Arsenic in Rice Bran Products: In Vitro

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Food Safety and Toxicology

Arsenic in Rice Bran Products: In Vitro Oral Bioaccessibility, Arsenic Transformation by Human Gut Microbiota, and Human Health Risk Assessment Naiyi Yin, Pengfei Wang, Yan Li, Huili Du, Xiaochen Chen, Guo-Xin Sun, and Yanshan Cui J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02008 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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Journal of Agricultural and Food Chemistry

Arsenic in Rice Bran Products: In Vitro Oral Bioaccessibility, Arsenic Transformation by Human Gut Microbiota, and Human Health Risk Assessment Naiyi Yin,a,b Pengfei Wang,a,b Yan Li,a,b Huili Du,a,b Xiaochen Chen,c Guoxin Sun,b Yanshan Cui*a,b a

College of Resources and Environment, University of Chinese Academy of Sciences,

Beijing 101408, People’s Republic of China b

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,

Beijing 100085, People’s Republic of China c

College of Environment and Resources, Fuzhou University, Fujian, Fuzhou 350116,

People's Republic of China

Corresponding author: Yanshan Cui Yanshan Cui Tel: +86 10 69672968 Fax: +86 10 69672968 E-mail: [email protected] 1

ACS Paragon Plus Environment


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ABSTRACT (147 words)

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Despite rice consumption, rice bran as a byproduct of rice milling contains

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higher arsenic (As). The present study evaluated the metabolic potency of in vitro

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cultured human colon microbiota toward As from 5 rice bran products with

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0.471-1.491 mg As/kg. Arsenic bioaccessibility ranged from 52.8% to 78.8% in the

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gastric phase and a 1.2-fold increase (66.0-95.8%) was observed upon the small

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intestinal phase. Subsequently, a significant decline of As bioaccessibility

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(11.3-63.6%) and a high methylation percentage of 18.5-79.8% were found in the

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colon phase. The predominant As species in the solid phase was always As(V)

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(49.6-63.4%) and As-thiolate complexes increased by 10% at the end of colon

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incubation. Human gut microbiota could induce As bioaccessibility lowering and As

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transformation in rice bran, which illustrated the importance of food-bound As

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metabolism in the human body. This will result in better understanding of health

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implications associated with As exposures.

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KEYWORDS: rice bran, arsenic, speciation, bioaccessibility, colon, Simulator of the

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Human Intestinal Microbial Ecosystem, health risk.

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INTRODUCTION

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Arsenic (As) exposure for humans, a toxic contaminant distributed in the

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environment and food has been associated with a variety of adverse health effects

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including cancers, neurological and cardiovascular effects.1 Typically, human

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exposure to As includes inhalation, ingestion, and dermal contact.2 Ingestion exposure

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can occur via the consumption of contaminated food or water, and inadvertent

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non-dietary ingestion of soil or dust. For several countries, the predominant staple

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food is rice, and rice intake was recognized as a source of As exposure beyond

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drinking water.3 Therefore, rice consumption is an important contributor to the dietary

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As intake.

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Rice bran is a byproduct of the rice milling process containing the vitamins,

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antioxidants, and important dietary fiber, and could be a new “super food”.4-7

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Stabilized rice bran is an emerging food ingredient and has been used in food aid

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programs for malnourished children.4,8 The concentrations of total As and inorganic

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As in brown rice from various countries exceeded those of the corresponding polished

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rice, and inorganic As species were predominantly present in both rice and bran.8-11

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Sun et al. (2008)8 determined As concentrations in five rice bran products, ranging

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from 0.71 to 1.98 mg/kg, with inorganic As of 0.48-1.88 mg/kg. In Thailand,

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concentrations of total and inorganic As in polished rice were 85.5-262.0 and 41.7 to

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156 μg/kg, lower than those found in the corresponding brown rice with 116-300 and

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73.1-193 μg/kg, respectively.10 Mean concentrations of total and inorganic As in rice 3



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bran from China reached 412 and 404 μg/kg, being about 5 times greater than those

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values in rice.11 Arsenic speciation is a key issue in terms of bioavailability and

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toxicity. Speciation and distribution of As in rice grains have been determined based

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on some advanced techniques of high-performance liquid chromatography coupled

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with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and X-ray

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absorption near edge spectroscopy (XANES). In comparison with endosperm, total As

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concentrations are also higher in rice bran, and inorganic As was predominant As

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species with a proportion of dimethylarsinic acid [DMA(V)] for rice bran products

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and obtained in milling process.8,12 The safety of rice bran consumption is of

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significant health concern.

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Recently, human health risk assessment associated with As exposures, especially

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from As-contaminated rice, has drawn increasing attention.13-16 Diet is considered one

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primary exposure pathway for inorganic arsenic (iAs), and mean iAs exposures from

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rice were 1.4 μg/d for U.S. population and 2.8 μg/d for population from other

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regions.17 Bioavailable As is one critical parameter to better estimate rice-As exposure,

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being the fraction of As that can be absorbed into the systemic circulation. In vivo

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studies using swine and mice, determined As bioavailability in rice samples and

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indicated that accurate risk characterization depended on As bioavailability especially

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(iAs) in rice.18,19 The bioavailability of As and other metals for foods has been

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evaluated through Caco-2 cells.15,20 Absorption of different As species in rice varied,

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and the absorption percentage (28%) of DMA(V) was lower.21 In addition, As 4


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bioavailability can be predicted by in vitro bioaccessibility method, as a fast and

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simple risk assessment method. Bioaccessible As is the fraction of As that is soluble

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in the gastrointestinal environment of humans and available for absorption into the

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circulatory system. In vivo bioaccessibility evaluated through mass balance with a

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controlled dietary experiment over 10 days has demonstrated that, ingested As from

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cooked rice could be excreted in urine (mostly DMA) with the percentages of 58-69%

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for two volunteers.14 Large variations in As bioaccessibility ranging from 43-96% in

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the gastrointestinal phases through in vitro methods, have been reported for raw or

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cooked rice.16,22 Bioaccessible As species were predominantly inorganic forms being

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63-99% including arsenite [As(III)] and arsenate [As(V)],15,23 whereas DMA(V) was

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dominant described previously.21

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Gut microbiota are important as “second brain” for the host health, and there is

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much current interest in the interaction between gut microbiota with xenobiotics.24

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The metabolic potency of human gut microbiota has been demonstrated toward

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metal(loid)s (e.g. arsenic, selenium, and antimony) and organic pollutants (polycyclic

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aromatic hydrocarbons).25-27 The in vitro exploration associated with dietary intake

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from rice, indicated the effect of human gut microbiota on As bioaccessibility and

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speciation.13,21,23 The bioacessible As fraction was decreased to about 30-60% by the

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end of colon incubation, when comparing the values of the small intestinal phase. The

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important observation was speciation change of As induced by human gut microbiota;

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consequently, it has been found to be the occurrence of monomethylarsonous acid 5



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[MMA(III)] and monomethylmonothioarsonic acid [MMMTA(V)].

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The research on rice bran products has received very little attention and the

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metabolic potency of in vitro cultured human colon microbiota toward As from rice

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bran products is unknown. In the present study, we investigated metabolic potency of

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human gut microbiota toward As from rice bran products in different countries. Using

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in vitro method, we examined the differences in As bioaccessibility between five rice

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bran products and between the gastrointestinal phases. Daily As intake was calculated

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using As bioaccessibility to assess As exposure associated with the consumption of

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rice bran. Speciation analysis of As was evaluated to understand As transformation by

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human gut microbiota and As distribution post colon incubation. It could uncover the

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importance of As metabolism when evaluating risks upon dietary As exposure.

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MATERIALS AND METHODS

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Sample Preparation. A total of 5 rice bran products were obtained from four

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different countries (including China, USA, Germany, and Japan). Rice bran samples

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were oven-dried at 80 °C until constant weight. All samples were analyzed in

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triplicate. The analysis of total As concentrations and As speciation in rice bran

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samples was carried out by microwave digestion.8,28 In the digestion process, the

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blank samples and the rice flour standard reference material SRM 1568b from NIST

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(Gaithersburg, MD, USA) were included to ensure the accuracy and recovery.

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Human Gut Microbiota of SHIME. The in vitro colon microbial community 6


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was cultured in a dynamic SHIME reactor (Simulator of the Human Intestinal

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Microbial Ecosystem), including five compartments simulating the stomach, small

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intestine, ascending colon, transverse colon and descending colon. Fresh fecal

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microorganisms were inoculated into three colon compartments of the SHIME reactor,

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from one 28-year-old healthy male volunteer without antibiotic treatment for one year.

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Feeding was provided in the Supporting Information. The temperature (37 °C) and

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anaerobic environment was maintained, and the pH (5.6-5.9 for the ascending colon,

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6.15-6.4 for the transverse colon, and 6.7-6.9 for the descending colon) and

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continuous stirring were automatically controlled. When community composition and

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microbial fermentation activity were in excellent agreement with previous description

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of the SHIME,26-27 the microbial community was stable after four weeks of adaptation

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and used for in vitro experiments.

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In Vitro Bioaccessibility. The oral As bioaccessibility from the five rice bran

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were investigated using a combination of physiologically based extraction test (PBET,

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gastric and small intestinal phases) combined with SHIME (colon phase) as

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previously described.29 In brief, 3.0 g of the rice bran sample was added into 30 mL of

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the simulated fluid (30 mL) at a solid/solution (s/s) ratio of 1:10 for the gastric (pH

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1.5 and 1 h) and small intestinal (pH 7.0 and 4 h) phases. Following the small

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intestinal phase, the simulated fluid was transferred into a 100-mL anaerobic serum

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bottle without loss and was mixed with the colon suspension (30 mL) at a ratio (s/s)

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of 1:20 from the descending colon compartment of the SHIME. Subsequently, each 7



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bottle was capped with a butyl rubber stopper and immediately flushed with nitrogen

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gas for 30 min to ensure anaerobic conditions. The duration of the colon phase was 48

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h. Samples taken at the end of each phase were centrifuged (4000 g) for 20 min. The

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supernatant was filtered (0.22 μm) and stored at -20 °C until analysis. The in vitro

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experiment was shaken (150 rpm) at 37 °C and conducted in triplicate.

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Liquid-Phase Arsenic. Deionized water (Millipore, Billerica, MA, USA) was

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prepared from a Milli-Q reference system (Millipore, Billerica, MA, USA). All glass

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and plastic-ware were cleaned by soaking in 10% (v/v) nitric acid for a minimum of

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24 h, followed by thorough rinsing with Milli-Q water.

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Total As concentrations were determined by inductively coupled plasma-mass

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spectrometry (ICP-MS, 7500a; Agilent Technologies, Santa Clara, CA, USA), and the

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recoveries were 95.5-104.6% (mean value 99.9%) with a standard solution of 20 μg/L.

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Arsenic speciation analysis in rice bran extraction solution, the small intestinal and

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colon digests was accomplished by high-performance liquid chromatography coupled

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with ICP-MS (HPLC-ICP-MS) as described previously with the detection limit of 0.1

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μg/L.27,30 The PRP-X100 anion-exchange column (250 mm × 4.1 mm, 10 μm), used

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in separation system was from Hamilton with a pre-column (11.2 mm, 12-20 μm).

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The mobile phase was a mixture of 10 mM (NH4)2HPO4 and 10 mM NH4NO3 at pH

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6.2. The flow rate was 1 mL/min. Arsenic species were identified by comparing their

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retention times with four standards [As(III), MMA(V), DMA(V), and As(V)] as

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quantified by external calibration curves with peak areas. In this study, the 8


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bioaccessible As was the sum of all As species in the filtrates (0.22 μm) observed

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chromatographically.

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XANES Analysis. All absorption spectra were collected at the XAS beamline

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1W1B (multi-pole wiggler) at the Beijing Synchrotron Radiation Facility (BSRF) in

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Beijing, China. The electron storage ring operated at 2.5 GeV with a ring current of

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200 mA. Energy calibration of the XANES spectra was accomplished by

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simultaneous measurement of the Au metal foil reference.31 Rice bran samples and

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residual solids at the end of in vitro experiments were freeze-dried in vacuum and

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preserved in oxygen-free vials for XAS analysis. The XANES spectra were collected

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in fluorescence mode with a solid-state 19-element Ge detector. Reference As

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compounds were recorded in fluorescence mode using a Lytle detector: arsenite

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(NaAsO2), arsenate (Na2HAsO4·7H2O), MMA(V) (monomethylarsonic acid), and

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DMA(V) (dimethylarsinic acid). The spectrum for As(III) glutathione [As(GSH)3]

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was freshly synthesized as described in detail,31 and prepared in 30% glycerol to

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avoid crystal formation (Figure S1). XANES spectra were normalized and analyzed

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using linear combination fitting (LCF) using Athena software (Demeter 0.9.20).32 The

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normalized spectra were fitted in the range -30 to +60 eV from the absorption edge, a

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combination of up to four standards was allowed, and the standards were not

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constrained to use a single E0 shift. The weighting factors were constrained to

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between 0 and 1, and the weights were not forced to sum to 1. It should be

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emphasized here that the LCF approach is not free of uncertainties and limitations. 9



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The As(GSH)3 was used as a surrogate for As-phytochelatin and As-metallothionein

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complexes, but it cannot be excluded that if the spectra of these complexes were used

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in the LCF procedure, different results could have been obtained.12,28

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Daily Arsenic Intake Calculation. Daily As intake (DI, μg As/kg bw/d) was

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calculated in adults with body weight of 60 kg and rice bran consumption rate of 30 g

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per day.5 Daily intake for rice bran products was calculated based on total As

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concentration (DItotal) and in vitro oral bioaccessibility (DIbioaccessible) as follows: C × CR × BA BW C × CR × BA DIbioaccessible = BW DItotal =

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where, C represents total As concentration (μg/kg) in rice bran, CR represents the

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daily consumption rate of rice bran (30 g/d), BW represents body weight (60 kg) of

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adults, and BA represents the As bioaccessibility measured using in vitro method.

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Statistical Analysis. One-way analysis of variance (ANOVA) was applied to

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detect possible differences in total As concentrations, As bioaccessibility, and

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concentrations of As species in the small intestinal and colon digests. A significant

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level of P < 0.01 was adopted for all comparisons. Statistical analysis was performed

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using SPSS software (version 20.0, IBM, USA).

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RESULTS

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Arsenic in Rice Bran Products. Total As concentrations in rice bran samples

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were in the range of 0.471-1.491 mg/kg (Table 1). One rice bran product from Japan 10


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is produced in Aichi-ken (JA1), and another with 0.471 mg As/kg is from organic rice

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in Kumamoto-ken (JA2). Most samples exceeded the Chinese maximum contaminant

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level which is set at 0.5 mg/kg As in cereals and 0.2 mg/kg inorganic As in rice.33,34

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There is also a limit of 1 mg/kg As for cereals such as rice in Australia. In the rice

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flour NIST 1568b, As concentration was 0.282 ± 0.018 mg/kg with the recovery of 99

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± 6% (n = 5); subsequently, As species was analyzed after extraction with 2% v/v

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nitric acid and was in agreement with certified mass fraction values (Table S1). The

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concentrations of different As species were observed in rice bran products (Table S2),

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and the percentage of inorganic As species ranged from 85-95%.

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Arsenic Bioaccessibility and Health Risk Assessment. Statistical analysis

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shows that there are significant differences in As bioaccessibility between the

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gastrointestinal phases and between rice bran samples (P < 0.01). The bioaccessibility

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of As for 5 rice bran samples ranged from 52.8% to 78.8% with the mean value for

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66.9% in the gastric phase (Table 1). Upon 4 h of the small intestinal digestion, an

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increase was observed in As bioaccessibility with 66.0-95.8% (80.0% of mean value),

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about 1.2 times higher than that of the gastric phase. Subsequently, the bioaccessible

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As fraction significantly decreased to 11.3-63.6% (31.3% of mean value) upon 48 h of

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the colon incubation, being only 40% of that in the small intestinal phase.

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As Speciation in the Small Intestinal and Colon Digests. This study consisted

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of As speciation analysis in the small intestinal and colon digests after the

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gastrointestinal incubation of the five rice bran samples. The As(V) was 11



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predominantly present in the small intestinal digests from 45.9 μg/L to 89.7 μg/L with

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the percentages of 86.6-93.3%. Whereas, inorganic As was the only As species in the

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small intestinal digest of rice bran JA2 (Figure 1).

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After the 48‑hr incubation with active fecal microbiota, the concentrations of As

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species varied greatly in the colon digests. The methylation percentage for colon

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digests of rice bran JA1 (79.8%) and JA2 (52.0%) exceeded that of inorganic As, with

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JA1 displaying MMA(V) of 24.0 μg/L and DMA(V) of 2.7 μg/L, and JA2 displaying

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only MMA(V) of 8.0 μg/L. The concentrations of only DMA(V) of 2.3 and 2.1 μg/L

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was less than that of As(III) (5.9 and 2.7 μg/L) and As(V) (3.6 and 2.4 μg/L) for rice

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bran US and GE, respectively (P < 0.01). The only As species were As(III) (3.1 μg/L)

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and As(V) (2.6 μg/L) in the colon digest of rice bran CH as well.

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Incubation with sterilized fecal microbiota resulted in significant As(V)

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reduction to As(III), probably due to the highly reducing conditions. The As(III)

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concentrations reached 13.1-28.4 μg/L (55.5-71.4%), being 5-11 folds higher than that

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with active fecal microbiota. In contrast, lower methylation percentages of 4.4-9.5%

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were observed displaying 1.1 μg/L MMA(V) only for GE and DMA(V) (1.8-2.3 μg/L)

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for all rice bran samples except JA2 where no methylated As species were detected in

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the colon digests.

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Arsenic Speciation in the Solid Phase. Synchrotron-based measurement of As

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speciation by XANES confirmed the change in the proportion of As(III), As(III)-GSH,

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As (V), MMA, and DMA for the rice bran and residual solids (Figure 2, Figure S2 12


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and Table S3). XANES results showed that As(V) with 40-65% was predominantly

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present in the rice bran, and the percentages of As(III)-GSH and organic As species

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ranged from 11-26% and 26-36%, respectively.

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At the end of 48-h incubation with active fecal microbiota, As(III)-GSH

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significantly increased up to 18-37%, whereas the percentage of organic As species

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has been reduced to 5-14% and the proportion of DMA went up. Finally, in the rice

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bran products the dominant As species was similar to As(V). In addition, the rice bran

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JA2 has not been included in the XANES analysis due to the low total As

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concentration. It should be noted in this study that, As(III)-thiolate complexes are

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probably in the form of As-glutathionine, As-phytochelatin, or As-metallothionein

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which are present naturally in rice grains.12 As(GSH)3 is simply an analogue of these

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species that is often used as the As-thiolate standard spectra.9

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Human Health Risk Assessment. Daily As intake was calculated for rice bran

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products (Table 2), assuming daily rice bran consumption rate of 30 g for an adult of

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60 kg body weight. On the basis of As bioaccessibility in the small intestinal phase,

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Daily As intake of 0.23-0.48 μg As/kg bw/d, contributed to 7.5-16.1% of the

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benchmark dose at 3 μg/kg bw/d.35 However, when taking colon As bioaccessibility

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into consideration, the daily As intake and the corresponding contribution were

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reduced to 0.06-0.33 μg As kg-1 bw d-1 and 1.9-11.1%, respectively. Rice-based

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consumers need realize that consumption of rice bran is an important contributor to

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dietary As intake and increases potential health risk. 13



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DISCUSSION

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In this study, for the first time, the metabolic potency of human colon microbiota

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toward As from rice bran products was investigated. Significant differences (P < 0.01)

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in As bioaccessibility were observed between rice bran samples and between the

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gastrointestinal phases. Arsenic bioaccessibility was higher in the small intestinal

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phase, and human gut microbiota resulted in the significant reduction of As

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bioaccessibility upon the colon incubation. Speciation analysis of solid-liquid phase

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indicate that generally, the methylation percentage for colon digests was higher

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ranging from 18.5-79.8%, and large proportion of As(III)-GSH on the residues was

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known as the presence of As(III)-thiolate complexes.

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Total As concentrations of rice bran samples were higher than the values of rice.

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Our data of 0.471-1.491 mg/kg were comparable to only available studies which

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reported As concentrations of 0.16-1.98 mg/kg in rice bran products from different

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countries.8,10,11 However, As concentrations in bran for freshly milled samples were

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higher, ranging from 1.63-6.24 mg/kg.8,12 When considering As concentrations for

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most of rice bran samples exceeded the recommended levels,36 it came out the

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urgency of our study to evaluate risks upon dietary As exposure. Xue et al. (2010)37

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estimated dietary iAs exposure is 0.05 µg/kg/d with 17% from rice, being

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approximately two times higher than mean iAs exposure from drinking water. For U.S.

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population, mean iAs exposures were 4.2 μg/d and 1.4 μg/d from drinking water and 14


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rice, respectively.17 About 0.5-3.7 µg iAs/kg/d could be delivered to consumers

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through rice ingestion.38 The daily consumption of rice was 350-432 g dry weight/d

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for an average body weight of 60 kg,18,19,38 being much higher than the rate of rice

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bran (30 g/d).5 On the basis of As speciation in small intestine and colon, daily iAs

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intake of 0.43 μg/kg bw/d, contributed to about 15% of the benchmark dose at 3

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μg/kg bw/d,35 which poses a potential risk to consumers. Arsenic metabolism by

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human gut microbiota should be taken into account human health risk assessment

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associated with As exposures.

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The finding of As bioaccessibility upon gastrointestinal digestion was reported

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for rice bran products. Arsenic bioaccessibility in the small intestinal phase was

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consistently higher than the corresponding gastric and colon bioaccessibility values

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(Table 1) obtained with the combination of PBET and SHIME method. This is

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consistent with other in vitro exploration associated with rice intake, reporting that

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colon bioaccessibility went down to 30-60% in comparison to the data of small

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intestinal phase.13,23 Alava et al. (2015)21 found that Asian type diet (fiber rich)

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induced a great drop in colon As bioaccessibility in rice compared with a Western

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type diet (fat and protein rich). Significant differences in As bioaccessibility between

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rice bran products from different countries and between the gastric, small intestinal

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and colon phases were observed probably due to the large variation in nutrients. The

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composition of rice bran has been reported previously,5 and rice bran is rich in

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proteins, fiber, and vitamins, which can influence As bioaccessibility in rice and 15



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soils.21,39,40 Previous in vitro findings about rice reported that differences in As

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bioaccessibility were observed between the rice types, and the As bioaccessibility was

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higher for the rice with high fibre content.13 It should be further investigated what

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constituents in rice bran play a role in As release. In addition, the main As species was

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As(V) of 86.6-93.3% in the small intestinal digests probably due to iAs species

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predominantly in rice bran products, according with earlier studies,15,23 whereas

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DMA(V) was dominant described previously due to high levels of DMA(V) in rice.21

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The acidic environment of the gastric digests would induce the release of As from rice

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by inactivation of proteins.41 The digestive enzymes of pancreatin and bile salts could

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play a key role in the release of protein-bound As, resulting in the higher As

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bioaccessibility in the small intestinal phase.41 The bioaccessibility of As in rice and

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soil is also affected through the metabolism of the colon microbiota.13,27 In addition,

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the difference in As bioaccessibility might be attributed to rice types of total As

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concentrations and the origin.22

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The present study demonstrates the metabolic potency of human gut microbiota

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toward rice bran associated As. The iAs species was more easily released from rice

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than DMA(V) during gastrointestinal digestion.21 MMA(V) with high concentrations

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in colon digests of JA1 and JA2 was due to the methylation of As(III). After

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incubation of rice bran with active fecal microbiota, bioaccessible As fraction

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significantly decreased but As(V) was largely transformed to As(III) (5.7-48.3%) and

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organic As species (18.5-79.8% of bioaccessible As). Except for four common As 16


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species, MMA(III), and even DMA(III) were not stable and have been detected in the

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colon digests.13,23 The significant reduction of As(V) and lower methylation were

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found after incubation with sterilized fecal microbiota, probably because of the highly

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reducing conditions. It also gave an evidence that these compounds of colon digest

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could only result in As reduction but the methylation should be due to human gut

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microbiota. Arsenic bioavailability is highly dependent on its speciation. Through in

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vivo studies using animals, As bioavailability (89%) in rice dominated by inorganic

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As far exceeded the value (33%) from rice with mainly DMA(V).18 However, relative

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bioavailability of inorganic As in cooked rice ranging from 26.2% to 49.5% (mean

325

39.9%) was higher than DMA(V) with 27.0% (13.2-53.6%), which indicated the

326

importance of inorganic As was higher especially resulting in accurate risk assessment

327

associated with rice consumption.19 In vivo bioaccessibility study revealed that DMA

328

was predominantly present in urinary excretion (58-69%) after a 10-day dietary

329

experiment with cooked rice.14

330

The significant finding of As speciation on the residues made clear of As

331

distribution by the end of colon incubation. XANES measurement confirmed that

332

about half of total As was As(V) as main As species pre and post the experiment.

333

Interestingly, As(GSH)3 as the standard spectra of As-thiolate complexes after colon

334

incubation was 31.5%, higher than 19.9% for original rice bran products (Table S3).

335

The formation of thiolated arsenicals could be resulted from the thiolation of MMA(V)

336

by human gut microbiota and the role of sulfate-reducing bacteria has been 17



337

demonstrated in the production pathway of MMMTA(V).21,42 Higher sulphur playing

338

a great role in As thiolation was distributed in the external region of the endosperm

339

being part of rice bran,12 and these thiolated arsenicals could be easily immobilized by

340

nutrient component of rice bran. The proportions of MMA(V) and DMA(V) were

341

relatively small, and their decline could be also an explanation for an increase of

342

thiolated arsenicals. Ingested As was mostly accumulated in most organs as DMA and

343

finally excreted via feces and urine.43 Speciation analysis including XANES

344

technique is essential when revealing As transformation and translocation, and further

345

evaluating health risks associated with exposure to As from rice consumption.

346

The metabolic potency of in vitro cultured human gut microbiota toward As in

347

rice bran has been evaluated. Significant differences in As bioaccessibility were

348

observed. We found higher As bioaccessibility in the small intestinal phase and a

349

significant decline in colon bioaccessibility. The As-thiolate complexes increased

350

from As thiolation and were absorbed on the solid phase. Human gut microbiota

351

lowered As bioaccessibility and induced As transformation in rice bran. The present

352

study has two limitations that may be improved to better evaluate As exposure from

353

rice bran consumption in the future. First, more data should be needed to determine

354

the bioaccessibility and speciation of arsenic using in vitro methods for a larger

355

sample size of rice bran. Second, in vivo study using animal models need to be

356

developed to determine arsenic bioavailability in rice bran and to examine the relation

357

between in vitro bioaccessibility and in vivo bioavailability. 18


Page 18 of 33

Page 19 of 33


358 359

AUTHOR INFORMATION

360

* (Y.S.C) [email protected]

361

ORCID

362

Yanshan Cui: 0000-0002-7805-1567

363

Funding

364

This work was supported by the National Natural Science Foundation of China (No.

365

21637002), the project of National Postdoctoral Program for Innovative Talents

366

funded by China Postdoctoral Science Foundation (No. BX20180299), and the China

367

Postdoctoral Science Foundation (No. 2018M641453).

368

Notes

369

The authors declare no conflict of interest.

370 371

ACKNOWLEDGMENT

372

The authors thank the Beijing Synchrotron Radiation Facility (BSRF) for the valuable

373

beamtime. We thank Dr. Lirong Zheng for technical support of XANES analysis in

374

Institute of High Energy Physics, Chinese Academy of Sciences.

375 376

Supporting Information

377

Table S1. Linear combination fitting (LCF)-XANES analysis of rice bran (CH, US

378

GE and JA1) and the corresponding residual solids at the end of colon incubation 19



379

(CHC, USC, GEC and JA1C) yielding information on As speciation.

380

Figure S1. 1H NMR spectrum of a mixed solution of AsO33- and GSH (Reduced

381

glutathione).

382

Figure S2. Normalized XANES spectra of standard compounds.

383 384

REFERENCES

385

(1) Carlin, D. J.; Naujokas, M. F.; Bradham, K. D.; Cowden, J.; Heacock, M.;

386

Henry, H. F.; Lee, J. S.; Thomas, D. J.; Thompson, C.; Tokar, E. J.; Waalkes, M. P.;

387

Birnbaum, L. S.; Suk, W. A. Arsenic and environmental health: state of the science

388

and future research opportunities. Environ. Health Perspect. 2016, 124, 890-899. DOI:

389

10.1289/ehp.1510209.

390

(2) United States Environmental Protection Agency (U.S. EPA). Exposure Factors

391

Handbook. Washington, D.C., 2011. https://www.epa.gov/expobox/about- exposure-

392

factors-handbook.

393

(3) Meharg, A. A.; Williams, P. N.; Adomako, E.; Lawgali, Y. Y.; Deacon, D.;

394

Villada, A.; Cambell, R. C. J.; Sun, G.; Zhu Y. G.; Feldmann, J.; Raab, A.; Zhao, F. J.;

395

Islam, R.; Hossain, S.; Yanai, J. Geographical variation in total and inorganic arsenic

396

content of polished (white) rice. Environ. Sci. Technol. 2009, 43, 1612-7. DOI:

397

10.1021/es802612a.

398 399

(4) Brahic, C. High levels of arsenic in food-aid rice drinks. New Phytol. 2008, 199, 7. DOI: 10.1016/S0262-4079(08)62149-1. 20


Page 20 of 33

Page 21 of 33


400

(5) Friedman, M. Rice brans, rice bran oils, and rice hulls: Composition, food and

401

industrial uses, and bioactivities in humans, animals, and cells. J. Agric. Food Chem.

402

2013, 61, 10626-10641. DOI: 10.1021/jf403635v.

403

(6) Natural Superfoods for Optimal Nutrition, Weight Loss & Nutrition.

404

http://naturalsuperfoodsblog.com/category/natural-superfood-products/stabilized-rice-

405

bran/.

406 407

(7) Prakash, J. Rice bran proteins: Properties and food uses. Crit. Rev. Food Technol. 1996, 36, 537-552. DOI: 10.1080/10408399609527738.

408

(8) Sun, G. X.; Williams, P. N.; Carey, A. M.; Zhu, Y. G.; Deacon, C.; Raab, A.;

409

Feldmann, J.; Islam, R. M.; Meharg, A. A. Inorganic arsenic in rice bran and its

410

products are an order of magnitude higher than in bulk grain. Environ. Sci. Technol.

411

2008, 42, 7542-7546. DOI: 10.1021/es801238p.

412

(9) Meharg, A. A.; Lombi, E.; Williams, P. N.; Scheckel, K. G.; Feldmann, J.; Raab,

413

A.; Zhu, Y, G.; Islam, R. Speciation and localization of arsenic in white and brown

414

rice grains. Environ. Sci. Technol. 2008, 42, 1051-1057. DOI: 10.1021/es702212p.

415

(10) Ruangwises, S.; Saipan, P.; Tengjaroenkul, B.; Ruangwises, N. Total and

416

inorganic arsenic in rice and rice bran purchased in Thailand. J. Food Prot. 2012, 75,

417

771-774. DOI: 10.4315/0362-028X.JFP-11-494.

418

(11) Dai, S.; Yang, H.; Mao, X.; Qiu, J.; Liu, Q.; Wang, F.; Wang, M. Evaluation of

419

arsenate content of rice and rice bran purchased from local markets in the People's

420

Republic

of

China.

J.

Food

Prot.

2014,

21


77,

665-669.

DOI:


421

Page 22 of 33

10.4315/0362-028x.jfp-13-344.

422

(12) Lombi, E.; Scheckel, K. G.; Pallon, J.; Carey, A. M.; Zhu, Y. G.; Meharg, A. A.

423

Speciation and distribution of arsenic and localization of nutrients in rice grains. New

424

Phytol. 2009, 184, 193-201. DOI: 10.1111/j.1469-8137.2009.02912.x.

425

(13) Alava, P.; Tack, F.; Du Laing, G.; Van de Wiele, T. Arsenic undergoes

426

significant speciation changes upon incubation of contaminated rice with human

427

colon

428

10.1016/j.jhazmat.2012.05.042.

microbiota.

J.

Hazard.

Mater.

2013,

262,

1237-1244.

DOI:

429

(14) He, Y.; Zheng Y. Assessment of in vivo bioaccessibility of arsenic in dietary

430

rice by a mass balance approach. Sci. Total Environ. 2010, 408, 1430-1436. DOI:

431

10.1016/j.scitotenv.2009.12.043.

432

(15) Laparra, J. M.; Velez, D.; Barbera, R.; Farre, R.; Montoro, R. Bioavailability

433

of inorganic arsenic in cooked rice: Practical aspects for human health risk

434

assessments. J. Agric. Food Chem. 2005, 53, 8829-8833. DOI: 10.1021/jf051365b.

435

(16) Zhuang, P.; Zhang, C. S.; Li, Y. W.; Zou, B.; Mo, H.; Wu, K. J.; Wu, J. T.; Li,

436

Z. A. Assessment of influences of cooking on cadmium and arsenic bioaccessibility in

437

rice, using an in vitro physiologically-based extraction test. Food Chem. 2016, 213,

438

206-214. DOI: 10.1016/j.foodchem.2016.06.066.

439

(17) Mantha, M.; Yeary, E.; Trent, J.; Creed, P. A.; Kubachka, K.; Hanley, T.;

440

Shockey, N.; Heitkemper, D.; Caruso, J.; Xue, J.; Rice, G.; Wymer, L.; Creed, J. T.

441

Estimating inorganic arsenic exposure from U.S. rice and total water intakes. Environ. 22


Page 23 of 33

442


Health Perspect. 2017, 125, 057005. DOI: 10.1289/EHP418.

443

(18) Juhasz, A. L.; Smith, E.; Weber, J.; Rees, M.; Rofe, A.; Kuchel, T.; Sansom, L.;

444

Naidu, R. In vivo asseasment of arsenic bioavailability in rice and its significance for

445

human health risk assessment. Environ. Health Perspect. 2006, 114, 1826-1831. DOI:

446

10.1289/ehp.9322.

447

(19) Li, H. B.; Li, J.; Zhao, D.; Li, C.; Wang, X. J.; Sun, H. J.; Juhasz, A. L.; Ma, L.

448

Q. Arsenic relative bioavailability in rice using a mouse arsenic urinary excretion

449

bioassay and its application to assess human health risk. Environ. Sci. Technol. 2017,

450

51, 4689-4696. DOI: 10.1021/acs.est.7b00495.

451

(20) Cai, X. L.; Chen, X. C.; Yin, N. Y.; Du, H. L.; Sun, G. X.; Wang, L. H.; Xu, Y.

452

D.; Chen, Y. Q.; Cui, Y. S. Estimation of the bioaccessibility and bioavailability of Fe,

453

Mn, Cu, and Zn in Chinese vegetables using the in vitro digestion/Caco-2 cell model:

454

the influence of gut microbiota. Food Funct. 2017, 8, 4592-4600. DOI:

455

10.1039/c7fo01348e.

456

(21) Alava, P.; Du Laing, G.; Tack, F.; De Ryck, T.; Van de Wiele, T. Westernized

457

diets lower arsenic gastrointestinal bioaccessibility but increase microbial arsenic

458

speciation changes in the colon. Chemosphere 2015, 119, 757-762. DOI:

459

10.1016/j.chemosphere.2014.08.010.

460

(22) Althobiti, R. A.; Sadiq, N. W.; Beauchemin, D. Realistic risk assessment of

461

arsenic

in

rice.

Food

462

10.1016/j.foodchem.2018.03.015.

Chem.

2018,

23


257,

230-236.

DOI:


463

(23) Sun, G. X.; Van de Wiele, T.; Alava, P.; Tack, F.; Du Laing, G. Arsenic in

464

cooked rice: Effect of chemical, enzymatic and microbial processes on

465

bioaccessibility and speciation in the human gastrointestinal tract. Environ. Pollut.

466

2012, 162, 241-246. DOI: 10.1016/j.envpol.2011.11.021.

467

(24) Koppel, N.; Maini Rekdal, V.; Balskus, E. P. Chemical transformation of

468

xenobiotics by the human gut microbiota. Science 2017, 356, 2770. DOI:

469

10.1126/science.aag2770.

470

(25) Diaz-Bone, R. A.; Van de Wiele, T. Biovolatilization of metal(loid)s by

471

intestinal microorganisms in the simulator of the human intestinal microbial

472

ecosystem. Environ. Sci. Technol. 2009, 43, 5249-5256. DOI: 10.1021/es900544c.

473

(26) Van de Wiele, T.; Vanhaecke, L.; Boeckaert, C.; Peru, K.; Headley, J.;

474

Verstraete, W.; Siciliano, S. Human colon microbiota transform polycyclic aromatic

475

hydrocarbons to estrogenic metabolites. Environ. Health Perspect. 2005, 113, 6-10.

476

DOI: 10.1289/ehp.7259.

477

(27) Yin, N. Y.; Zhang, Z. N.; Cai, X. L.; Du, H. L.; Sun, G. X.; Cui, Y. S. An in

478

vitro method to assess soil arsenic metabolism by human gut microbiota: Arsenic

479

speciation and distribution. Environ. Sci. Technol. 2015, 49, 10675-10681. DOI:

480

10.1021/acs.est.5b03046.

481

(28) Maher, W.; Foster, S.; Krikowa, F. Measurement of inorganic arsenic species

482

in rice after nitric acid extraction by HPLC-ICPMS: Verification using XANES.

483

Environ. Sci. Technol. 2013, 47 (11), 5821-5827. DOI: 10.1021/es304299v. 24


Page 24 of 33

Page 25 of 33


484

(29) Cai, X. L.; Chen, X. C.; Yin, N. Y.; Du, H. L.; Sun, G. X.; Wang, L. H.; Xu, Y.

485

D.; Chen Y. Q.; Cui, Y. S. Estimation of the bioaccessibility and bioavailability of Fe,

486

Mn, Cu, and Zn in Chinese vegetables using the in vitro digestion/Caco-2 cell model:

487

the influence of gut microbiota. Food Funct. 2017, 8, 4592-4600. DOI:

488

10.1039/c7fo01348e.

489

(30) Zhu, Y. G.; Sun, G. X.; Lei, M.; Teng, M.; Liu, Y. X.; Chen, N. C.; Wang, L.

490

H.; Carey, A. M.; Deacon, C.; Raab, A.; Meharg, A. A.; Williams, P. N. High

491

percentage inorganic arsenic content of mining impacted and nonimpacted Chinese

492

rice. Environ. Sci. Technol. 2008, 42, 5008-5013. DOI: 10.1021/es8001103.

493

(31) Smith, P. G.; Koch, I.; Gordon, R. A.; Mandoli, D. F.; Chapman, B. D.; Reimer,

494

K. J. X-ray absorption near-edge structure analysis of arsenic species for application

495

to biological environmental samples. Environ. Sci. Technol. 2005, 39, 248-254. DOI:

496

10.1021/es049358b.

497

(32) Ravel, B.; Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis

498

for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron. Radiat. 2005, 12,

499

537-541. DOI 10.1107/S0909049505012719.

500 501 502 503 504

(33) Australia and New Zealand Food Standards Code, Standard 1.4.1. Contaminants and Natural Toxicants, 2016. (34) China Food and Drug Administration. Maximum Levels in Contaminants in Food, GB 2762-2017. (35) World Health Organization (WHO). Evaluations of Certain Contaminants in 25



Joint

FAO/WHO

Expert

Committee

on

Page 26 of 33

505

Food.

Food

Additives,

2011.

506

http://apps.who.nt/food-additives-contaminants-jecfa-database/chemical.aspx?chemI

507

D=1863.

508

(36) Islam, S.; Rahman, M. M.; Rahman, M. A.; Naidu, R. Inorganic arsenic in rice

509

and rice-based diets: Health risk assessment. Food Control 2017, 82, 196-202. DOI:

510

10.1016/j.foodcont.2017.06.030.

511

(37) Xue, J.; Zartarian, V.; Wang, S. W.; Liu, S. V.; Georgopoulos, P. Probabilistic

512

modeling of dietary arsenic exposure and dose and evaluation with 2003-2004

513

NHANES

514

10.1289/ehp.0901205.

data.

Environ.

Health

Perspect.

2010,

118,

345-350.

DOI:

515

(38) Islam, S.; Rahman, M. M.; Duan, L.; Islam, M. R.; Kuchel, T.; Naidu, R.

516

Variation in arsenic bioavailability in rice genotypes using swine model: An animal

517

study.

518

10.1016/j.scitotenv.2017.04.215.

Sci.

Total

Environ.

2017,

599-600,

324-331.

DOI:

519

(39) Wang, P. F.; Yin, N. Y.; Cai, X. L.; Du, H. L.; Li, Z. J.; Sun, G. X.; Cui, Y. S.

520

Nutritional status affects the bioaccessibility and speciation of arsenic from soils in a

521

simulator of the human intestinal microbial ecosystem. Sci. Total Environ. 2018, 644,

522

815-821. doi.org/10.1016/j.scitotenv.2018.07.003.

523 524 525

(40) Yokoyama, W. Nutritional properties of rice and rice bran. In Rice Chemistry and Technology; Champagne, E. T., Ed.; AACC: St. Paul, MN, 2004; pp 595-609. (41) Goodman, B. E. Insights into digestion and absorption of major nutrients in 26


Page 27 of 33

526


humans. Adv. Physiol. Educ. 2010, 34, 44-53. DOI: 10.1152/advan.00094.2009.

527

(42) Rubin, S. S. C. D.; Alava, P.; Zekker, I.; Du Laing, G.; Van de Wiele, T.

528

Arsenic thiolation and the role of sulfate-reducing bacteria from the human intestinal

529

tract. Environ. Health Persp. 2014, 122, 817-822. DOI 10.1289/ehp.1307759.

530

(43) Lewchalermvong, K.; Rangkadilok, N.; Nookabkaew, S.; Suriyo, T.;

531

Satayavivad, J. Arsenic speciation and accumulation in selected organs after oral

532

administration of rice extracts in Wistar rats. J. Agric. Food Chem. 2018, 66,

533

3199-3209. DOI: 10.1021/acs.jafc.7b05746.

27



Figure captions Figure 1. Concentrations (μg/L) of As species in the small intestinal digests (I) and colon digests with active (C) and sterilized (CS) colon microbiota. Figure 2. Normalized XANES spectra of rice bran (CH, US GE and JA1) and the corresponding residual solids at the end of colon incubation (CHC, USC, GEC and JA1C).

28


Page 28 of 33

Page 29 of 33


Table 1. Total As in Rice Bran Products and As Bioaccessibility in the Gastric, Small Intestinal and Colon Phases (n = 3). Rice Bran

Total As

G-BA (%)

I-BA (%)

C-BA (%)

(mg/kg) China (CH)

1.001 ± 0.055b

52.8 ± 3.4dB

66.0 ± 0.6bA

11.3 ± 1.7dC

USA (US)

1.065 ± 0.042b

78.8 ± 1.5aB

82.9 ± 1.8aA

22.1 ± 1.5cC

Germany (GE)

0.973 ± 0.110b

77.9 ± 1.9aA

90.4 ± 1.5aA

14.8 ± 0.5cdB

Japan (JA1)

1.491 ± 0.100a

58.7 ± 1.3cA

64.8 ± 7.1bA

44.8 ± 2.2bB

Japan (JA2)

0.471 ± 0.036c

66.4 ± 1.0bB

95.8 ± 1.7aA

63.6 ± 4.7aA

G-BA, I-BA, and C-BA: As bioaccessibility in the gastric, small intestinal and colon phases. Means marked with the same letter indicate that data are not significantly different (P > 0.01). Small letter: values between rice bran samples; Capital letter: As bioaccessibility between gastric, small intestinal, and colon phases.

29



Page 30 of 33

Table 2. Daily As Intake Calculation for Rice Bran Products. Daily intake (μg As/kg bw/d)a Rice Bran

totalb

small

colonc

Contribution (%)d totalb

intestinec

small

colonc

intestinec

China (CH)

0.50

0.48

0.06

16.7

16.1

1.9

USA (US)

0.53

0.44

0.12

17.8

14.7

3.9

0.49

0.44

0.07

16.2

14.7

2.4

Japan (JA1)

0.75

0.33

0.33

24.8

11.0

11.1

Japan (JA2)

0.24

0.23

0.15

7.9

7.5

5.0

Germany (GE)

a

Daily As intake for an adult with body weight (bw) of 60 kg and rice bran

consumption rate of 30 g/d. bBased on total As concentration of rice bran product. c

Based on bioaccessible As concentration in the small intestinal and colon phases.

d

Contribution of the benchmark dose at 3 μg/kg bw/d.30

30


Page 31 of 33


Figure 1

31



Figure 2

32


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Page 33 of 33


TOC Graphic

33


Arsenic in Rice Bran Products: In Vitro

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