Arsenic Metabolism and Toxicity Influenced by Ferric Iron in

Cluster analysis of gut microbiota in ascending, transverse and descending ...... software for describing and comparing microbial communities Appl. En...
0 downloads 0 Views 989KB Size
Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Environmental Science & Technology

1

Arsenic metabolism and toxicity influenced by ferric iron in

2

simulated gastrointestinal tract and the roles of gut microbiota

3

Haiyan Yu, Bing Wu, Xu-Xiang Zhang, Su Liu, Jing Yu, Shupei Cheng, Hong-qiang

4

Ren, Lin Ye

5

State Key Laboratory of Pollution Control and Resource Reuse, School of the

6

Environment, Nanjing University, Nanjing, 210023, P.R. China

7

*Corresponding Author:

8

School of the Environment, Nanjing University

9

NO.163 Xianlin Road, Nanjing, P.R. China

10

Tel. & Fax: 0086-25-89680720

11

E-mail: [email protected] (B Wu) or [email protected] (L Ye)

1

ACS Paragon Plus Environment

Environmental Science & Technology

12

ABSTRACT

13

Iron (Fe) is a common trace element in drinking water. However, little is known about

14

how environmental concentrations of Fe affect the metabolism and toxicity of arsenic

15

(As) in drinking water. In this study, influence of Fe at drinking water-related

16

concentrations (0.1, 0.3 and 3 mg Fe (total)/L) on As metabolism and toxicity, and the

17

roles of gut microbiota during this process were investigated by using in vitro

18

Simulator of the Human Intestinal Microbial Ecosystem (SHIME). Results showed

19

that Fe had ability to decrease bioaccessible As by co-flocculation in small intestine.

20

0.1 and 0.3 mg/L Fe significantly increased As methylation in simulated transverse

21

and descending colon. Gut microbiota played an important role in alteration of As

22

species, and Fe could affect As metabolism by changing the gut microbiota.

23

Bacteroides, Clostridium, Alistipes and Bilophila had As resistance and potential

24

ability to methylate As. Cytotoxicity assays of effluents from simulated colons

25

showed that the low levels of Fe decreased As toxicity on human hepatoma cell line

26

HepG2, which might be due to the increase of methylated As. When assessing the

27

health risk of As in drinking water, the residual Fe should be considered.

28

Keywords: Arsenic; Iron; Toxicity; Metabolism; Gut microbial community;

29

Simulator of the Human Intestinal Microbial Ecosystem

2

ACS Paragon Plus Environment

Page 2 of 33

Page 3 of 33

Environmental Science & Technology

30

TOC Art

31

3

ACS Paragon Plus Environment

Environmental Science & Technology

Page 4 of 33

INTRODUCTION

32 33

Arsenic (As) is a widely distributed metalloid on Earth. Epidemiological studies

34

and clinical observations have indicated that As is associated with many kinds of

35

human cancers and noncancerous diseases 1. Drinking water is the main source of As

36

exposure

37

around the world, such as Bangladesh, India, Mongolia, China, etc. 4, 5 Toxicity of As

38

is largely determined by its species, which include inorganic arsenic (iAs),

39

monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Generally, iAs is

40

more toxic than organic As (MMA and DMA),and the toxicity of AsIII is higher than

41

AsV 6. Besides species, As toxicity can also be affected by some compounds and

42

elements. For instance, vitamins can reduce As toxicity by increasing As methylation

43

and antioxidative enzymes to antagonize oxidative stress which is thought to be main

44

mechanism of As toxicity 7, 8. Among the factors which can affect As toxicity, iron (Fe)

45

is a common element. In the past few years, several studies have been conducted to

46

investigate the toxicity of co-existence of As and Fe. Chandrasekaran et al.,

47

that a combination of As and Fe could cause synergistically hepatic damage in rats by

48

gavage. Our previous studies showed that co-exposure of As and Fe could cause a

49

synergetic effect based on HepG2 cell lines. However an antagonistic effect was

50

found in the animal experiment, which indicates that Fe could change the metabolism

51

and bioaccessibility of As in gastrointestinal tract and further alter its toxicity 10, 11. It

52

is noticed that very high Fe concentrations (5~200 mg/L) have been applied in

53

abovementioned researches. To our knowledge, few studies have been conducted to

54

investigate how the normal environmental concentrations of Fe affect the

55

bioaccessibility and toxicity of As, which is very important to characterize the actual

56

risk of As in drinking water.

2, 3

. Safety of As in drinking water is a major concern in many countries

4

ACS Paragon Plus Environment

9

found

Page 5 of 33

Environmental Science & Technology

57

In order to characterize the metabolism and bioacessibility of xenobiotics in

58

gastrointestinal tract, many kinds of in vitro simulated system have been developed.

59

Simulator of the Human Intestinal Microbial Ecosystem (SHIME) is one of these

60

simulated systems which can dynamically simulate the digestive processes of stomach,

61

small intestine as well as ascending, transverse, and descending colon of human

62

gastrointestinal tract

63

unique advantages in simulating the gut microbial community and functions in

64

different regions of the human colon, therefore, it was widely used to study

65

metabolism and bioaccessibility of xenobiotics and the underlying roles of gut

66

microbiota

67

and bioaccessibility in human gastrointestinal tract 3, 17, 18.

12-14

. Compared with other in vitro simulations, SHIME offers

3, 15,16

. Recently, SHIME has also been applied to explore As metabolism

68

The aim of this study is to explore the influence of Fe at environmental level on

69

As metabolism, bioaccessibility and toxicity, and the roles of gut microbiota during

70

this process in SHIME. High performance liquid chromatography and inductively

71

coupled plasma mass spectrometry (HPLC-ICP-MS) was applied to analyze As

72

species, and the atomic absorption spectrometry (AAS) was used to determine Fe

73

concentrations in different parts of SHIME. High-throughput sequencing was

74

conducted to characterize the microbial community. Cytotoxicity experiments in

75

human hepatoma cell line HepG2 were conducted to evaluate toxicity of bioaccessible

76

As in gastrointestinal tract. HepG2 cells are a suitable in vitro model system for the

77

study of hepatotoxicity, which have been widely used to analyze the metabolism and

78

toxicity of As 19, 20. This study improves our understanding on the As metabolism and

79

provides insights into the health risk assessment of As and residual Fe in drinking

80

water.

81

MATERIALS AND METHODS 5

ACS Paragon Plus Environment

Environmental Science & Technology

82

Page 6 of 33

SHIME

83

The SHIME, consisting of five double-jacketed vessels maintained at 37 oC, was

84

fed three times a day using a nutritional medium as described by Van de Wiele et al, 21.

85

1L of the nutritional medium contained 1 g arabinogalactan, 2 g pectin, 1 g xylan, 4 g

86

starch, 0.4 g glucose, 3 g yeast extract, 1 g peptone, 4 g mucin, and 0.5 g cystein. The

87

five vessels simulate stomach, small intestine, ascending colon, transverse colon and

88

descending colon, respectively. Pancreatic juices were pumped into small intestine 22.

89

Fecal microbiota was obtained from a volunteer who did not receive antibiotic

90

treatment in the 6 months before the study. The fecal sample was disposed and

91

transferred into the last three vessels of simulator whose pH values were controlled in

92

the ranges of 5.5–5.9, 6.0–6.4 and 6.6–6.9, respectively

93

experiments, the SHIME was fed with nutritional medium for two weeks to stabilize

94

the microbial community

95

medium (CK), nutritional medium + 100 µg/L As, nutritional medium + 600 µg/L As,

96

nutritional medium + 600 µg/L As + 0.1 mg/L Fe, nutritional medium + 600 µg/L

97

As+0.3 mg/L Fe, and nutritional medium + 600 µg/L As+3mg/L Fe. The exposure

98

concentrations of Fe were selected according to actual concentration of Fe in drinking

99

water and National Standards for Drinking Water Quality of China (GB5749-2006). In

100

normal drinking water, the concentration of Fe is about 0.1mg/L. The limit of Fe

101

concentration in drinking water shown in GB5749-2006 is 0.3mg/L. The 3mg/L as the

102

high control concentration of Fe was also applied in this study. Since As has been

103

detected to be > 100 µg/L in drinking water in some regions, such as China, Argentina

104

and Chile

105

was iAsIII, which is present in a significant amount under reducing conditions in

106

groundwater

12, 22

. Prior to exposure

21, 23

. The SHIME was sequentially exposed to nutritional

4, 5

, 100 µg/L and 600 µg/L As were chosen in this study. The exposed As

24, 25

. The As2O3 solution and FeCl3 powder was purchased from O2si 6

ACS Paragon Plus Environment

Page 7 of 33

Environmental Science & Technology

107

(USA) and Adamas reagent (China), respectively. In purchased As2O3 solution, As2O3

108

was dissolved in 2% nitric acid solution. The exposure duration was set to 7 days for

109

each condition according to the previous studies 26, 27. At the end of each condition 30

110

mL sample was taken from each vessel of SHIME. Then the samples were centrifuged

111

at 10400×g for 10 min. The separated supernatants and pellets were flash frozen with

112

liquid nitrogen, and stored at -80 oC for further analyses 18.

113

Determination of As species and Fe

114

Concentrations of As species in supernatants were measured. The supernatants

115

were diluted with ultrapure water, and then filtered through 0.22 µm polyether sulfone

116

(PES) membranes with 5-mL syringes for further analyses. Four As species, including

117

iAsIII, iAsV, MMA and DMA, were measured by HPLC-ICP-MS (PerkinElmer), and

118

three repetitions for one sample were performed. Separation of different As species

119

was performed on a PRP-X100 HPLC column (Hamilton, UK). The chromatographic

120

mobile phase was a solution of 8 mM NH4NO3 and 8 mM NH4H2PO4, and the pH was

121

adjusted to 7.2 before use. Flow rate of mobile phase was 1.2 mL/min, and sample

122

injection volume was 50 µL. The retention time were 1.9 min, 3.0 min, 4.1 min, and

123

8.8 min for iAsIII, DMA, MMA, and iAsV, respectively (Figure S1). To ensure the

124

stability of signals, germanium standard liquid (O2si, USA) was used as an internal

125

standard.

126

The Fe concentration in supernatants was determined by AAS (Hitachi, Japan) 28

127

according to the methods described by Andrade et al.

. Before measurement, the

128

supernatants were filtered through 0.45 µm PES membranes with 5-mL syringes.

129

DNA extraction and 16S rRNA gene sequencing

130

Genomic DNA of gut microbiota was extracted from the samples by using Fast

131

DNA SPIN Kit for Soil (MP Biomedicals, USA). Concentration and quality of the 7

ACS Paragon Plus Environment

Environmental Science & Technology

Page 8 of 33

132

extracted DNA were determined by NanoDropND-2000 (NanoDrop Technologies,

133

Wilmington, DE) and gel electrophoresis. Subsequently, the genomic DNA was

134

amplified with barcoded primers targeting the V1-V2 region of 16S rRNA gene. The

135

forward primer is 5’-AGAGTTTGATYMTGGCTCAG-3’, and the reverse primer is

136

5’-TGCTGCCTCCCGTAGGAGT-3’. PCRs were conducted in a reaction system (50

137

µL) containing 2 µL template DNA (20 ng/µL), 2 µL forward primer and 2 µL reverse

138

primer (10 µM), 19 µL ddH2O, and 25 µL 2×EasyTaq PCR supermix. The protocol for

139

PCR amplification is: 98 oC for 5 min; 20 cycles of 94 oC for 30 s, 50 oC for 30 s, 72

140

o

141

MiniBest DNA Fragment Purification Kit Ver.4.0 (TaKaRa, Japan), the PCR products

142

of different samples were mixed in equimolar amounts and submitted for sequencing

143

on an Illumina Miseq sequencer.

C for 40 s, with a final elongation step at 72 oC for 10 min. After purification with

144

After Miseq sequencing, Sickle tool was used to perform the original quality

145

filtering to remove reads with averages quality score less than 20 or with any

146

unknown bases. Then, the quality-filtered reads were processed using Mothur

147

Forward and reverse sequences were joined into contigs by the “make.contigs”

148

command. And then, the further quality filtering process was conducted using the

149

“trim.seqs” command including four steps: trimming off the adapters, barcodes and

150

primers; removing the low quality reads and the reads containing ambiguous “N”;

151

removing the reads shorter than 300 bp. After quality filtering, chimeras were

152

removed using chimera slayer, and then sequence numbers of each sample were

153

normalized to the same to achieve same sequencing depth. OTU (operational

154

taxonomic units) picking was conducted by using the uclust method in QIIME (v

155

1.9.1)

156

the representative sequences was performed using RDP classifier

30

29

.

with a sequence similarity threshold of 0.97. The taxonomic assignment of

8

ACS Paragon Plus Environment

31

with 80%

Page 9 of 33

Environmental Science & Technology

157

confidence level.

158

Cytotoxicity assay

159

Cytotoxicity of As and Fe in effluent of each vessel in SHIME was measured

160

based on HepG2 cell lines which were obtained from keyGEN Biotech (China).

161

HepG2 was maintained in Dulbecco’s Modified Eagles Medium (DMEM) with 10%

162

fetal bovine serum under standard cell culture conditions (37 oC, and 5% CO2). For

163

exposure experiment, the HepG2 at a density of 10000 cells per well was seeded in a

164

96-well microplate. The cells were incubated for 24 h, and then the exposure solution

165

obtained from the effluents of SHIME was added. Before exposure, the supernatants

166

were freeze-dried and DMEM was added, and then filtered for sterilization. The

167

exposure time was 24 h. Cell viability after exposure was determined by cell counting

168

kit-8 (CCK-8, Dojindo Molecular Technologies, Inc. Japan) method, which was

169

presented by the ratio of absorbance of treated sample to absorbance of non-treated

170

HepG2 cell sample. The absorption wavelength for CCK-8 was 460 nm. Three

171

microplate (6 wells in one microplate) for each condition were applied, and there were

172

18 absorbance values in all for each condition.

173

Statistical analysis

174

Difference among groups was evaluated using one-way analysis of variance

175

(ANOVA) test followed by Tukey’s post-hoc test. All analyses were performed on

176

Graphpad Prism 5. A p value