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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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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
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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
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Graphical abstract
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Introduction
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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,
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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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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
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(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).
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Arsenic quantification
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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
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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
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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
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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
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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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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
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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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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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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
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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
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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
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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
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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
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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
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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
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consumption, also considering the interindividual variability of the human digestive
533
microbiome.
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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