volume was 50 µL. The retention time were 1.9 min, 3.0 min, 4.1 min, and
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8.8 min for iAsIII, DMA, MMA, and iAsV, respectively (Figure S1). To ensure the
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stability of signals, germanium standard liquid (O2si, USA) was used as an internal
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standard.
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The Fe concentration in supernatants was determined by AAS (Hitachi, Japan) 28
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according to the methods described by Andrade et al.
. Before measurement, the
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supernatants were filtered through 0.45 µm PES membranes with 5-mL syringes.
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DNA extraction and 16S rRNA gene sequencing
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Genomic DNA of gut microbiota was extracted from the samples by using Fast
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DNA SPIN Kit for Soil (MP Biomedicals, USA). Concentration and quality of the 7
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extracted DNA were determined by NanoDropND-2000 (NanoDrop Technologies,
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Wilmington, DE) and gel electrophoresis. Subsequently, the genomic DNA was
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amplified with barcoded primers targeting the V1-V2 region of 16S rRNA gene. The
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forward primer is 5’-AGAGTTTGATYMTGGCTCAG-3’, and the reverse primer is
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5’-TGCTGCCTCCCGTAGGAGT-3’. PCRs were conducted in a reaction system (50
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µL) containing 2 µL template DNA (20 ng/µL), 2 µL forward primer and 2 µL reverse
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primer (10 µM), 19 µL ddH2O, and 25 µL 2×EasyTaq PCR supermix. The protocol for
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PCR amplification is: 98 oC for 5 min; 20 cycles of 94 oC for 30 s, 50 oC for 30 s, 72
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o
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MiniBest DNA Fragment Purification Kit Ver.4.0 (TaKaRa, Japan), the PCR products
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of different samples were mixed in equimolar amounts and submitted for sequencing
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on an Illumina Miseq sequencer.
C for 40 s, with a final elongation step at 72 oC for 10 min. After purification with
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After Miseq sequencing, Sickle tool was used to perform the original quality
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filtering to remove reads with averages quality score less than 20 or with any
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unknown bases. Then, the quality-filtered reads were processed using Mothur
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Forward and reverse sequences were joined into contigs by the “make.contigs”
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command. And then, the further quality filtering process was conducted using the
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“trim.seqs” command including four steps: trimming off the adapters, barcodes and
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primers; removing the low quality reads and the reads containing ambiguous “N”;
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removing the reads shorter than 300 bp. After quality filtering, chimeras were
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removed using chimera slayer, and then sequence numbers of each sample were
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normalized to the same to achieve same sequencing depth. OTU (operational
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taxonomic units) picking was conducted by using the uclust method in QIIME (v
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1.9.1)
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the representative sequences was performed using RDP classifier
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with a sequence similarity threshold of 0.97. The taxonomic assignment of
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confidence level.
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Cytotoxicity assay
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Cytotoxicity of As and Fe in effluent of each vessel in SHIME was measured
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based on HepG2 cell lines which were obtained from keyGEN Biotech (China).
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HepG2 was maintained in Dulbecco’s Modified Eagles Medium (DMEM) with 10%
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fetal bovine serum under standard cell culture conditions (37 oC, and 5% CO2). For
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exposure experiment, the HepG2 at a density of 10000 cells per well was seeded in a
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96-well microplate. The cells were incubated for 24 h, and then the exposure solution
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obtained from the effluents of SHIME was added. Before exposure, the supernatants
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were freeze-dried and DMEM was added, and then filtered for sterilization. The
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exposure time was 24 h. Cell viability after exposure was determined by cell counting
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kit-8 (CCK-8, Dojindo Molecular Technologies, Inc. Japan) method, which was
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presented by the ratio of absorbance of treated sample to absorbance of non-treated
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HepG2 cell sample. The absorption wavelength for CCK-8 was 460 nm. Three
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microplate (6 wells in one microplate) for each condition were applied, and there were
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18 absorbance values in all for each condition.
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Statistical analysis
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Difference among groups was evaluated using one-way analysis of variance
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(ANOVA) test followed by Tukey’s post-hoc test. All analyses were performed on
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Graphpad Prism 5. A p value
