Arsenic Speciation and Accumulation in Selected Organs after Oral

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Food Safety and Toxicology

Arsenic speciation and accumulation in selected organs after oral administration of rice extracts in Wistar rats Kittima Lewchalermvong, Nuchanart Rangkadilok, Sumontha Nookabkaew, Tawit Suriyo, and Jutamaad Satayavivad J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05746 • Publication Date (Web): 10 Mar 2018 Downloaded from http://pubs.acs.org on March 11, 2018

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Arsenic speciation and accumulation in selected organs after oral administration of rice

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extracts in Wistar rats

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Kittima Lewchalermvong1, Nuchanart Rangkadilok2,3,4, Sumontha Nookabkaew 3, Tawit

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Suriyo3,4, Jutamaad Satayavivad2,3,4

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Affiliations:

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1) Applied Biological Sciences Program, Chulabhorn Graduate Institute (CGI),

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Chulabhorn Royal Academy, Kamphaeng Phet 6, Laksi, Bangkok 10210, Thailand

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2) Environmental

Toxicology

Program,

Chulabhorn

Graduate

Institute

(CGI),

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Chulabhorn Royal Academy, Kamphaeng Phet 6, Laksi, Bangkok 10210, Thailand

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3) Laboratory of Pharmacology,Chulabhorn Research Institute (CRI), Kamphaeng Phet

11 12 13

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6, Laksi, Bangkok 10210, Thailand 4) Center of Excellence on Environmental Health and Toxicology (EHT), Ministry of Education, Bangkok 10400, Thailand Corresponding Author:

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Assoc.Prof. Jutamaad Satayavivad, Ph.D.

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Address: Laboratory of Pharmacology, Chulabhorn Research Institute (CRI),

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Kamphaeng Phet 6, Laksi, Bangkok 10210, Thailand

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Phone: 6625538555

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Email: [email protected]

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21

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Abstract

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Despite its nutritional values, rice also contains arsenic. There has been increasing

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concern about health implications associated with exposure to arsenic through rice

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consumption. The present study evaluated arsenic accumulation and its speciation in selected

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organs of Wistar rats after 28-day repeated oral administrations of polished or unpolished rice

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and their control arsenic compounds (sodium arsenite or dimethylarsinic acid). Only the

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treatment of sodium arsenite (2 µg/kg body weight), significantly increased total arsenic

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concentrations in blood when compared to the distilled water control group. In all groups,

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total arsenic concentrations were highest in kidney (1.54-1.90 mg/kg) followed by liver

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(0.85-1.52 mg/kg), and the predominant arsenic form in these organs was DMA. However,

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there was no significant difference in arsenic accumulation in the measured organs among the

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control and rice-treated groups. Therefore, the repeated 28-day administration of arsenic-

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contaminated rice did not cause significant arsenic accumulation in the animal organs.

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Keywords: rice extract, arsenic-contaminated rice, arsenic accumulation, arsenic speciation

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Introduction

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Rice is a staple food and the main economic crop grown across South-East Asia,

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especially in Thailand. While white rice is the most common consumable type, rice also

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exists in different colors, which is un-milled or partly milled. Rice with colors such as black,

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purple, and red contains twice as many nutrients as ordinary white rice1. Colored rice has

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high levels of several phytochemicals such as gamma-aminobutyric acid (GABA), oryzanol,

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polyphenols, tocopherols, and anthocyanin1, 2. It has also been found that colored rice may

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enhance brain functions and reduce levels of lipids and glucose in blood3, 4.

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Arsenic is found in the environment as a natural substance or as a result of human

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activities such as mining, metal smelting, combustion of fossil fuels, and use of pesticide in

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agriculture. It is generally found in water, soil, and food5. Groundwater contaminated with

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arsenic is used for drinking and also for crop irrigation, particularly for paddy rice, in some

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areas of South and South-East Asia6. The use of groundwater contaminated with arsenic in

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rice cultivation results in a high deposition of arsenic in topsoil and uptake into rice grains.

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This is a serious threat to the sustainable agriculture in Asia. Arsenic can be easily

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accumulated in rice which is typically cultivated in flooded soils. In the anaerobic condition

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together with excessive water, arsenic in its soluble form can be absorbed into the roots and

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shoots of plants resulting in an elevated arsenic accumulation in the grains7. In addition,

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cooking rice with arsenic-contaminated water could also increase the arsenic burden in the

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cooked rice 6. Bangladesh and West Bengal (India) are the hot spots for arsenic-contaminated

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groundwater8. In Bangladesh, arsenic has been detected in rice grains grown in the regions

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where arsenic was found in the soil8. The problem of arsenic contamination in groundwater is

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not just restricted to Bangladesh but also in specific areas of other countries in South and

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South-East Asia such as West Bengal (India), Vietnam, Thailand, Nepal, and Taiwan9.


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Arsenic found in food is present in several forms. Different forms of arsenic have

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different degree of toxicities. Inorganic arsenics [arsenite – As(III) and arsenate – As(V)] are

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the most toxic forms of arsenic present in food. Arsenic trivalent is a class-1 carcinogen10 ,

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and it binds to proteins in red blood cells which could be accumulated in both liver and

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kidney11. Meanwhile, organic arsenic forms, such as monomethylarsonic acid (MMA) and

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dimethylarsinic acid (DMA), are the metabolites of inorganic arsenic and they are less toxic.

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However, these organic forms have been classified as a class-2B, possibly carcinogenic to

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humans10.Arsenic in both organic and inorganic forms can be found in rice. In addition, rice

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has different levels of inorganic arsenic depending on methods of cultivation such as cultivar,

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growing area, and fertilizer etc. Previous studies demonstrated that the major arsenic species

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detected at high levels in 44 different rice samples (white, parboiled white, brown, parboiled

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brown, parboiled organic and organic white) from different Brazilian regions were As(III),

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As(V), and DMA12. Red and brown rice also contained higher inorganic arsenic and total

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arsenic than white rice13.

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There has been increasing concern about the health implications regarding exposure

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to inorganic arsenic through rice consumption14. Recently, the European Commission has

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established the maximum limits for inorganic arsenic in rice and rice products. (Commission

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Regulation (EU) 2015/1006). This regulation sets up the maximum levels of inorganic

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arsenic at 0.20 mg/kg for polished rice (PR), and 0.25 mg/kg for unpolished rice (UR) or

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husk rice. A lower level at 0.10 mg/kg is set for products intended for children, and a

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maximum level at 0.30 mg/kg for certain puffed rice products. Thus, analysis of arsenic

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speciation both in food and human body should be conducted to assess the health risk of

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arsenic exposure from food consumption. The bioavailability of arsenic species in food and

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human body would be useful for the risk assessment of different arsenic species in terms of

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consumption of food containing arsenic, leading to more accurate estimation for the daily



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intake15,16. The exposure to arsenic from different types of rice may result in the

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accumulation of different arsenic species in the body. Little was known about the arsenic

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speciation and accumulation in human organs after arsenic-contaminated rice consumption.

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Most previous reports in human could determine total arsenic concentrations and speciation

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only in blood, urine or feces after consumption of food containing arsenic17-19. Benramdane

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et al.20 reported a first case of arsenic speciation in human organs following fatal acute

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intoxication by arsenic trioxide. Their results showed that liver and kidneys had the highest

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concentrations of total arsenic and skin had the lowest. They also reported that As(III) is the

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predominant species in all organs and MMA is more concentrated than DMA. In fact, arsenic

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compounds vary in their metabolism and disposition, depending on the speciﬁc compound,

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the exposure route, and the animal specie21. These factors are signiﬁcant in determining the

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differences in toxicity between the various arsenic compounds and between the toxic effects

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produced in the different animal species. However, since there is a limitation for operating

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experimental study of arsenic toxicity in human, the use of laboratory animals appears to be

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easier to undertake and control in the experiment. In addition, large doses of compounds can

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be applied to the tested animals to evaluate the possible toxic effects and accumulation in

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organs for short term treatment. Therefore, this study aimed to determine arsenic speciation

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and its accumulation in selected organs of rats after 28-day repeated oral administration of

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different types of Thai rice extracts. Two types of rice extracts, polished and unpolished rice,

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containing different concentrations of inorganic and organic (DMA) arsenic species and at

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different doses calculated as human rice consumption were used in this study for comparison

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on the accumulation and possible toxicity from arsenic exposure.

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Material and methods

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Preparation of rice extracts


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Because of poor nutrition leading to the development of chronic disease, an increase

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in healthy food consumption may be one practice for maintaining good health. Comparison to

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polished rice (white rice), unpolished rice or colored rice is found to contain more fiber

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content, minerals and vitamins, and bioactive compounds22. Therefore, consumption of

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unpolished rice may reduce the risk of diseases. Apart from beneficial compounds, these two

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types of rice also contained different concentrations of arsenic. In the present study, polished

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rice (PR) and unpolished rice (UR) were purchased from local markets in Thailand in July,

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2015. Arsenic concentrations in these two rice types were measured. PR and UR containing

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the highest concentrations of inorganic and organic arsenic were selected for the experiment.

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Both PR and UR were extracted with hot water (80-90°C, 1:15 ratio, w/v) on hot plate for 1

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h. After centrifugation at 3000 × g for 30 min, the supernatant was collected and filtered with

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Whatman® No.4 filter paper (GE Healthcare UK limited, Buckinghamshire, UK). The

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extracts were kept at 4 ◦C until freeze drying. Rice extracts were freeze-dried using Freeze

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Dryer (Labconco, MO, USA). The % yields of PR and UR extracts were 4.09 and 10.56 %,

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respectively. Arsenic species were determined in both rice extracts for the dose calculation.

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These two rice extracts were used in the present animal study as they were concentrated and

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contained higher concentrations of arsenic than raw rice and it can be controlled for the doses

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when administration to each rat in the treatment groups. Rice extracts were dissolved in 90°C

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deionized water and the solution was left to cool down before administration.

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Animal experiment

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Dose calculation

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Administered doses for the test animals were estimated based on equivalent surface

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area dosage conversion factors. Doses of rice extracts used in the experiments were converted

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from human doses to rats23. Low dose of rice extract was equivalent to 50 g of raw rice, and



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high dose was equivalent to 150 g of raw rice. The table of quantity of reference serving size

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of cereals and grains was used for calculation (Notification of the Thai Ministry of Public

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Health; No. 182/1998) and then it was converted to raw rice doses in rat; 1 serving size at

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5,850 and 3 serving sizes at 17,550 mg/kg body weight of rat, for low and high doses,

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respectively. The doses of freeze-dried extracts for all rat groups were calculated from

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percentages of extraction yields and raw rice doses in rat (freeze-dried rice extract dose = raw

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rice dose × percent extraction yield). Doses for PR extract were 240 and 720 mg/kg body

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weight while those of UR were 620 and 1860 mg/kg body weight.

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The concentration of arsenic species in freeze-dried rice extracts was determined and

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the highest concentrations of inorganic and organic arsenic species in two freeze-dried

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extracts were used as arsenic control groups in this experiment. Inorganic arsenic, As(III),

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was found at the highest concentration in UR at 0.616 mg/kg, and it was calculated as sodium

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arsenite at the dose of 2 µg/kg body weight . DMA was detected at the highest concentration

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in PR at 0.095 mg/kg rice, and it was calculated as DMA at the dose of 0.1 µg/kg body

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weight.

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Animals

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Thirty-five young adult male Wistar rats with the weight of 320–350 g (7-week old)

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were used. All animals were obtained from the Chulabhorn Research Institute - Laboratory

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Animal Unit (CRI-LAU). The CRI-LAU has been accredited by the Association for

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Assessment and Accreditation of Laboratory Animal Care International (AAALAC

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International). The care and use of animals were in accordance with the ethical principles and

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guidelines for the use of animals for scientific purposes (National Research Council of

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Thailand, Ministry of Science and Technology, and CRI’s Animal Care and Use Program).

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The study was approved by CRI- Institutional Animal Care and Use Committee (Project


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number: 2015-04 and approval date: 20/08/2015). All animals were housed under controlled

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housing conditions (temperature 24 ± 1°C, humidity 55 ± 10%) with a 12:12 h light: dark

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cycle). They were fed with commercial animal feed (C.P. 082) and water ad libitum. These

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animal feed and water were collected to determine arsenic concentrations during experiment.

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Animals were acclimatized for 7 days prior to the study. Clinical signs were observed in all

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animals during the experiments.

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Repeated dose 28-day oral administration of rice extracts

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Rats were randomly divided into 7 groups (5 rats/group) and they received 0.5 ml of

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the extract per 100 g of the total body weight. Rats in each group were treated daily as

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following:

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Group 1 - control (distilled water);

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Group 2 - freeze-dried PR dissolved in distilled water at 240 mg/kg body weight (low dose

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polished rice: LPR);

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Group 3 - freeze-dried PR dissolved in distilled water at 720 mg/kg body weight (high dose

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polished rice: HPR);

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Group 4 - freeze-dried UR dissolved in distilled water at 620 mg/kg body weight (low dose

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unpolished rice: LUR);

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Group 5 - freeze-dried UR dissolved in distilled water at 1860 mg/kg body weight (high dose

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unpolished rice: HUR).

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Group 6 - inorganic arsenic (sodium arsenite, NaAsO2) control group at 2 µg/kg body weight

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[As(III)]

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Group 7 - organic arsenic (dimethylarsenic acid) control group, at 0.1 µg/kg body weight

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(DMA)



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All groups received treatments by oral gavages. All animals were fed using feeding

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tube every day in the morning with tested compounds for 28 days. Body weight was

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measured daily. At day 0 and day 14, rats were fasted overnight, and then blood was

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collected by tail vein puncture to determine arsenic and glucose baseline levels. After 28

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days, all rats were fasted overnight and blood glucose was measured via tail nicking before

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euthanasia. The rats were euthanized by carbon dioxide inhalation. Blood was collected via

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cardiac puncture. Before analysis, the collected blood was left at room temperature for 2 h

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and the clotted blood was centrifuged at 3500×g for 10 min at 25°C. Serum was collected and

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immediately stored at -80°C. Serum samples were then sent to Bangkok R.I.A. (BRIA LAB,

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Thailand) for hematology and clinical biochemistry analyses. The anticoagulated blood

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(EDTA) was stored at -80°C until further use. Selected organs including liver, kidney,

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pancreas, testis, and blood vessels at descending aorta were collected and stored at -80°C

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until freeze-dried and further use for the determination of total arsenic concentrations and

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arsenic speciation.

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Determination of arsenic concentrations in samples

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Total arsenic concentration

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Rice grains and animal feed were grounded into powder then weighed at 0.20 g.

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Animal organs, liver, pancreas, kidney, testis, and aorta were freeze-dried and grounded into

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powder. Dried powders of organ samples were weighed at 10.00 mg of pooled group for

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aorta, 20.00 mg of pancreas, kidney, and testis and 100.00 mg of liver. The samples were

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digested with concentrated nitric acid (HNO3, 65%) (Suprapur, Merck) by using microwave

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digestion system (MARS6, CEM Corporation, NC, USA). The two replications for each

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sample were subjected to the same conditions. The conditions setting for microwave

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digestion were maximum power 1200W, pressure 180 psi, temperature 190°C, ramp time 25


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min and maintained at these conditions for 30 min. After microwave digestion, the digested

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solutions were diluted to 10-50 g with high purified water.

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Blood, 25 µL, was transferred into 5 mL tube, and then 600 µL of 65% HNO3 was

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added into the samples. They were heated in heat box at 90°C for 1 h in fume hood. The

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digested sample was diluted to 5 g with high purity water. Water samples were acidified with

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10% HNO3.

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The total arsenic concentrations in all digested samples were then analyzed by using

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inductively coupled plasma mass spectrometry; ICP-MS (7500C and 8800QQQ, Agilent,

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Tokyo, Japan). The excitation power of the plasma was 1500 W; the gas flow rates for

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plasma gas, carrier gas, and makeup gas were 15.0, 0.9, and 0.3 L/min, respectively. Helium

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(He) gas was used as a collision gas at the flow rates of 5.0 mL/min. Rhodium (Rh) was used

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as an internal standard.

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Arsenic speciation

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Freeze-dried rice samples and organs (liver and kidney) were weighed at 0.25 g and

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40 mg, respectively and then transferred into 50 mL of polypropylene centrifuge tubes. 0.15

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M HNO3 (Suprapur, Merck), 10 mL, was added into the samples. The samples were then

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placed in a shaking water bath at 95 °C for 2 h. After that, the samples were centrifuged at

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3490×g for 5 min. The supernatants were filtered through a 0.20 µm syringe-type PVDF

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membrane filter. Separation of arsenic species in rice was performed with C18 column (X-

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Select, Charged Surface Hybrid; CSH; 4.6 × 150 mm of id and 5 µm of particle size) (Water

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Corporation, Milford, MA, USA). The mobile phase solutions were 7.5 mM

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tetrabutylammonium hydroxide and 10 mM ammonium phosphate monobasic at pH 8.25

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(Solvent A) 95% and methanol (Solvent B) 5%, at a flow rate of 1.0 mL/min. Separation of

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arsenic species in organs was performed with C18 column (X-Select, Charged Surface



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Hybrid; CSH; 4.6 × 250 mm of id and 5 µm of particle size) (Water Corporation). The mobile

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phase solutions were 7.5 mM tetrabutylammonium hydroxide and 10 mM ammonium

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phosphate monobasic at pH 8.50 (Solvent A) 95% and methanol (Solvent B) 5%, at a flow

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rate of 1.0 mL/min.

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The effluent from the HPLC column connected directly into ICP-MS nebulizer

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through PFA (perfluoroalkoxy) tubing to analyze the arsenic species. To ascertain possible

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chloride interference in m/z 75 (75As, 40Ar35Cl), m/z 35 (35Cl) was monitored.

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Quality assurance and quality control

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The accuracy and precision of the method were tested with three certified reference

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materials (CRMs), SRM1568b Rice flour, SRM1566b Oyster tissue (US Department of

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Commerce National Institute Standards and Technology; NIST, Gaithersburg, MD, USA)

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and DORM2 Dogfish muscle (National Research Council, Canada). The %recoveries of total

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arsenic and arsenic species found in all CRMs were in the ranges of 85.4-101.1% (Table 1).

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Although DMA and inorganic arsenic were not certified in DORM2, our results found that

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DMA was detected at the concentration of 0.32 mg/kg and inorganic arsenic was not detected

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in this CRM. These present results of analytical performances showed that the method was

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suitable for the determination of arsenic concentrations in the tested samples.

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Statistical analysis

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Most of data were expressed as mean ± standard error of mean (SEM). Only data in

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Table 2 were expressed as mean ± standard deviation (SD). Differences were analyzed for

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statistical significance by using one-way analysis of variance (ANOVA) and multiple

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comparisons between groups were performed by using Tukey test. Kruskal-Wallis test was

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used for non-parametric data. Time-course series data were tested by two-way ANOVA


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followed by a multiple comparison using Turkey test. Data with statistical values of p DMA > As(V) > MMA. The percentages of each arsenic species per total arsenic in

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UR were As(III) 84%, DMA 8%, and As(V) 8%. MMA was not detected in UR extract.

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These results indicated that UR extract contained inorganic arsenic (As(III)+As(V))

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accounted for 92% with organic arsenic (MMA+DMA) only 8%. In comparison, all arsenic

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species were found in PR extract with As(III) 71%, DMA 15%, As(V) 12%, and MMA 2%

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(inorganic arsenic accounted for 83% and organic arsenic for 17%). Notably, the predominant

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arsenic species found in both rice types were As(III) followed by DMA. The higher

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concentration of As(III) was found in the rice extract of UR (0.616 mg/kg) than that found

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in PR (0.452 mg/kg). In contrast to As(III), the DMA concentration was found to be higher in

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PR than UR. Therefore, As(III) and DMA were selected to be used as arsenic control groups

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in the next experiments.

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Animal experiment

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Total arsenic concentration in animal feed and water

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To determine the possible arsenic exposure from the other sources, standard diet and

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water samples were collected during the experiment for total arsenic determination. Feed



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samples contained total arsenic at the concentration of 0.200 ± 0.017 mg/kg while total

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arsenic in water samples was

Arsenic Speciation and Accumulation in Selected Organs after Oral

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