In Vitro Reduction of Arsenic Bioavailability Using Dietary Strategies

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In vitro Reduction of Arsenic Bioavailability Using Dietary Strategies M. J. Clemente, V. Devesa, and D. Velez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05234 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 20, 2017

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Journal of Agricultural and Food Chemistry

In vitro Reduction of Arsenic Bioavailability Using Dietary Strategies

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Clemente, M.J., Devesa V., Vélez, D.*

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Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), C/. Agustín Escardino

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7, 46980 Paterna (Valencia), Spain.

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* To whom correspondence should be addressed (telephone (+34) 963 900 022; fax (+34)

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963 636 301; e-mail: [email protected]).

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1 ACS Paragon Plus Environment


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ABSTRACT

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The main route of human exposure to inorganic arsenic (As) is through consumption of

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food and water. Continued exposure to inorganic As [As(III) and As(V)] may cause a

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variety of diseases, including various types of cancer. Removal of As from these sources is

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complex, especially for food. One way to decrease As exposure could be by reducing

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intestinal absorption of it. The aim of this study is to seek dietary strategies (pure

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compounds, extracts, or supplements) that are capable of reducing the amount of As that is

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absorbed and reaches the systemic circulation. Standard solutions of As(III) and As(V) and

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bioaccessible fractions of food samples with or without the dietary strategies to be tested

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were added to colon-derived human cells (NCM460 and HT-29MTX) to determine the

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apparent permeability (Papp) of As. Results show that transport across the intestinal

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monolayers is substantial, and passage of As(III) (Papp = 4.2 × 10–5 cm/s) is greater than that

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of As(V) (Papp = 2.4 × 10–5 cm/s). Some of the treatments used (iron species, cysteine,

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grape extract) significantly reduce transport of both inorganic As standards across the

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intestinal monolayer, thus decreasing absorption of them. In food samples, the effect of the

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dietary compounds on inorganic As bioavailability was also observed, especially in the

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cases of curcumin and cysteine. Compounds that proved effective in these in vitro assays

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could be the basis for intervention strategies aimed at reducing As toxicity in chronically

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exposed populations or regular consumers of food products with high As contents.

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Keywords: rice, seaweed, arsenic, bioavailability reduction, dietary strategies, human

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intestinal cells.


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INTRODUCTION

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Drinking water and food are the main paths by which inorganic arsenic [As(III) and

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As(V)] enters the organism. These species of arsenic and the methylated trivalent

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metabolites formed during their metabolism are the most toxic forms of As.1 Chronic

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exposure to inorganic As through drinking water, which affects millions of people in

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various parts of the world, is linked to greater prevalence of certain types of cancer,

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cardiovascular and cerebrovascular pathologies, type 2 diabetes, and non-cancerous skin

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lesions.2

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The presence of inorganic As in food is well characterized. Foods such as rice, hijiki

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seaweed (Hizikia fusiforme), and some seafood products, especially bivalves, present the

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highest concentrations of inorganic species. Noteworthy quantities have been found in

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Hizikia fusiforme (up to 88 mg/kg dry weight),3 and for this reason food agencies in

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countries such as Canada and the UK recommend avoiding its consumption.4,5 Because of

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the high consumption of rice, it is one of the foods that contributes most to intake of

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inorganic As, including intake by the infant population. The European Food Safety

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Authority (EFSA) stated that consumption of three portions (90 g/day) of rice-based infant

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food could represent an important source of inorganic As (1.6–2.0 µg/kg body weight per

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day),6 close to the benchmark dose proposed by the Joint FAO/WHO Expert Committee on

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Food Additives (BMDL0.5: 2-7 µg/kg b.w. per day)7 and EFSA (BMDL 0.1: 0.3-8 µg/kg

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b.w. per day)6. Furthermore, in countries with high concentrations of inorganic As in water,

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the foods ingested have high concentrations resulting from the cooking process.8 Therefore,

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in addition to drinking water, there are food matrices that represent a food safety problem

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because of their inorganic As content.



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Studies have been conducted to determine the mechanisms of accumulation of inorganic

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As in some of these matrices, especially in rice, and the conditions that favor this process. It

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is known that in rice the uptake of As from the soil is carried out by transporters in the

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aquaporin family, specifically a transporter for silicic acid (Lsi1).9 Although silicon has not

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been recognized as an essential element for higher plants from a physiological viewpoint, it

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is required for healthy growth and high production,10 so mutants deficient in Lsi1 are not a

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suitable solution to achieve the aim of reducing As accumulation. Attempts to change

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growing conditions have also been investigated. Minimization management strategies to

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reduce As accumulation in rice include more aerobic cultivation practices, avoiding

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flooding. However, rice is typically grown under flooded conditions, which helps to control

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the build-up of pathogens and can also increase nutrient availability,11 and therefore it is not

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easy to modify the growing conditions. The difficulty of altering production conditions

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could also apply in the case of other food matrices with high levels of inorganic As.

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An alternative way of avoiding high exposure through food and drinking water is to

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reduce the quantity of the toxin that reaches the systemic circulation after ingestion

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(bioavailability). Some studies indicate lower bioavailability of As when it forms part of a

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food matrix,12 which indirectly suggests that food components may modulate absorption.

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The process of absorption may be affected by the contaminant binding with compounds

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that reduce its solubility or its passage through the epithelium, or by competition between

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the contaminant and dietary compounds for the mechanisms of entry into the epithelium.13

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The aim of this study is to seek dietary strategies (pure compounds, plant extracts, and

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supplements) that are capable of reducing the amount of As that is absorbed and reaches the

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systemic circulation. For this purpose, standard solutions of As(III) and As(V) and the

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bioaccessible fractions of food samples (rice and seaweed), with or without the dietary 4 ACS Paragon Plus Environment

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strategies to be tested, were added to cultures of NCM-460 and HT29-MTX colon-derived

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human cells.

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MATERIALS AND METHODS

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Arsenic Standard Solutions. The standard of As(V) (1000 mg/L, As2O5) was obtained

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from Merck (VWR, Spain). The standard of As(III) (1000 mg/L) was prepared by

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dissolving 1.320 g of As2O3 (Riedel de Haën, Spain) in 25 mL of 20% (m/v) KOH

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(Panreac, Spain), neutralizing with 20% (v/v) H2SO4 (Merck, Spain), and diluting to 1 liter

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with 1% (v/v) H2SO4.

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Bioaccessible Food Fractions. The samples used for the bioaccessibility assays were

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the seaweed Hizikia fusiforme (hijiki) and white rice. Seaweed samples were boiled in

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deionized water for 20 min (sample/water ratio: 1/4, w/w) following the instructions of the

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manufacturer, and the water that remained after the process was discarded. Rice samples

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were boiled in a standard solution of 1 mg/L of As(V) until dryness (sample/water ratio:

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1/4). No additional ingredients were used in any of the processes.

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The foods were subjected to gastrointestinal digestion following the protocol described

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by Jadán-Piedra et al.,14 with minor modifications. Ten grams of cooked food was weighed

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in an Erlenmeyer and 90 mL of deionized water was added. The pH of the mixture was

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adjusted to 2 with 6 mol/L HCl (Merck, Spain), and a solution of pepsin (0.1 g of

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pepsin/mL prepared in 0.1 mol/L HCl) was added in order to obtain 2 mg of pepsin/g of

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sample. After incubation for 2 h at 37 °C with continuous shaking (120 rpm), the digest

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was subjected to the intestinal stage. The pH was adjusted to 6.5 with 1 mol/L NH4CO3, 5 ACS Paragon Plus Environment


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and a solution of pancreatin and bile extract (0.004 g/mL of pancreatin and 0.025 g/mL of

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bile extract in 0.1 mol/L NH4CO3) was added to obtain a final concentration of 0.25 mg of

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pancreatin/g of sample and 1.5 mg of bile extract/g of sample. The mixture was incubated

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for 2 h at 37 °C with constant shaking (120 rpm).

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After the digestion, the samples were centrifuged at 10000 rpm (30 min, 4 °C), and the

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soluble (bioaccessible) fractions obtained were treated for the assay in cell culture. The

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fractions were heated for 4 min at 100 °C to inhibit sample proteases. Glucose (Sigma, 5

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mM final concentration) was then added to facilitate cell viability. Water or NaCl (Panreac)

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was added to adjust the osmolarity to 300 ± 25 mOsm/kg, using a freezing point osmometer

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(Löser Type 15 Automatic Micro-Osmometer, Löser Messtechnik, Germany).

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Cell Culture Conditions. The normal human colon mucosal epithelial cell line

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NCM460 was acquired from INCELL Corporation (USA). The cells were maintained in 75

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cm2 flasks to which 10 mL of supplemented Dulbecco’s Modified Eagle Medium (DMEM)

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with high glucose (4.5 g/L) and glutamine (0.87 g/L) was added at pH 7.2. The DMEM was

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supplemented with 10% (v/v) of fetal bovine serum (FBS), 10 mM N-2-

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hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 100 U/mL of penicillin, 0.1

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mg/mL of streptomycin, and 0.0025 mg/L of amphotericin B (NCM-DMEMc). The assays

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were performed with cultures between passages 14 to 25.

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The HT29-MTX cell line was kindly provided by Dr. Thécla Lesuffleur (Institut

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National de la Santé et de la Recherche Médicale, INSERM UMR S 938, Paris, France) and

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used between passages 22 and 34. Cell maintenance was done in DMEM high glucose,

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supplemented with 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL of

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penicillin, 0.1 mg/mL of streptomycin, and 0.0025 mg/L of amphotericin B (HT-DMEMc). 6 ACS Paragon Plus Environment

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The cells were incubated at 37 °C in an atmosphere with 95% relative humidity and a

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CO2 flow of 5%. The medium was changed every 3 days. When the cell monolayer reached

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80% confluence, the cells were detached with a solution of trypsin (0.5 g/L) and EDTA

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(ethylenediaminetetraacetic acid, 0.2 g/L) and reseeded at a density of 5–6 104 cells/cm².

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All the reagents used were obtained from HyClone (Fisher, Spain).

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In vitro Bioavailability Assays. The assays were performed in 12-well plates with

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polyester membrane inserts (12 mm diameter, pore size 0.4 µm, Transwell, Corning,

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Cultek, Spain). In this system the cells are seeded on the porous membrane of the insert that

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separates the well into two compartments: apical (upper) and basolateral (lower). The cells

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were seeded (1.8 105 cells/cm²) on the apical side to produce monocultures of NCM-460

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and co-cultures of NCM-460/HT29-MTX (80/20). Then 0.5 mL of NCM-DMEMc (for

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NCM-460) or HT-DMEMc (for co-cultures) was added to the apical chamber, and 1.5 mL

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of the same medium was added to the basolateral chamber. The cells were maintained in a

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humidified atmosphere of 5% CO2 at 37 °C, with a change of medium every 2–3 days until

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11 days post-seeding.

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The pure dietary compounds assayed (n = 25; Table 1) were selected on the basis of

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presenting characteristics that might favor reduction of transport of inorganic As across the

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cell monolayer for various reasons: a) formation of complexes with the inorganic forms of

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As; b) competition for the same transport mechanisms; and c) modification of opening of

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cell junctions. The plant extracts and supplements used (n = 7; Table 1) were selected on

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the basis of being important sources of the pure dietary components that have proved most

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effective in reducing the transport of As in this study.



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To evaluate the influence of the dietary strategies on As bioavailability, they were

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initially added to aqueous standard solutions of As(V) and As(III) (0.75 mg/L; 1 µM)

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prepared in Hanks’ Balanced Salt Solution (HBSS) (HyClone) with 10 mM HEPES, and

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then the mixture was added (0.5 mL) to the apical compartment of the well. In the

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basolateral compartment, 1.5 mL of HBSS-10 mM HEPES was placed, without As or

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dietary components. At stipulated times (30, 45, 60, and 120 minutes), an aliquot (1 mL)

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was taken from the basolateral compartment and replaced with an equal volume of HBSS-

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10 mM HEPES. After the assay, the cell monolayers were washed with phosphate buffered

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saline solution (PBS, HyClone), and detached with trypsin/EDTA solution. For co-cultures,

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prior to trypsinization, the mucus layer was recovered by treatment of the monolayers with

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0.5 mL of 10 mM of N-acetylcysteine (NAC, Sigma) at 37 °C for 30 min with constant

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shaking.

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Total As concentration was determined in the aliquots taken at each time, and in the cell

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monolayer, the mucus layer, and the apical medium collected at the end of the experiment.

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The apparent permeability coefficients (Papp) were calculated using Equation 1.

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Papp = (dC/dt) (Vr/AC0)

(Eq. 1)

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where:

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dC/dt is the flow (µg/s) determined by the linear slope of the equation that governs the

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variation in the As species concentrations, corrected for dilution, versus time.

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Vr is the volume of the receptor compartment (1.5 mL).

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A is the surface occupied by the cell monolayer (1.12 cm2).

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C0 is the As concentration added initially to the apical compartment (µg/L).

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The compounds that were effective in reducing As bioavailability in aqueous standards

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were tested in foods. For the transport assay using the inactivated bioaccessible food 8 ACS Paragon Plus Environment

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fraction (section 2.2), 0.5 mL of the fraction was added to the apical compartment and 1.5

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mL of HBSS-10 mM HEPES was added to the basolateral compartment. Papp was

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determined following the protocol described above.

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Evaluation of Cell Monolayer Integrity. For NCM-460 monocultures, the integrity of

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the monolayer after the assays was verified by visualization. The cells were washed with

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PBS, fixed in 4% paraformaldehyde (v/v in PBS) and permeabilized with 0.5% of Triton-

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X100 (Merck) for 5 min. Afterwards, the cells were stained with 10% crystal violet (Sigma)

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in 10% methanol for 15 min and washed twice with water. After being allowed to dry

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upside down, the stained cell monolayers were viewed under a Nikon Eclipse 90i

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microscope (Nikon Corporation, Japan). The assays were considered valid when continuous

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regular monolayers without apparent signs of damage were observed.

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For NCM-460/HT29-MTX co-cultures, the integrity was monitored by evaluating the

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Papp of Lucifer Yellow (LY), a fluorescent compound that is transported mainly by a

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paracellular route across intercellular junctions. LY was added at a concentration of 100

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µM to the apical compartment at the same time as the treatments. The fluorescence of the

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LY transported to the basolateral side was measured with a fluorescence microplate reader

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(PolarSTAR OPTIMA, BMG-Labtech, Germany) at excitation/emission wavelengths of

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485/520 nm. The assays were considered valid when the Papp values of LY were ≤1 10–5.

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At the end of each assay the number of viable cells was quantified, using the trypan blue exclusion technique (Trypan Blue Solution, 0.4%, Sigma).

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Determination of Total and Inorganic Arsenic. The analysis of total As

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concentrations in food samples, bioaccessible fractions, media, cells, and mucus was 9 ACS Paragon Plus Environment


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performed by flow injection-hydride generation-atomic absorption spectrometry (FI-HG-

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AAS) after a dry ashing step.15 Arsenic quantification was performed with an AAS (model

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3300, Perkin-Elmer, Spain) equipped with an autosampler (AS-90, Perkin-Elmer), a FI-HG

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system (FIAS-400, Perkin-Elmer), and an electrothermally heated quartz cell. A rice flour

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reference material (SRM1568a), obtained from the National Institute of Standards and

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Technology (NIST), with a certified As concentration of 0.29 ± 0.03 mg/kg was used for

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quality control of the method.

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Inorganic As was analyzed in the food and bioaccessible fractions. The analysis was

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performed by acid digestion, solvent extraction, and detection by FI-HG-AAS.15 Rice flour

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SRM 1568a was analyzed with each series of samples, and the quality assurance/quality

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control of the measurement was checked by comparing the values found (0.105 ± 0.003

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µg/g dw) with the range reported in the literature for this material (0.080–0.110 µg/g dw).16

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Statistical Analysis. All assays were performed at least in triplicate in independent

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cultures. The results were statistically analyzed by one-factor analysis of variance

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(ANOVA) with Tukey’s HSD post hoc multiple comparison or using the Student t-test

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(SigmaPlot, version 12.0). Differences were considered significant for p < 0.05.

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RESULTS

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Apparent Permeability Coefficients (Papp) of As(III) and As(V) Standard Solutions

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in NCM-460 and NCM-460/HT29-MTX Cultures. Table 2 shows the apparent

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permeability coefficients and the retention in cells and mucus (in the case of the co-

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cultures) of the inorganic forms of As. In both cell models, Papp was greater for As(III) than 10 ACS Paragon Plus Environment

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for As(V) (1.6 times in monocultures and 1.3 times in co-cultures), a similar trend to that

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observed by other authors in Caco-2 monocultures and Caco-2/HT29-MTX co-cultures.17-19

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Cellular accumulation was low (

In Vitro Reduction of Arsenic Bioavailability Using Dietary Strategies

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