Salivary and gut microbiomes play a significant role in in vitro oral

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Ecotoxicology and Human Environmental Health

Salivary and gut microbiomes play a significant role in in vitro oral bioaccessibility, biotransformation and intestinal absorption of arsenic from food Marta Calatayud, Chan Xiong, Gijs Du Laing, Georg Raber, Kevin A. Francesconi, and Tom Van de Wiele Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 07 Nov 2018 Downloaded from http://pubs.acs.org on November 7, 2018

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Environmental Science & Technology

1

Salivary and gut microbiomes play a significant role in in vitro oral

2

bioaccessibility, biotransformation and intestinal absorption of arsenic from

3

food

4 5

Marta Calatayuda†, Chan Xiongb†*, Gijs Du Laingc, Georg Rabera, Kevin Francesconib

6

and Tom van de Wiele a*

7

a Center for Microbial Ecology and Technology (CMET), Ghent University, Coupure Links

8

653, 9000 Ghent, Belgium

9

b Institute of Chemistry, NAWI Graz, University of Graz, 8010 Graz, Austria

10

c Department of Green Chemistry and Technology, Ghent University, 9000 Ghent, Belgium

11

† Both authors contributed equally to this work

12

* Corresponding authors:

13

Tom Van de Wiele, Phone: +32 9 264 59 76. Fax: + 32 9 264 62 48. E-mail:

14

[email protected]

15

Chan Xiong, Phone: +43 (0)316 380-5318, E-mail: [email protected]

16 17 18 19

1 ACS Paragon Plus Environment


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150–200-word abstract

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The release of a toxicant from a food matrix during the gastrointestinal digestion is a crucial

22

determinant of the toxicant’s oral bioavailability. We present a modified setup of the human

23

simulator of the gut microbial ecosystem (SHIME), with four sequential gastrointestinal

24

reactors (oral, stomach, small intestine, and colon), including the salivary and colonic

25

microbiomes. Naturally arsenic-containing rice, mussels and nori seaweed were digested in

26

presence of microorganisms and in vitro oral bioaccessibility, bioavailability and metabolism

27

of arsenic species were evaluated following analysis by using HPLC/mass spectrometry. When

28

food matrices were digested with salivary bacteria, the soluble arsenic in the gastric digestion

29

stage increased for mussel and nori samples, but no coincidence impact was found in the small

30

intestinal and colonic digestion stages. However, the simulated small intestinal absorption of

31

arsenic was increased in all food matrices (1.2-2.7 fold higher) following digestion with

32

salivary microorganisms. No significant transformation of the arsenic species occurred except

33

for the arsenosugars present in mussels and nori. In those samples, conversions between the

34

oxo arsenosugars were observed in the small intestinal digestion stage whereupon the thioxo

35

analogs became major metabolites. These results expand our knowledge on the likely

36

metabolism and oral bioavailabiltiy of arsenic during the human digestion, and provide

37

information valuable for future risk assessments of dietary arsenic.

38

Keywords: Gastrointestinal digestion, in vitro, microbiome, food, arsenic, cell culture

39


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Graphical abstract

42 43



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Introduction

45

Humans are exposed to arsenic, a highly toxic element, primarily through dietary sources such

46

as drinking water, rice and seafood 1. Epidemiological and toxicological research over many

47

years has established the toxic nature of inorganic arsenic (iAs), even at low levels, and led to

48

regulations in many countries that set maximum permissible concentrations of arsenic in water

49

and rice 1-6.

50

In vitro oral bioaccessibility testing has been adopted as a conservative estimator of

51

contaminant bioavailability. Most of the current in vitro methods incorporate physicochemical

52

parameters (e.g. temperature, pH, enzymatic activity) 7-11, however, the impact of the microbial

53

component, widely present in the colonic, but also in the oral environments, is not typically

54

considered in in vitro models. Earlier work showed that anaerobic microbiota from mouse or

55

human origin converted aqueous standards or iAs in soils into simple methylated oxyarsenicals

56

and thioxo analogs 12-14. Thioxo-arsenicals show toxicity towards human cells 15-18, in particular,

57

thioxo-DMA has shown strong cytotoxic effects in cultured human bladder cells

58

Furthermore, thioxo-arsenosugars have higher intestinal bioavailability compared to the oxo-

59

arsenosugars 17. Thus, a risk assessment of dietary arsenic should consider not only the amount

60

of arsenic and its chemical form, but also its bioaccessibility and metabolism. Moreover, the

61

gut microbiome is involved in maintaining the intestinal barrier integrity, which is an essential

62

factor in the arsenic bioavailability process 19. Thus, the gut microbiome through its interaction

63

with the food matrix and with the host, may play a substantial role in arsenic toxicokinetics.

64

The main aim of this research was, therefore, to optimize in vitro gastrointestinal model

65

systems by incorporating microbiota from the oral and colonic environment and evaluate to

18.


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what extent microbial presence is an important determinant of oral As bioaccessibility,

67

bioavailability and As speciation profiles. We use HPLC/mass spectrometry to follow the

68

biotransformation of the natural arsenic compounds in the foods in order to evaluate the

69

metabolic potential of the microbiome and the influence of microbiota as determinants of oral

70

arsenic bioaccessibility and intestinal transport.

71 72

Cell cultures

73

Caco-2 (ECACC 86010202) and HT29-MTX-E12 (ECACC 12040401) cells were obtained

74

from the European Collection of Authenticated Cell Cultures. Cell maintenance was carried

75

out routinely as described in Calatayud et al. 2011

76

passages 50 and 60.

77

Cell differentiation and the posterior tests were carried out in double chamber wells (Corning®

78

HTS Transwell®-24 well, pore size 0.4 µm; Costar Corp., NY, USA). The cells were seeded

79

at a density of 7.5 x 104 cells/cm² in a proportion of 90/10 and 70/30 Caco-2/HT29-MTX for

80

resembling the small intestine and colon epithelium, respectively, and maintained with

81

Dulbecco's Modified Eagle's Medium - high glucose (4.5 g/L) (DMEM), supplemented with

82

10% (v/v) heat-inactivated Fetal Bovine Serum (iFBS, Greiner Bio-One, Wemmel, Belgium),

83

1%

84

penicillin/streptomycin (Life Technologies, Merelbeke, Belgium) until differentiation (15

85

days). Then, DMEM was removed and cells were washed twice with 0.2 mL of Hanks Balanced

86

Salt Solution (HBSS). The cell monolayer resembling the colon was covered by 50 µL of a

87

biosimilar mucus layer prepared as described in Boegh et al. 2014 21.

(v/v)

GlutaMAX™

(Gibco,

Life

20.

All the cultures were used between

Technologies

Europe

BV),

and

1%



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88

Apparent permeability coefficient (Papp) of arsenic and arsenic cellular uptake

89

One mL of HBSS was added to the basolateral compartment and the filter-sterilized

90

supernatants from the small intestine and colonic digestion were diluted in HBSS (v/v) 1:2 (rice

91

samples, small intestine), 1:5 (rice samples, colon), 1:10 (nori and control samples, small

92

intestine), 1:20 (nori, mussel and control samples, colon), and added to the apical chambers

93

(0.2 mL).

94

The control condition consisted of the simulated digestion fluids (small intestine or colon), not

95

containing any dietary matrix, and in absence or presence of salivary bacteria, spiked with AsV

96

100 µg/L.

97

For permeability assays, samples from the basolateral compartment (0.5 mL) were obtained at

98

30, 60 and 120 min (small intestine) and at 60, 240, 1440 min (colon), and replaced with HBSS.

99

During the transport assays, cell monolayers were kept under stirring conditions (60 rpm) in a

100

shaker (ROCKER 3D basic, IKA, Belgium), at 37° C, 90% humidity and 10% CO2. At the end

101

of the permeability assay (small intestine model: 2 hours; colon model: 24 hours), cell

102

monolayers were washed twice with HBSS. The apical and basal media and the cells were

103

recovered, digested with 65% HNO3/30% H2O2 (2:1 v/v) 90°C, 4h, in a proportion to the

104

sample of 1:1 (v/v), and filtered (0.45 µm, PTFE; Metrohm Belgium N.V.) before analysis.

105

Tests were evaluated independently at least in triplicate. Percentage of cell uptake and cell

106

uptake + transport (total uptake) was calculated with respect to the total arsenic content added

107

to the apical compartment. The Papp of arsenic was calculated as described in Calatayud et al.,

108

2010

109

quantified in the apical, basal, and cellular compartments at the end of the assays, and referred

22.

The recovery of As (mass balance) was calculated as the percentage of total As


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to the total As in the filter-sterilized food digests. Recoveries and relative percent differences

111

(RPD) are reported in Table S6.

112 113

Assessment of the epithelial barrier function: TEER and Papp of LY

114

During the period of growth and differentiation, cell monolayer integrity was monitored every

115

2–3 days, measuring the transepithelial electrical resistance (TEER) with a Millicell®-ERS

116

(Merck KGaA, Darmstadt, Germany). The reported values were calculated as described by

117

Srinivasan et al., 2015 23. The cell monolayer was considered completely formed when stable

118

TEER values were obtained (≥ 80 Ω cm2). Monolayer integrity was also evaluated by

119

calculating the Papp of the paracellular transport marker Lucifer Yellow (LY) as described in

120

Calatayud et al., 2010

121

Microplate Reader, Molecular devices, Orleans, CA).

122

Results of TEER are expressed as a percentage of TEER after 2 hours (small intestine) or 24

123

hours (colon) of exposure to different digests, compared to the initial TEER values in the

124

individual wells.

22,

using a microplate fluorescence reader (Spectramax Gemini XS

125 126

Materials and methods

127

Reagents used in the investigation were of analytical or reagent grade. Reagents used in this

128

research were purchased from Merck KGaA, Darmstadt, Germany, unless otherwise stated.

129

Water used for experiments was purified (18.2 MΩ cm) with a Millipore purification system

130

(Millipore GmbH, Vienna, Austria or Millipore Inc., Belgium).

131 7 ACS Paragon Plus Environment


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Arsenic quantification

133

Plastic and glassware material was treated with 10% HNO3 (v/v) for 24 h, and then rinsed with

134

deionized water before use. Reagents and standard solutions of arsenic used for identification

135

and quantification of arsenic species are described in methods S1. For quality control, we used

136

the certified reference material (CRM) IAEA 407 (homogenized fish tissue) from International

137

Atomic Energy Agency (Vienna, Austria), CRM 7405-a (Hijiki) from National Metrology

138

Institute of Japan (Tsukuba, Ibaraki, Japan), and ERM-BC211 (rice flour) from Sigma-Aldrich

139

(Vienna, Austria) (Table S1).

140

Total arsenic measurements were performed on the microwave-assisted acid mineralised

141

samples (UltraCLAVE IV Microwave Reactor; MLS GmbH, Leutkirch, Germany) by using an

142

Agilent 7900 ICP-MS (Agilent Technologies, Waldbronn, Germany). Arsenic speciation was

143

performed by HPLC (Agilent 1260 Infinity HPLC system) coupled in parallel with an

144

inductively coupled plasma mass spectrometer (ICP-MS, Agilent 7900) and electrospray

145

ionization tandem mass spectrometer (ESI-MS-MS, Agilent 6460). Detailed information about

146

determination of total arsenic, extraction procedure, and determination of arsenic species is

147

provided as Methods S2-S4. For the structures of typical arsenic species see Figure S1. Full

148

details of the operating conditions for the analyses by HPLC-ICP-MS/ESI-MS-MS were

149

provided in Table S2. MRM transitions and optimum conditions for determining arsenic

150

species by MS/MS analysis were listed in Table S3.

151


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Food samples

153

Three batches of brown rice (Oriza sativa), mussels (Mytilus edulis), and laminated nori

154

seaweed (Porphyra tenera) were purchased at different supermarkets in Ghent (Belgium). Rice

155

was washed twice with 1:10 (w/w) water and once drained, it was cooked in a stainless steel

156

stewpot with 1:3 (w/w) water for 30 min. Mussels were rinsed with water (1:10, w/w) two

157

times and steamed in a stainless steel stewpot for 10 min without adding cooking water. Non-

158

edible portions (valves) were removed and liquid and edible parts were mixed. Nori seaweed

159

was roasted 15 sec in a pre-heated Teflon pan. After cooking, food matrices were homogenized

160

by using a Thermomix food processor (Vorwerk, Spain M.S.L, S.C) at highest speed until

161

obtaining a soft texture (mussels, rice) or a powder (nori).

162

In vitro gastrointestinal digestion and fermentation

163

Foods were digested using a semi-continuous simulation of the digestion process, modified

164

from the SHIME with four stages: oral, gastric, small intestine, and colon. A schematic

165

representation of the in vitro digestion is given in the Figure S2.

166

The prepared samples of rice (90 g), mussels (50 g) and nori (2.5 g) were added to double-

167

jacketed reactors maintained at 37°C under continuous stirring. As a control, sterile water

168

samples (50 g) were used. The amount of food was estimated on representative quantities of

169

daily intakes for the European population 24. The in vitro digestions were run in parallel and

170

tested in duplicate. Samples were mixed with a simulated salivary fluid (SSF) in a ratio of 1:1

171

(w/w) for rice, mussels and control, and in a ratio of 1:20 (w/w) for nori. The higher ratio of

172

SSF/sample for nori was selected to mimic the dilution of the 2.5 g of sample with digestive

173

fluids in a real theoretical scenario. Oral digestion was performed for 2 min. The SSF was used 9 ACS Paragon Plus Environment


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either supplemented with salivary bacteria or not supplemented. SSF with bacteria was

175

obtained by re-suspending the pellet obtained after centrifugation 25, 26 (15 min, 9000 g) of 100

176

mL of saliva from a pool of 5 donors with 100 mL of SSF.

177

To emulate the gastric stage of the digestion, the pH was adjusted to 3 with 1 M HCl by using

178

a pH electrode coupled to a pH controller (Consort R301) and a Master Flex pump drive (Cole-

179

Parmer Instrument Company, LLC). Then, gastric simulated fluid (GSF) was added (1:1, w/w).

180

The gastric digestion was maintained for 2 hours. For the intestinal digestion, the pH value was

181

raised to 6.5 by addition of 1 M NaOH. Then, the simulated intestinal fluid (SIF) was added

182

(1:1, w/w). Incubation at 37 °C and continuous stirring were maintained for 2 h. Colonic

183

digestion was performed by adding anaerobic nutritional medium (1:1, v/v) and flushing the

184

reactors with N2 for 15 min to create anaerobic conditions. After flushing, fecal inoculum (20%,

185

w/v) diluted in anaerobic PBS (pool of 5 donors, the same as for the saliva pool) was added to

186

the vessels in a proportion of 1:10 (v/v). Information about the origin of the salivary and fecal

187

samples is showed in Table S4. Detailed methodology for inocula preparation is described in

188

method S5.

189

The pH was adjusted to 5.6-5.9 with 1 M HCl and maintained in this range by adding 0.5 M

190

HCl or 0.5 M NaOH. The system was flushed with N2 for 15 min more and incubated 24 h at

191

37°C and constant stirring.

192

Detailed composition of SSF, SGF, SSF, and nutritional medium for colonic fermentation is

193

described in Methods S6.

194

At the end of each of the four digestion step, samples were obtained and pH values recorded.

195

Samples were centrifuged 9509 g/10 min at 4 °C and the supernatant was filtered (Whatman® 10 ACS Paragon Plus Environment

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qualitative filter paper, Grade 1, 11 µm, Millipore, Belgium). Samples for bioaccessibility and

197

arsenic biotransformation assays were freeze-dried (Heto Powerdry PL3000 Thermo,

198

Denmark) for further analysis. We defined bioaccessible arsenic as the amount of arsenic

199

released from the matrix and which is soluble after the centrifugation and filtration steps. We

200

calculated the % of bioaccessible arsenic considering the total arsenic quantification. The

201

values above 100% may be most likely caused by the complexity of the experimental model,

202

combined human samples and food matrices in a complex mixture of gastrointestinal fluids.

203

The sampling, subsampling, and processing (e.g. freezing dry) could introduce an experimental

204

variation which causes the observed deviations.

205

Samples for cell culture assays were filter sterilized (0.22 µm; Millipore, Belgium), and pH

206

and osmolarity were adjusted to 7.2 ± 0.2 and 290 ± 15 mOsM/kg, respectively. All

207

gastrointestinal digestion stages containing salivary bacteria will be referred to as bacteria-

208

conditioned and those without salivary bacteria as non-bacteria conditioned. Control samples

209

will be considered as digestive fluids without food matrices.

210 211

Cell cultures

212

Caco-2 (ECACC 86010202) and HT29-MTX-E12 (ECACC 12040401) cells were obtained

213

from the European Collection of Authenticated Cell Cultures. Cell maintenance was carried

214

out routinely as described in Calatayud et al. 2011

215

passages 50 and 60.

216

Cell differentiation and the posterior tests were carried out in double chamber wells (Corning®

217

HTS Transwell®-24 well, pore size 0.4 µm; Costar Corp., NY, USA). The cells were seeded

20.

All the cultures were used between



Page 12 of 49

218

at a density of 7.5 x 104 cells/cm² in a proportion of 90/10 and 70/30 Caco-2/HT29-MTX for

219

resembling the small intestine and colon epithelium, respectively, and maintained with

220

Dulbecco's Modified Eagle's Medium - high glucose (4.5 g/L) (DMEM), supplemented with

221

10% (v/v) heat-inactivated Fetal Bovine Serum (iFBS, Greiner Bio-One, Wemmel, Belgium),

222

1%

223

penicillin/streptomycin (Life Technologies, Merelbeke, Belgium) until differentiation (15

224

days). Then, DMEM was removed and cells were washed twice with 0.2 mL of Hanks Balanced

225

Salt Solution (HBSS). The cell monolayer resembling the colon was covered by 50 µL of a

226

biosimilar mucus layer prepared as described in Boegh et al. 2014 21.

(v/v)

GlutaMAX™

(Gibco,

Life

Technologies

Europe

BV),

and

1%

227 228

Apparent permeability coefficient (Papp) of arsenic and arsenic cellular uptake

229

One mL of HBSS was added to the basolateral compartment and the filter-sterilized

230

supernatants from the small intestine and colonic digestion were diluted in HBSS (v/v) 1:2 (rice

231

samples, small intestine), 1:5 (rice samples, colon), 1:10 (nori and control samples, small

232

intestine), 1:20 (nori, mussel and control samples, colon), and added to the apical chambers

233

(0.2 mL).

234

The control condition consisted of the simulated digestion fluids (small intestine or colon), not

235

containing any dietary matrix, and in absence or presence of salivary bacteria, spiked with AsV

236

100 µg/L.

237

For permeability assays, samples from the basolateral compartment (0.5 mL) were obtained at

238

30, 60 and 120 min (small intestine) and at 60, 240, 1440 min (colon), and replaced with HBSS.

239

During the transport assays, cell monolayers were kept under stirring conditions (60 rpm) in a 12 ACS Paragon Plus Environment

Page 13 of 49


240

shaker (ROCKER 3D basic, IKA, Belgium), at 37° C, 90% humidity and 10% CO2. At the end

241

of the permeability assay (small intestine model: 2 hours; colon model: 24 hours), cell

242

monolayers were washed twice with HBSS. The apical and basal media and the cells were

243

recovered, digested with 65% HNO3/30% H2O2 (2:1 v/v) 90°C, 4h, in a proportion to the

244

sample of 1:1 (v/v), and filtered (0.45 µm, PTFE; Metrohm Belgium N.V.) before analysis.

245

Tests were evaluated independently at least in triplicate. Percentage of cell uptake and cell

246

uptake + transport (total uptake) was calculated with respect to the total arsenic content added

247

to the apical compartment. The Papp of arsenic was calculated as described in Calatayud et al.,

248

2010 22.

249

Assessment of the epithelial barrier function: TEER and Papp of LY

250

During the period of growth and differentiation, cell monolayer integrity was monitored every

251

2–3 days, measuring the transepithelial electrical resistance (TEER) with a Millicell®-ERS

252

(Merck KGaA, Darmstadt, Germany). The reported values were calculated as described by

253

Srinivasan et al., 2015 23. The cell monolayer was considered completely formed when stable

254

TEER values were obtained (≥ 80 Ω cm2). Monolayer integrity was also evaluated by

255

calculating the Papp of the paracellular transport marker Lucifer Yellow (LY) as described in

256

Calatayud et al., 2010

257

Microplate Reader, Molecular devices, Orleans, CA).

258

Results of TEER are expressed as a percentage of TEER after 2 hours (small intestine) or 24

259

hours (colon) of exposure to different digests, compared to the initial TEER values in the

260

individual wells.

22,

using a microplate fluorescence reader (Spectramax Gemini XS

261 13 ACS Paragon Plus Environment


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Mitochondrial metabolic activity assay

263

The cells were grown in Transwell inserts and exposed to the supernatants from the small

264

intestine and colonic digestion as previously described. After 2 hours (small intestine model)

265

or 24 hours (colon model) the cells were washed with phosphate buffered saline (PBS) (Gibco,

266

Belgium). Resazurin test (7-hydroxy- 3H-phenoxazin-3-one-10-oxide sodium salt) was

267

performed as described by Calatayud et al. 2013 27 and a Spectramax Gemini XS Microplate

268

Reader was used for quantification of resorufin (560Ex/590Em) in the apical media. The results

269

were expressed as percentages of resazurin reduction with respect to the fluorescence from

270

cells exposed to the simulated digestion fluids without food matrix.

271 272

Protein quantification

273

The cellular protein content was evaluated by the Bradford dye-binding method (BioRad,

274

Belgium), following the instructions of the manufacturer.

275

Statistical analysis

276

The statistical analysis was performed on SigmaPlot 13 software (Systat Software Inc., UK).

277

The significance level was set at 0.05. Normality of the data set was tested with the

278

Kolmogorov-Smirnov test. In case of normality, mean values of two different groups were

279

compared with an independent samples t-test. Significant differences between treatments were

280

tested with one way ANOVA in case of normality. Homogeneity of variances was tested with

281

the Modified Levene test. Depending on the outcome of the Levene test, Bonferroni or Dunnett

282

T3 were used as post hoc tests to determine p-values. In case of non-normal distributions,

283

differences were tested with non-parametric Mann-Whitney U test. Pearson Product Moment 14 ACS Paragon Plus Environment

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Correlation coefficient was calculated to assess the possible linear correlation between different

285

variables.

286 287

Results and discussion

288

Salivary bacteria significantly modify arsenic bioaccessibility dependent on food matrix

289

and digestion stage

290

Mussels

291

The total arsenic content found in the mussel sample (23.4 ± 3.0 mg/kg dry weight, dw) was

292

higher than previously reported values 9.15-17.48 µg g-1 dw

293

contained mainly arsenobetaine and arsenosugars (Table 1, Figure S3 and S4), which was the

294

expected arsenic species pattern based on many previous studies 29-31. High concentrations of

295

iAs (up to 5.8 mg kg-1 ww) and other organic arsenicals (e.g thio-arsenosugars) have also been

296

described occasionally in fresh and processed mussel samples 29, 32.

297

Arsenic bioaccessibility values for digested mussels in the absence of salivary bacteria ranged

298

from 23 to 54% (Figure 1). The presence of salivary bacteria significantly (p < 0.05) increased

299

the arsenic bioaccessibility in the oral (29 ± 0.3%), gastric (55 ± 3%), and colon (72 ± 9%)

300

reactors by a factor of 1.3, 1.6 and 1.7 (Figure 1). Independent of the effect of the salivary

301

bacteria, the high solubility of arsenic in the oral digestion (14 to 42%) indicates a significant

302

release from food at an early digestion stage. Our findings agree with Leufroy et al., 2012 who

303

demonstrated that the arsenic released by saliva represents at least half of the bioaccessible

304

arsenic in seafood certified reference materials and real seafood samples 33. Despite the fast

28.

The initial mussel sample



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305

transit time of the food in the oral cavity, high bioaccessibility values in the mouth could lead

306

to increased absorption in the proximal sections of the gastrointestinal tract.

307

Mussels contain collagenous molecules 34, which can be partially degraded by α-amylase. α-

308

Amylase is one of the principal enzymes of the saliva

309

glycosaminoglycan from various connective tissues 36. The removal of glycoproteic fraction of

310

the collagen is required for further collagenase digestion. We removed the host α-amylase

311

during the centrifugation process but previous research has reported α-amylase activity in

312

different bacterial strains from human and environmental samples 37, 38. The initial process of

313

the mussels digestion in the oral reactor may affect food matrix structure and further digestion

314

in the lower compartments. Moreover, recent research has shown that human salivary amylase

315

gene copy number impacts oral and gut microbiomes 35, supporting the hypothesis of a close

316

interplay between the oral cavity and gut microbiomes and the host.

317

It has been reported that mussel consumption could result in the provisional tolerable weekly

318

intake for iAs being exceeded 29. Despite the high arsenic concentration in the tested mussels,

319

we found the % of iAs to be quite low (0.43% of total arsenic). An increased release of

320

arsenobetaine in the oral cavity may thus cause a larger absorption in the small intestine, but

321

there are no known consequences for risk assessment up to now.

322

Nori seaweed

323

In our study, the total arsenic content in nori samples was 18.3 ± 2.9 mg/kg dw. The percentage

324

of total arsenic bioaccessible varied from 30 ± 2% (oral reactor, bacteria-conditioned) to 136

325

± 0.7% (colon reactor, non-conditioned) (Figure 1). Previous research reported in vitro total

326

arsenic bioaccessibiltiy in Porphyra spp. between 67-87% (raw) and 80-106% (cooked) 39, 40.

35

and that this enzyme releases acid


Page 17 of 49


327

For dialyzable arsenic in the red alga (Porphyra umbilicalis), however, García-Sartal et al. 33

328

observed much lower values of 17.0% (raw) and 15.3 % (cooked).

329

Interestingly, the arsenic bioaccessibility from nori was increased by salivary bacteria in the

330

stomach and small intestine from ~ 60 to ~ 100% (Figure 1).

331

Previous research has reported carbohydrate active enzymes in the human gut 41, 42, involved

332

in the agarolytic pathway 43 which allows for agarose saccharification into 3-O-β-D-galactose

333

(GAL) and 4-O-α-3,6-anhydro-L-galactose (AHG). Despite the bacterium containing this

334

specific enzymatic activity was only isolated in Japanese and Chinese individuals, it is possible

335

that this enzymatic activity was also present in the microbiomes of one or multiple donors of

336

this study. The degradation of complex polysaccharides from nori to GAL and AHG may cause

337

the release of arsenic in the form of inorganic arsenic.

338

Moreover, in the presence of salivary bacteria, the amount of iAs in the gastric, small intestine

339

and colon compartments in nori samples increased 8, 2 and 3 times respectively (Table 2). It

340

has been shown that iAs induces oxidative stress, inflammation, cell cycle alterations, changes

341

in protein expression in intestinal cells, and epithelial barrier impairment 27, 44, besides being a

342

well-recognized human carcinogen 45.

343

Previous data showed that conventional mice methylate and absorb iAs to the same extent as

344

germfree mice 46, thus gut microorganisms may have a negligible effect on biotranformation

345

and absorption of iAs in an in vivo mice model. However, the bioaccessible arsenic in the

346

lumen of the stomach, small intestine, and colon can be affected by the presence of

347

microorganisms. Taking into account the small intestine as the main site of arsenic absorption,



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348

the increase in bioaccessibility may result in higher internal exposure after nori intake, but also

349

in a higher local exposure of intestinal cells and the mucosal niche to different arsenic species.

350

Previous research has shown that As(III) induced erosion of bacterial biofilms adjacent to the

351

mucosal lining and changes in the diversity and abundance of morphologically distinct species

352

indicated changes in microbial community structure 47. The effect of other arsenical species in

353

the host-microbiome interface is still unknown.

354

Rice

355

Total arsenic content in the rice used in our studies was 0.22 ± 0.01 mg/kg dw. The percentage

356

of arsenic bioaccessible at different digestion stages varied from 14 ± 3%(oral reactor, bacteria

357

conditioned) to 117 ± 3% (colon rector, non-bacteria conditioned). Trenary et al. 2012 found

358

that the bioaccessible arsenic in cooked brown rice during synthetic gastrointestinal extraction

359

ranged from 58% to 64% 48; Laparra et al. 2005 found that the bioaccessible fraction accounted

360

for more than 90% of the total arsenic content of cooked whole grain rice after simulated

361

gastrointestinal digestion 49; and He et al. 2012 found the extractable arsenic ranged from 53%

362

to 102% after rice was treated with in vitro artificial gastrointestinal fluid 50.

363

In the presence of salivary bacteria along the gastrointestinal digestion, the % of arsenic

364

bioaccessible decreased from 36 ± 2% to 14 ± 3% (oral), and from 117 ± 3% to 89 ± 10%

365

(colon), a trend that was also observed in nori samples (Figure 1). Salivary bacteria did not

366

affect the As bioaccessibility in the gastric and small intestinal digestion. A possible

367

explanation for our findings is that iAs has chemical similarity to substrates of membrane

368

transporter proteins of bacterial cells. The uptake of iAs by aquaglyceroproteins (e.g. GlpF) 51

369

or phosphate transporters (e.g. Pit and Pst) has been previously described 52. The centrifugation 18 ACS Paragon Plus Environment

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370

of the samples (9509 g, 10 min) can remove the microorganisms containing arsenic from the

371

bioaccessible fraction, which results in lower bioaccessibility values. In a highly dense (> 108

372

viable cells/ml) and complex community as the saliva or fecal microbiome, the “trapping”

373

effect caused by the bacterial cells could be significant. To corroborate this hypothesis, a batch

374

test incubating 100 µg/L of arsenate with saliva and fecal samples was performed, and the

375

percentage of arsenic retention by the bacterial cells was found to be quite substantial, ranging

376

from 35-54% (Method S7; data not shown). Sun et al., 2012 also observed a significant drop

377

in arsenic bioaccessibility from rice in a simulated colon digestion model. Authors suggested

378

that this drop is probably due to the higher amount of organic matter that is introduced in the

379

colon suspension under the form of microbial biomass 53.

380

Other factors related to food sample preparation, as the cooking processes, may affect

381

bioaccessibility of arsenic. Laparra et al., 2004 that bioaccessible inorganic arsenic in raw

382

seaweed (54-67%) increased after cooking (78-84%)

383

values of arsenic species in raw and cooked seaweed 55. These both studies used boiling water

384

as the cooking method, which may cause a different effect on the food matrix than the roasting

385

process applied in this research. Zhuang et al., 2006 observed that cooking process of rice (30

386

min in water, 2:1 w/v ratio) reduced bioaccessibility of arsenic 56.

387

Moreover, Alava et al., 2013 showed that the particle size of rice had a major influence on As

388

extraction from the food matrix 57. In this research, the grinding process and the solid to volume

389

ratios might affect the As solubility from the food matrices.

390

Thus, the presence of salivary and colonic bacteria decreases the bioaccessible arsenic

391

(potentially available for intestinal absorption) in rice and nori, possibly acting as a

54.

Other study reported comparable



Page 20 of 49

54-56, 58

392

symbiotic/mutualistic protective mechanism against internal arsenic exposure.

393

disruption of the microbial ecosystems by antibiotic intake or infections could change the

394

behavior of arsenic in the gastrointestinal tract, and such an outcome would require further

395

research.

396

Individual microbial signatures of salivary and stool samples have previously been

397

demonstrated 59-61 but have not been considered in this study because of the use of a single spot

398

sample of pooled saliva and feces from 5 donors. The effect of the inter-individual variability

399

on arsenic bioaccessibility and speciation may require further research. Moreover, sample

400

processing may affect the salivary and fecal microorganisms, reducing the representativeness

401

of the microbiomes. Regarding the saliva, flow cytometry counts of viable cells before and

402

after the centrifugation steps gave similar results ( ~ 108 viable cells/mL). Fecal samples are

403

routinely used to investigate the intestinal microbiome and have been demonstrated to be a

404

useful proxy of distal colon microbiome

405

affect the viability of strictly anaerobic microorganisms.

62,

The

although preparation of the fecal inoculum can

406

407 408

Figure 1. Effect of salivary bacteria in arsenic bioaccessibility from food matrices. Bars

409

represent the percentage of arsenic bioaccessibility from mussels, nori and rice in the different

410

digestion steps [oral, stomach, small intestine (SI), and colon] in absence (white bars) or 20 ACS Paragon Plus Environment

Page 21 of 49


411

presence (grey bars) of salivary bacteria. The percentage of arsenic solubilized from the food

412

matrix was calculated with respect to the total arsenic content in the original sample (mean ±

413

standard deviation; n=2). Significant differences (p < 0.05) comparing the absence and

414

presence of salivary bacteria are marked by an asterisk (*).

415 416

Biotransformation of arsenic from food during the gastrointestinal passage

417

Arsenic speciation analyses were performed on extracts of the initial samples and on the fluid

418

from each of the gastrointestinal digestive compartment by HPLC/MS under both anionic and

419

cationic chromatographic conditions (Figure S3).

420

Mussels

421

The in vitro digestion did not transform the arsenobetaine present in the original sample; the

422

soluble arsenobetaine initially constituted 60% of total arsenic, and the values were 51-53%

423

for all three gastrointestinal digested fluids (gastric, small intestinal and colonic) for non-

424

bacteria and bacteria-conditioned treatments (Table 1). This result is consistent with metabolic

425

studies of arsenobetaine with mice

426

excreted mostly unchanged in the urine after oral intake.

427

The original pattern of arsenosugars slightly changed in the gastrointestinal digestive

428

compartments with the oxo-form generally becoming more dominant (Figure 2A). We did find

429

small amounts of thioxo arsenosugars in the small intestinal and colonic stages, as well as in

430

the initial mussels but not in the gastric digestion stage (Table 1; Figure S3 and S4). There was

431

no clear change in the ratio between the glycerol arsenosugar and the phosphorylated

432

arsenosugar going from initial mussel extract to the colon fluid.

63

and humans

64,

which showed that arsenobetaine is



Page 22 of 49

433

Few studies have assessed the toxicity and toxicokinetics of arsenosugars. To our knowledge,

434

the only study in vivo showed that high doses (20-50 mg synthetic oxo-arsenosugar-glycerol/kg

435

body weight) induced blood and brain oxidative stress, DNA damage and neurobehavioral

436

impairments

437

genotoxic to Caco-2 cells 66.

438

Thioxo-AsSug-Glycerol was detected by Molin et al. 2012 in human urine after blue mussel

439

consumption, which accounted for about 1.5% of the total excreted arsenic species

440

metabolism of arsenosugars from mussels might occur in the gastrointestinal tract due to

441

biotransformation by the human tract microbiota. Previous research supports these findings, as

442

the anaerobic microbiota of mouse cecum material can produce sulfur-containing analogs of

443

arsenosugars

444

found to be twice as high as its oxo- analog, the pre-absorptive metabolism in the digestive

445

tract can change the toxicokinetics and toxicodynamics of arsenic, but also affect the

446

gastrointestinal barrier functionality 66.

447

The minor arsenicals found in mussel (e.g. DMA, TETRA) were also present at comparable

448

levels in the digest fluids, a result suggesting that these compounds, like arsenobetaine, were

449

not greatly affected by the gastrointestinal digestive process or by the presence of salivary

450

bacteria.

65.

Few in vitro studies showed that oxo-arsenosugars were non-cytotoxic or

68, 69.

67.

The

Because of the in vitro intestinal permeability of thio-AsSugar-Gly was

451 452

Nori seaweed

453

By far, the major compounds in initial samples were the thioxo (62% of total arsenic) and oxo

454

(30%) forms of the phosphorylated arsenosugar (Table 2; Figure S3 and S6). This result is 22 ACS Paragon Plus Environment

Page 23 of 49


70,

455

consistent with previous studies on nori

456

phosphorylated) arsenosugar-328 was present in trace amounts only, whereas it usually occurs

457

at levels comparable to the phosphorylated compound. For example, Li et al. 2003 found that

458

the distribution of Oxo-AsSug-Phosphorylglycerol and Oxo-AsSug-Glycerol (Figure S6) in

459

five red alga samples (Porphyra) obtained in Beijing varied from 13%-68% and 19%-86%,

460

respectively

461

dominant compound. Although thioxo forms of arsenosugars are commonly reported in algae

462

71,

463

in our experiments was briefly roasted before gastrointestinal digestion, and this treatment may

464

have influenced the observed speciation pattern.

465

Quite different from the outcome with mussels, in nori samples we observed marked changes

466

in the ratios of both the oxo/thioxo forms and the glycerol/phosphoryl arsenosugars, which also

467

depended on the absence or presence of oral bacteria (Table 2, Figure 2B). In the gastric fluids,

468

the oxo form predominated - a clear change from the initial nori, which contained mainly the

469

thioxo arsenosugar. In the colon, the phosphorylated arsenosugars originally present in the nori

470

were completly degraded to the glycerol arsenosugar, which was then mainly present as the

471

thioxo form. This large change was not evident after the gastric and small intestinal stages, and

472

thus had been elicited only at the colon stage. In the colon environment, microbial sulfate

473

reduction to hydrogen sulfide is a common process which can trigger the formation of

474

thioarsenosugars 13, 68. Conklin et al. 2006 reported that the anaerobic microflora from mouse

475

gastrointestinal tract can readily convert an oxo-arsenosugar to its thioxo analogue

31.

but differs in two ways. First, the related (de-

Second, the thioxo, rather than the oxo form of the arsenosugar was the

they are usually minor compounds compared to the oxo analogs. We note that the nori used

68.

In



Page 24 of 49

476

contrast, Chavez-Capilla et al. 2016 showed that oxo-arsenosugars were not changed when

477

seaweed was exposed to physiologically based extraction 72.

478

The presence or absence of salivary bacteria did not appear to play a significant role in the

479

observed transformations, except for the small intestine fluid where the thioxo form was

480

present only in trace amounts in the bacteria-conditioned treatments.

481

Sulphate reducing bacteria (SRB) can produce H2S, which is necessary to induce As thiolation

482

73.

483

Desulfovibrio fairfieldensis, Desulfovibrio desulfuricans and Raoultella ornithinolytica as

484

SRB inhabitants of the oral cavity 74. It is feasible that the saliva samples used in this research

485

also contain SRB.

486

Because of the high oral bioavailability of arsenosugars and the cellular toxicity of their

487

metabolites

488

Experiments with the Caco-2 intestinal barrier model, which mimics human intestinal

489

absorption, indicated that the thioxo-arsenosugars have higher bioavailability and toxicity as

490

compared to the oxo-arsenosugars

491

samples during the gastrointestinal digestive process suggest that the composition of the food

492

matrix might be a significant factor in the transformation of arsenosugars. Previous research

493

shows that diet has a significant impact in shaping the gut microbiome, even after short term

494

(24 h) of dietary alterations 77. The same research showed that foodborne microbes can survive

495

the transit through the digestive system and be present in a metabolically active form in the

496

distal gut. This phenomenon described in vivo, can occur in the in vitro system, affecting the

497

microbial communities in the colonic reactor and, in consequence, the metabolic potency

Heggendorn et al., 2013 have detected SRB in human saliva samples and identified

75, 76,

the possible risks from ingesting arsenosugars cannot be fully excluded.

16, 18.

The observed differences between mussel and nori


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498

towards arsenic. Lu et al., 2014 observed that the metabolic profile of As in urine significantly

499

differ in dysbiotic mice induced by IL-10 knockout, compared to wild type, supporting the

500

relevance of gut microbiome impact on As biotransformations 78. Transformations between the

501

various forms of arsenosugars under simulated gut conditions could have direct implications

502

for toxicity studies and risk assessment.

503

In our in vitro system, both the digestion stage and the food source influenced the

504

transformation between the two major arsenosugars and between their oxo- and thioxo forms

505

(Figure 2C).

506 507

Figure 2. Panel A and B: transformation of the arsenosugars in initial samples and

508

gastrointestinal digested fluids of mussel and nori. The bar graphs are colour-coded to

509

represent the two arsenosugars (blue and green) as their oxo (dark shading) and thioxo (light 25 ACS Paragon Plus Environment


Page 26 of 49

510

shading) forms. Panel C: overview of the interrelationships between the four major

511

arsenosugars found in this study.

512 513

Rice

514

The rice in our study contained mainly iAs (ca 70% of extractable arsenic) and DMA (8%);

515

about 20% was not extractable and hence could not be assigned by our method (Table 3; Figure

516

S3). There were no clear differences between the non-bacteria or bacteria-conditioned

517

treatments for any of the arsenic species. A study by Sun et al. 2012 found similar results for

518

iAs bioaccessibility in cooked white rice where the soluble iAs stayed between 77% and 87%

519

in the stomach, intestine and colon treatments 79. Contrarily to this study, Sun et al., 2012 found

520

biotransformation of iAs from rice to MMA(III) in a simulated colonic fermentation.

521

Differences in rice composition, microbial inoculum, composition of the media used for the

522

digestion can be some of the factors causing discrepancies between studies.

523

The in vitro model applied in this research allows the investigation of arsenic transformations

524

occurring in specific simulated gastrointestinal digestive processes, and when combined with

525

HPLC/mass spectrometry it can provide a more complete profile of the bioaccessibility of the

526

various arsenic species found in food. Our results indicate that salivary bacteria could increase

527

the soluble arsenic species, especially in the gastric digestion, and show that arsenosugars

528

readily interchange between their oxo and thioxo forms during the different stages of the

529

gastrointestinal simulation. This signals a possible modification in the risk estimation of arsenic

530

exposure when considering salivary microorganisms in vitro. The system is ideal to further

531

investigate the effects of salivary treatment, and the possible risk of arsenosugar-rich food 26 ACS Paragon Plus Environment

Page 27 of 49


532

consumption, also considering the interindividual variability of the human digestive

533

microbiome.



535

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Table 1 Arsenic species [arsenic in μg; % (arsenic μg of each species / sum of species)] in initial and gastrointestinal digested mussel samples Food matrix Stage Salivary bacteria Unit AB Oxo-AsSug-Glycerol Unknown(1) AC Unknown(2) TETRA DMA Oxo-AsSug-Phosphorylglycerol As(V) Unknown(3) Thioxo-AsSug-Glycerol Unknown(4) Thioxo-AsSugPhosphorylglycerol Sum of species

Initial mussel (244 µg As)

Gastrointestinal digested mussel Gastric

µg 61 10 2 1 2 2 2 8 1 1 4 2

% 60 9 2 1 2 2 2 8 1 1 4 2

Without μg % 40 53 11 15 1 1 1 1 2 3 3 4 2 2 11 15 1 1 1 1

Salivary and gut microbiomes play a significant role in in vitro oral

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