Arsenic Relative Bioavailability in Rice Using a Mouse Arsenic Urinary

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Arsenic relative bioavailability in rice using a mouse arsenic urinary excretion bioassay and its application to assess human health risk Hong-bo Li, Jie Li, Di Zhao, Chao Li, Xuejiao Wang, Hong-Jie Sun, Albert L. Juhasz, and Lena Q. Ma Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00495 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 23, 2017

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Environmental Science & Technology

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Arsenic relative bioavailability in rice using a mouse arsenic urinary

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excretion bioassay and its application to assess human health risk

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Hong-Bo Li,†,# Jie Li,†,‡, # Di Zhao,† Chao Li,† Xue-Jiao Wang,† Hong-Jie Sun,† Albert L.

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Juhasz,§ and Lena Q. Ma*,†,ǁ

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†

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Nanjing University, Nanjing 210023, China

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‡

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

China-Russia Joint Laboratory of Plasma Technologies, Laser Institute of Shandong

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Academy of Science, Jining 27000, China

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§

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ǁ

Future Industries Institute, University of South Australia, Mawson Lakes, SA 5095, Australia

Soil and Water Science Department, University of Florida, Gainesville, FL 32611, USA

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#

The authors contributed equally

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*

Corresponding author, State Key Laboratory of Pollution Control and Resource Reuse,

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School of the Environment, Nanjing University, Nanjing 210023, China; Tel./fax: +86 025

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8968 0637, E-mails: [email protected]

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ACS Paragon Plus Environment


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TABLE OF CONTENT

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ABSTRACT: A steady-state mouse model was developed to determine arsenic (As) relative

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bioavailability (RBA) in rice to refine As exposure in humans. Fifty-five rice samples from

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15 provinces of China were analyzed for total As, with 11 cooked for As speciation and

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bioavailability assessment. Arsenic concentrations were 38–335 µg kg–1, averaging 133 µg

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kg–1, with AsIII being dominant (36-79%), followed by DMA (18-58%) and AsV (0.5-16%).

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Following oral doses of individual As species to mice at low As exposure (2.5-15 µg As per

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mouse) over a 7-d period, strong linear correlations (R2=0.99) were observed between As

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urinary excretion and cumulative As intake, suggesting the suitability and sensitivity of the

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mouse bioassay to measure As-RBA in rice. Urinary excretion factor for DMA (0.46) was

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less than inorganic As (0.63-0.69). As-RBA in cooked rice ranged from 13.2±2.2% to

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53.6±11.1% (averaging 27.0±12.2%) for DMA and 26.2±7.0% to 49.5±4.7% (averaging

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39.9±8.3%) for inorganic As. Calculation of As intake based on total inorganic As in rice

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overestimated As exposure by 2.0-3.7 fold compared to that based on bioavailable inorganic

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As. For accurate assessment of the health risk associated with rice consumption, it is

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important to consider As bioavailability especially inorganic As in rice.

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INTRODUCTION Arsenic (As) is a ubiquitous contaminant present in food and the environment. Chronic

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exposure to As causes various adverse health effects including cancers, skin disorders,

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vascular disease, and diabetes mellitus.1,2 Humans are exposed to As via dietary and

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non-dietary pathways, with rice consumption being recognized as an important contributor to

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dietary As intake, particularly in Asian countries where rice consumption contributes ~70%

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of the food intake.3,4 Therefore, As exposure from rice is of global health concern.5

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Current research on As in rice has mostly focused on As concentration and speciation.

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Large variations in As concentrations have been reported for rice from different geographic

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locations (32–1,830 µg kg–1).6,7 Arsenic speciation is another important consideration as As

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toxicity varies with As species, with generally higher toxicity for inorganic than organic As

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species.8,9 Rice from different locations differs in As speciation due to differences in paddy

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soils, rice cultivars, water managements, and As species in irrigation water.10,11 Rice from

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China is dominated by inorganic As species (57–96%) including arsenate (AsV) and arsenite

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(AsIII),12-14 while organic As species including dimethylarsinic acid (DMAV) are dominant in

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US rice.10,15,16

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While As concentration and speciation are important factors to assess its health risks

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associated with rice consumption, bioavailable As is another critical parameter to better

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estimate As exposure from rice consumption.17 Bioavailable As from rice is the fraction of As

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that can be absorbed into the systemic circulation following consumption. When assessing

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the health risks associated with As exposure via rice consumption, dietary As intake is often

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calculated using rice consumption rate and total As or total inorganic As concentration in rice

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without considering its bioavailability.3-7,12,16 For example, using country-specific rice

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consumption data and inorganic As concentration in rice from 10 countries, Merhag et al.10

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estimated daily inorganic As intake across a large geographical distribution, with cancer risks

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ranging from 0.7 per 10,000 for Italians to 22 per 10,000 for Bangladeshis. While it provides

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a conservative estimate of health risks, conceivably not all As in rice is absorbed due to

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bioavailability constraints. Therefore, exposure assessment based on total As in rice may 4


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overestimate the risk from rice consumption. Reliable assessment of rice As exposure and the

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associated health risks should consider As bioavailability in rice.

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In vivo bioassays using animals such as swine and mice have been developed as a

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surrogate of human exposure to measure bioavailability of environmental contaminants.18

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Although numerous studies have assessed As concentrations and speciation in rice, only one

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study by Juhasz et al.19 determined the As absolute bioavailability (relative to intravenous

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doses of As species) in two rice samples using a swine bioassay with area under blood As

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concentration time curve (AUC) as the endpoint of As exposure. In rice with mainly

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inorganic As, As bioavailability was significantly higher than rice with predominantly organic

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As (89 vs. 33%). This was because that following oral ingestion, the absolute bioavailability

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of inorganic As (AsIII at 104% and AsV at 93%) was significantly higher than organic As

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(MMAV at 17% and DMAV at 33%).19

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Although valuable, Juhasz et al.19 utilized a limited rice sample size (n=2, one

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laboratory-grown and one spiked sample), which might not provide an accurate estimate of

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As bioavailability in rice. Since As concentration and speciation vary considerably with rice

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cultivar and agricultural management, a systemic study on As bioavailability in rice from a

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large geographic distribution is warranted to accurately assess As exposure in humans from

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rice consumption. In addition, the swine blood AUC bioassay used by Juhasz et al.19 is

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expensive and operationally complicated as it requires surgery and intravenous injection for

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swine. More importantly, for practical application for health risk assessment, it is difficult to

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extend As absolute bioavailability data to humans as As metabolism in humans is different

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from that in animals. However, to a certain extent, this drawback could be overcome by

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determining As relative bioavailability (RBA, relative to absorption of As species following

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oral consumption). Therefore, developing an in vivo bioassay that is low in cost, easy to

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undertake, and has the potential to be applied to larger sample sizes for rice As-RBA

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determination is important. One such method is the mouse steady state As urinary excretion

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bioassay, which has been used to determine As-RBA in contaminated soils.18,20 However, the

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mouse bioassay has previously been conducted at high As exposure levels (136–5224 µg As 5



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per mouse), while its suitability in determining As urinary excretion at low As exposure

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levels (2.5–15 µg As per mouse) comparable to rice As consumption has not been assessed.

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Therefore, the objectives of this study were to (1) develop a mouse steady state As

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urinary excretion bioassay suitable for determining As-RBA in rice at low levels of As

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exposure, (2) determine RBA of As in rice samples from a large geographic distribution

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across China, and (3) incorporate RBA of inorganic As into daily inorganic As intake

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calculations to refine exposure associated with rice consumption. This study has implications

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for accurate assessment of As exposure associated with rice consumption by including RBA

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using mouse bioassay data.

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MATERIALS AND METHODS

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Collection and Cooking of Rice. China is a primary rice production and consumption

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country in Asia, accounting for one-third of global production.14 To obtain representative rice

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samples from a large geographic distribution, 55 polished white rice samples (~500–1000 g

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each) were collected from local markets in 15 rice-producing provinces (Figure 1).

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Among the 55 samples, 11 subsamples with high As concentrations (140–335 µg kg–1)

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were randomly selected from 11 cities of 8 provinces in China, representing a large

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geographical distribution from southern to northern China (Figure S1A). They were cooked

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with Milli-Q water using an absorption method (rice:water ratio 1:2, w/w) following washing

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3 times (see Supporting Information, SI).7 The cooked samples were assessed for As

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bioavailability and the effect of cooking on As concentration and speciation. To determine the

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impact of As speciation on its bioavailability, one sample (#51) was randomly selected and

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cooked with water containing 750 µg L–1 AsV and AsIII (Table 1). This As concentration was

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selected because it is the highest As concentration reported in contaminated groundwater in

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China.21 In addition, to make a comparison between organic and inorganic As dominated rice,

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the sample was also cooked with water with same level of DMAV. Both raw and cooked

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samples were freeze-dried and stored at room temperature prior to analyses.

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Arsenic Concentration and Speciation Analyses. Freeze-dried rice samples were 6


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milled to a powder using a food mill (JYL-350 Model, Joyoung Co., Ltd, China). Total As

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concentrations in rice were determined using inductively coupled plasma-mass

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spectrophotometry (ICP-MS, NexIONTM300X, Perkin Elmer, USA) following digestion

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using USEPA 3050B Method (SI).

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In addition to total As analysis, selected paired raw and cooked rice samples were

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assessed for As speciation (Table 1). Arsenic in rice was extracted using 1% nitric acid in a

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microwave-accelerated reaction system,14 with the resulting supernatants being measured for

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As speciation using high performance liquid chromatography (HPLC, Waters e2695, USA)

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coupled with ICP-MS. Details of the As extraction procedure are provided in the SI. Arsenic

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species were separated using an anion exchange chromatography column (Hamilton,

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PRP-X100, 250 mm×4.1 mm, 10 µm particle size). The mobile phase was a mixture of 8.0

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mM (NH4)2HPO4 and 8.0 mM NH4NO3 (pH 6.2) at a flow rate of 1 mL min−1. Before

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separation, the column was equilibrated with the mobile phase for at least 0.5 h while the

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sample injection volume was 50 µL. Standard solutions (0–20 µg L–1) of As species (AsIII,

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AsV, MMAV, and DMAV) were prepared on the day of analysis from stock solutions

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containing 1000 mg As L–1.

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Certified reference materials including NIST 1568a (US long grain rice flour) and GBW

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10045 (Chinese rice flour) were used during analyses. The accuracy of the digestion method

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was confirmed by an As recovery of 294±10 and 109±3.0 µg kg–1 (n=3) from NIST 1568a

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(290±30 µg kg–1) and GBW 10045 (110±2.0 µg kg–1). During As analyses using ICP-MS,

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triplicate analyses, check standard, and spiked solutions were analyzed every 20 samples. The

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recovery of check standards was 96±1.5%, while the spike recovery was 94±3.6%. The

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relative standard deviation of triplicate analyses was 3.5%.

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For As speciation, the extraction efficiency (ratios of sum of As species to total As) of

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the 1% nitric acid method was 76.4–97.6%, averaging 90.7±5.56% (n=14). During As

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speciation analyses, blanks, blank spikes (0.1 g of 1000 µg As kg–1), and sample spikes (0.2 g

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rice flour, plus 0.1 g of 1000 µg As kg–1) for both AsIII and AsV were included with a batch of

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∼20 samples. Spike recoveries were 74–86%, suggesting the stability of As species during 7



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the process of As extraction. For NIST 1568a, concentrations of inorganic As (AsIII and AsV),

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DMAV, and MMAV were 112±2.8, 170±4.2, and 12±1.4 µg kg–1, consistent with Zhu et al.14

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and Meharg et al.22.

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Arsenic Relative Bioavailability Assessment. A mouse bioassay based on steady state

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As urinary excretion (SSUE) was used to determine As relative bioavailability (RBA) in 11

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randomly selected cooked rice samples (131–315 µg kg–1) in addition to one sample (#51)

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cooked with water containing 750 µg L–1 AsV, AsIII, or DMAV. Female Balb/c mice of 18–20

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g were acclimated in a 12/12 h light/dark photo cycle at 20–22°C, with free access to rice

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containing the lowest As concentration (rice #1, 38 µg As kg–1) and Milli-Q water over

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1-week period. Mouse basal feed was not used during the acclimation because the feed

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contained 100 µg kg–1 As, which would introduce higher background As values in mouse

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urine. Following acclimation, mice were fasted overnight and transferred to individual

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metabolic cages (4 mice per treatment group) before being introduced to cooked rice. The

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metabolic cages with stainless steel grid floor were used to separate urine from fecal pellets,

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with urine being collected in a urine cup located at the bottom of a metabolic cage (Figure

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S1B).

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Initially, urinary excretion of dominant As species in rice (AsV, AsIII, and DMAV) was

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determined. Arsenic species were spiked into the rice diet (rice #1) and fed to mice for 7 d

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with urine samples being collected daily, producing 3 levels of As concentration in spiked

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rice (~150, 300, and 600 µg kg–1), which corresponded with dose levels of ~0.5, 1, and 2 µg

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As d–1 for each As species. To determine As relative bioavailability (RBA) in rice samples,

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mice were fed with the rice for 7 d with daily collection of urine. Control mice receiving the

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rice basal diet (rice #1) during the 7-d period were used to determine the background urinary

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As excretion to adjust As excretion following As or rice consumption. The 7-d exposure

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period was used because it has been demonstrated that following rice consumption, As

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urinary excretion reaches steady state in the human body after 3–5 days.22 Cumulative rice

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consumption was determined as the weight difference in rice supplied and remaining at the

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end of exposure. 8


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During the 7-d exposure period, urine collected in the urine cups of metabolic cages

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were transferred to 50 mL centrifuge tubes every 24 h at 9:00 am and stored at –80°C. At the

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end of exposure, daily urine samples from the same mouse were processed to make a

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composite sample, with total volume being 5–10 mL. Arsenic concentrations in pooled urine

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samples were determined using ICP-MS following digestion using USEPA Method 3050B.

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Arsenic concentration in urine digest (1 ml of urine digested and diluted to 20 mL) ranged

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from 2.62–25.3 µg L–1, above the detection limit of ICP-MS (0.6 ng L–1). Cumulative urinary

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As excretion was calculated as the product of As concentration in the urine sample and its

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volume after subtracting background As urinary excretion from the basal rice diet

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consumption. Arsenic speciation in urine was not considered when calculating urinary

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excreted As. Regardless of its species, As in urine was considered bioavailable.

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Arsenic urinary excretion factor (UEF) for different As species or rice sample dosages

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was calculated by dividing cumulative As excreted in the urine (µg) by cumulative dietary

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intake of As (µg) (Eq. 1).

UEF = 189

Cumulative urinary excreted As Cumulative As intake

(1)

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To facilitate practical application of RBA data to refine As exposure in humans, we

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chose to determine relative bioavailability of organic and inorganic As in rice separately,

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instead of total As. As well demonstrated, DMAV in rice is of minor health concern while

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inorganic As is the major concern,16,23 though DMAV may undergo speciation transformation

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including demethylation.24 Due to As transformation following rice consumption, it is

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difficult to determine the fraction of As in urine comes from DMA or inorganic As intake

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from rice, making it difficult to determine UEF values for DMA and inorganic As intake

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separately for rice samples. One compromised method is to determine UEF for total As intake

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and proportion based on the contribution of organic and inorganic As to total As in rice.

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Therefore, we determined relative bioavailability of DMAV and inorganic As (AsV and AsIII)

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in rice individually by comparing As speciation data and UEF values for rice doses to UEF

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values for different As species (Eqs. 2 & 3). Relative bioavailability was not separately 9



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calculated for AsV and AsIII due to their similar UEF values (0.69 and 0.63). Although this

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was not the best way to determine RBA of DMAV and inorganic As in rice, it has an

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implication for practical application of bioavailability data to refine inorganic As exposure

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via rice.

RBA of DMA (%) = 206

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UEFrice × R DMA × 100 UEFDMA

 UEFrice × R iAs RBA of inorganic As (%) =  UEFiAs 

(2)

  × 100 

(3)

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where UEFrice, UEFDMA, and UEFiAs are UEF values for a rice, DMA, and inorganic As

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species dose; RDMA and RiAs are contribution of DMAV and inorganic As (AsIII and AsV) to

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total concentration of As in rice. The average of UEF values for AsV and AsIII doses (0.66)

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was set for UEFiAs. The method was similar to Juhasz et al.19 who calculated As absolute

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bioavailability in rice by using area under blood As concentration time curve data in swine

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and proportioning the contribution of As species. Since

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Daily Inorganic Arsenic Intake Calculation. Daily inorganic As intake (DI, µg As kg–1

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BW d–1) in adults with body weight of 60 kg and rice consumption rate of 350 g per day was

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calculated for the 14 samples that were measured for As-RBA. Daily intake was calculated

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based on total (DItotal) or bioavailable inorganic As (DIbioavailable) as followings:

DI total = 218

C × CR BW

DI bioavailable = 219

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(4)

C × CR × RBA BW

(5)

where, C represents total inorganic As concentration (µg kg–1) in rice; CR represents the

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daily rice consumption rate (350 g d–1); BW represents body weight (60 kg) of adults; and

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RBA represents the RBA of inorganic As in rice measured using in vivo mouse bioassay. 10


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Data Processing and Statistics. Animal experiments were performed with four

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replicates. Results are presented as mean values ± standard deviation. Statistical differences

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in As-RBA between rice samples were performed using variance analysis (ANOVA) based on

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Tukey's multiple comparisons using the software SAS version 9.1.3.

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RESULTS AND DISCUSSION

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Arsenic Concentrations and Speciation. In this study, 55 rice samples from 15

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important rice-producing provinces of China were assessed (Figure 1). Following

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determination of As concentrations, samples were numerically labeled (1–55) based on

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increasing As concentrations (Figure 2A). Arsenic concentrations in raw rice samples were

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38–335 µg kg–1, averaging 133 µg kg–1. Approximately 84% of samples (46 samples)

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contained As concentrations

Arsenic Relative Bioavailability in Rice Using a Mouse Arsenic Urinary

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