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Metabolism of a phenylarsenical in human hepatic cells and identification of a new arsenic metabolite Qingqing Liu, Elaine M. Leslie, Birget Moe, Hongquan Zhang, Donna N. Douglas, Norman M. Kneteman, and X. Chris Le Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05081 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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Metabolism of a phenylarsenical in human hepatic cells and identification of a new arsenic

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metabolite

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Qingqing Liu †, Elaine M. Leslie ‡,†, Birget Moe †, Hongquan Zhang †, Donna N. Douglas §,

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Norman M. Kneteman §, X. Chris Le *,†

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†

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Clinical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2G3

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‡

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Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, 10-102

Department of Physiology, Faculty of Medicine and Dentistry, 7-08A Medical Sciences

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§

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Sciences Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2B7

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* Corresponding author. Tel.: (780) 492-6416; Fax: 1 (780) 492-7800; E-mail: [email protected]

Department of Surgery, Faculty of Medicine and Dentistry, Walter C Mackenzie Health

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1 ACS Paragon Plus Environment

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ABSTRACT

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Environmental contamination and human consumption of chickens could result in potential

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exposure to Roxarsone (3-nitro-4-hydroxyphenylarsonic acid), an organic arsenical that has been

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used as a chicken feed additive in many countries. However, little is known about the

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metabolism of Roxarsone in humans. The objective of this research was to investigate the

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metabolism of Roxarsone in human liver cells and to identify new arsenic metabolites of

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toxicological significance. Human primary hepatocytes and hepatocellular carcinoma HepG2

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cells were treated with 20 or 100 µM Roxarsone. Arsenic species were characterized using a

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strategy of complementary chromatography and mass spectrometry. Results showed that

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Roxarsone was metabolized to more than 10 arsenic species in human hepatic cells. A new

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metabolite was identified as a thiolated Roxarsone. The 24-h IC50 values of thiolated Roxarsone

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for A549 lung cancer cells and T24 bladder cancer cells were 380 ± 80 µM and 42 ± 10 µM,

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respectively, which was more toxic than Roxarsone whose 24-h IC50 of A549 and T24 were 9300

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± 1600 µM and 6800 ± 740 µM, respectively. The identification and toxicological studies of the

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new arsenic metabolite are useful for understanding the fate of arsenic species and assessing the

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potential impact of human exposure to Roxarsone.

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INTRODUCTION

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Inorganic arsenic is known to be a significant environment hazard. Chronic exposure to

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inorganic arsenite (AsIII) through the ingestion of contaminated water and food results in lung,

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skin, and bladder cancers, and has a strong association with ischemic heart disease, diabetes, and

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respiratory disease 1. 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, Rox) is an organic

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arsenic species which has been widely used as a chicken feed additive for over 70 years 2, 3, for

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the purpose of infection control and growth promotion. Most of Rox taken up by chickens is

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excreted; and chicken waste contains as much as 30-60 mg/kg of Rox 4. The chicken litter, which

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contains Rox and other arsenic species 5, is often used as a farm fertilizer. The use of chicken

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litter as a fertilizer increases the arsenic concentration in the soil, crops, and water in the vicinity

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6-8

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. Small amounts of arsenic species, including inorganic arsenicals and Rox, have been

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detected in chicken meat and livers after feeding chickens with Rox 9, 10, 11. Residual arsenicals,

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albeit at trace concentrations, were detected in chicken meat seven days after the feeding of Rox

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was stopped 12. Arsenic species in chickens contribute to human dietary exposure to arsenic 2, 3.

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Therefore, there is considerable concern regarding human exposure and health risks posed by the

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poultry use of Rox 13. Although the European Union and the United States have stopped

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application of Rox in poultry industry 14, 15, many countries are still using Rox as an animal feed

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supplement 16.

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To understand the fate of Rox and the toxicological relevance of its metabolites in

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humans, we aimed to investigate the metabolism of Rox in human cells. Because liver is the

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primary site for the metabolism of inorganic arsenic 17, we chose to study metabolism of Rox in

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human liver cells. In the present work, we examined the hepatic biotransformation of Rox by


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using human hepatocellular carcinoma HepG2 epithelial cells and primary hepatocytes from

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human donors. We describe here results from analyses of arsenic species, identification of a new

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metabolite, and testing of its cytotoxicity. We demonstrate the identification of thiolated Rox as a

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new metabolite and show its higher toxicity than the original compound Rox.

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MATERIALS AND METHODS Materials. Stock solutions (1 mg/L) of arsenobetaine (AsB), AsIII, arsenate (AsV),

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monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), 3-amino-4-

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hydroxyphenylarsonic acid (3AHPAA), N-acetyl-4-hydroxy-m-arsanilic acid (NAHAA), and

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Rox were prepared from arsenobetaine (98% purity, Tri Chemical Laboratories Inc., Japan),

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sodium m-arsenite (97%, Sigma-Aldrich, St. Louis, MO), sodium arsenate (99.4%, Sigma-

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Aldrich), monosodium acid methane arsonate (99%, Chem Service, West Chester, PA),

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cacodylic acid (98%, Sigma-Aldrich), 3-amino-4-hydroxyphenylarsonic acid (Pfaltz and Bauer

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Inc., Waterbury, CT), N-acetyl-4-hydroxy-m-arsanilic acid (Pfaltz and Bauer Inc., Waterbury,

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CT), and 3-nitro-4-hydroxyphenylarsonic acid (98.1%, Sigma-Aldrich), respectively. The

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concentrations of the arsenic species were calibrated against a primary arsenic standard (Agilent

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Technologies, U.S.) and were determined using inductively coupled plasma mass spectrometry

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(ICPMS) Agilent 7500cs system (Agilent Technologies, Germany). Milli-Q18.2 MΩ·cm

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deionized water (Millipore Corporation, Billerica, MA) was used to prepare arsenic solutions.

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Phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Corning

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(Corning, NY). Eagle’s Minimum Essential Medium (EMEM) was purchased from American

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Type Culture Collection (ATCC, Manassas, VA). Fetal bovine serum (FBS), ammonium

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bicarbonate (NH4HCO3), and Hank’s balanced salt solution (HBSS) were purchased from



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Sigma-Aldrich (St. Louis, MO). Human recombinant insulin was purchased from Life

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Technologies (Carlsbad, CA). Trypsin-ethylenediaminetetraacetic acid (EDTA), phosphate

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buffered saline (PBS), nonessential amino acids (NEAA), penicillin-streptomycin solution

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(Penstrep), L-glutamine, Dextran, RIPA buffer [50 mM Tris-HCl, pH 8.0, with 150 mM sodium

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chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl

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sulfate] were purchased from Gibco (Burlington, ON, Canada). ITS+ (6.25 µg/mL insulin, 6.25

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µg/mL transferrin, and 6.25 ng/mL selenium) and Biocoat culture plates were purchased from

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BD Biosciences (Franklin Lakes, NJ). Methanol was from Thermo Fisher Scientific (Waltham,

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MA).

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Human primary hepatocytes. Ethical approval was obtained from the University of

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Alberta Faculty of Medicine Research Ethics Board and informed consent from all hepatocyte

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donors. Human hepatocytes were from two adult patients undergoing hepatic resection at the

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University of Alberta Hospital (Edmonton, Alberta, Canada) by qualified medical staff.

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Segments of human liver tissue (15–20 cm3) were obtained from the margin of normal tissue

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surrounding resected tumor specimens using a modified 2-step collagenase perfusion method 18

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and cultured on collagen coated plates. Hepatocytes were seeded on type I rat tail collagen

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Biocoat 6-well culture plates following the procedure of Roggenbeck et al. 19, except without

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collagen overlay.

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For hepatocytes, ~18 h after seeding, arsenicals (1 µM AsIII, 20 µM Rox, or 100 µM Rox)

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were added into the culture medium and then incubated for 24 hours. After 24 h, cells were

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washed twice with 1×PBS and lysed with 200 µL RIPA buffer.


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HepG2 cells. HepG2 cells (ATCC, Manassas, VA) were grown in EMEM containing 10% FBS and 1% Penstrep. HepG2 cells were used between passage 7 and 17, and incubated at 37 oC with 5% CO2. After 3 days seeding, 20 µM or 100 µM Rox was added to the culture medium and

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incubated for 24 h. The cells were washed twice with 1×PBS, trypsinized, and lysed with

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repeated freezing in liquid nitrogen and thawing.

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Preparation of cell samples for analysis. For both types of cells, the cell lysates were

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centrifuged at 8000×g for 5 min to remove the cell debris. Supernatant (300 µL) was filtered

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with a 3000 Dalton cut-off membrane ultracentrifugation unit (Millipore, Canada) at 14000×g

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for 30 minutes. The filtrates were used for arsenic speciation analyses. Another 10 µL of the

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supernatant was used for the determination of protein concentration by Bradford assay. The

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samples were stored in a -20 oC freezer.

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Synthesis of a thiolated Rox standard. A thiolated Rox standard was synthesized

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according to the previously reported method 20, 21. Briefly, Rox (10 mM) was mixed with Na2S

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(16 mM) in 10 mL deionized water. H2SO4 (16 mM) was added dropwise into the incubation

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mixture. The reaction mixture was left to stand for 1 h and the solution was diluted 104 times and

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filtered through a 0.45-µm membrane filter. The purity of the synthesized thiolated Rox was

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90%, which was determined on the basis of chromatographic peaks. The synthesis reaction is

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shown in Equation 1.

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(eq.1)

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Arsenic speciation analysis using high performance liquid chromatography (HPLC)

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coupled with ICPMS. An Agilent 1290 series HPLC system, an anion exchange column (PRP

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X110s, 150 mm × 4.6 mm, 7-µm particle size; Hamilton, Reno, NV) and a reversed phase

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column (ODS-3, 100 mm × 2 mm, 3-µm particle size; Phenomenex, Torrance, CA) were used

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for the separation of arsenicals. In the anion exchange separation, the mobile phase A contained

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5% methanol in water, and mobile phase B contained 60 mM NH4HCO3 and 5% methanol in

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water (pH 8.75). The flow rate was 2 mL/min. A 10 µL aliquot of a sample was injected and the

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separation was performed in a gradient. The mobile phase B was increased from 0% to 2% in the

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first 2 minutes, to 30% in the next 13 min, to 100% in the next 7 min, kept at 100% for 2 min,

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and then decreased to 0% in the next 1 min. The column was equilibrated in mobile phase A for

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4 min before the next analysis. A typical chromatogram obtained from the anion exchange

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HPLC-ICPMS analysis of arsenic species in water is shown in Figure 1a.

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In the reversed phase chromatographic separation, 0.2% formic acid in water was used as

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mobile phase A and methanol was used as mobile phase B. The flow rate was 0.15 mL/min. A

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10 µL aliquot of a sample was injected and the separation was performed in a gradient. The

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mobile phase B was linearly increased from 5% to 30% in the first 10 min, then increased from

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30% to 50% from 10 to 30 min and decreased to 5% in the next 1 min. The column was kept in

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95% mobile phase A and 5% mobile phase B for 9 min before the next analysis.


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The effluent from HPLC system was directly introduced into the nebulizer of a 7500cs

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ICPMS. Arsenic was monitored at m/z 75. When separation on HPLC was run in anion exchange

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mode, instrument parameters were shown in Supporting Information (SI) Table S1. When

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separation was run in reversed phase mode, 20% oxygen in argon gas was used as optional gas.

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Spray chamber temperature was decreased to -5 oC. Other parameters were kept the same as

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shown in SI Table S1.

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Characterization of arsenic species using HPLC coupled with electrospray

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ionization (ESI) hybrid quadrupole time-of-flight mass spectrometry (QqToFMS). The

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overall scheme for the characterization of arsenic species, including an unknown arsenic species,

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is shown in SI Figure S1. Reversed phase HPLC was hyphenated with ESI QqToFMS TripleToF

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5600 system (AB Sciex, Ontario, Canada). The separation condition was described above. The

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instrument parameters of QqToFMS are shown in SI Table S1. Two scans, MS and MS/MS,

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using the ToF mass spectrometer, were looped in one cycle 22, 23. These two ToF scans had the

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same source parameter, except that the first ToF scan had collision energy (CE) of -5 eV and the

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second one had CE of -45 eV. For the detection of arsenic species, the retention time of the

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arsenic fragments in the second ToF scan must be the same as that of the precursor ions in the

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first scan.

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The first step in extracting the data from the above MS and MS/MS scans was to search

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for the specific arsenic fragments in the second scan. Possible arsenic fragments were proposed

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on the basis of previous reports which have shown arsenic fragment ions 9, 24-28. Their retention

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time at which these fragment ions were detected was used to find the candidate precursors in the

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first scan. The “Non-Targeted Peak Finding” function in the “XIC Manager” which was an add-

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on for the PeakView™ 2.0 software (AB Sciex, Ontario, Canada) was used to find all peaks in



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the first scan. The peak finding criteria in this function were set as: ‘Approximate LC peak

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width’ 1 min, ‘Minimum intensity in counts’ 5 counts, and ‘Chemical noise intensity multiplier’

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1.5. Application of this function resulted in a table showing the detected ions at their respective

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retention time. The precursor ions detected at the retention times when the intensity of arsenic

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fragment ions was the highest were chosen. After candidate precursor ions were selected, a new

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product ion scan in QqToFMS was used to confirm that these ions could indeed generate the

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expected fragment ions.

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Arsenic speciation analysis using HPLC separation and simultaneous detection with

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ICPMS and ESIMS. ESI triple quadrupole mass spectrometry (QqQMS) and ICPMS were used

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simultaneously for HPLC detection 9, 29. A QTRAP 5500 system (AB Sciex, Ontario, Canada)

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was operated in the multiple reactions monitoring (MRM) mode. After HPLC separation, the

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effluent was split into the two mass spectrometers. The instrument parameters of the QqQMS are

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shown in SI Table S1.

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Cytotoxicity of thiolated Rox. Cytotoxicity of thiolated Rox, Rox, and AsIII was

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evaluated on human bladder epithelial cancer cell line T24 (ATCC, Manassas, VA) and human

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lung epithelial cancer cell line A549 (ATCC, Manassas, VA) using the real-time cell electronic

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sensing technique 30.

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RESULTS AND DISCUSSIONS

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Metabolism of Rox by HepG2 cells and human primary hepatocytes. We first

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investigated the Rox metabolism in HepG2 cells. The chromatogram obtained from anion

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exchange HPLC-ICPMS analysis of HepG2 exposed to 20 µM Rox is shown in Figure 1b. In

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addition to Rox, DMA, MMA, AsV, 3AHPAA, and NAHAA, several unknown arsenic species


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were detectable in HepG2 cells after exposure to 20 µM Rox for 24 h. HepG2 cells exposed to

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100 µM Rox for 24 h also showed the presence of these arsenic species (SI Figure S2).

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We then examined the metabolism of Rox by human primary hepatocytes (liver cells)

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isolated from two human liver samples. A typical chromatogram of arsenic species in the

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hepatocytes exposed to 20 µM Rox is shown in Figure 1c. These hepatocytes were from human

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donor 1. Rox, MMA, AsV, and some unidentified arsenic species were detectable in these

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hepatocytes after 24 hours incubation with 20 µM Rox. Chromatogram from HPLC-ICPMS

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analysis of hepatocytes exposed to 100 µM Rox also showed the presence of these arsenic

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species (SI Figure S2). These results demonstrate that the human primary hepatocytes were able

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to metabolize Rox.

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To test that the hepatocytes used in this study was metabolically active, we measured the

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methylation of AsIII as a control. Methylation of arsenic after treatment with AsIII has been

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previously characterized using plated human primary hepatocytes. According to Styblo et al. 31,

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when human hepatocytes were exposed to 1 µM AsIII, the apparent methylation rate (AMR),

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calculated as the amount of AsIII converted to MMA and DMA per hour per 106 cells, was 3.1-

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35.7 pmol/h/106 cells; and the ratio of DMA/MMA was 0.03-2.9 over 4 hepatocyte preparations,

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indicating considerable inter-individual variability in methylation. In our study, the plated

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hepatocytes exposed to 1 µM AsIII had AMR at 5 and 6.5 pmol/h/106 cells for the hepatocytes

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from human donor 1 and 2, respectively. The ratios of DMA/MMA were 0.8 and 1.7 for the

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hepatocytes from human donor 1 and 2, respectively. These results are at the same magnitude or

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within the range of the results reported by Styblo et al. 31, which showed that the hepatocytes in

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our study were metabolically active.



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The concentrations of arsenic species in HepG2 and human primary hepatocytes after 20

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µM and 100 µM Rox exposure are summarized in Table 1. When cells were exposed to 20 µM

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Rox, HepG2 cells produced more DMA, 3AHPAA, and NAHAA (not detected in hepatocytes)

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but less MMA and AsV (3-fold lower for HepG2) than hepatocytes from human donor 1. When

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cells were exposed to 100 µM Rox, the comparison between hepatocytes from human donor 1

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and HepG2 cells suggests that the primary hepatocytes have the potential to produce more MMA

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(200-fold), AsV (2-fold), and NAHAA (8-fold) than HepG2.

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Consistent with the previous study showing considerable inter-individual variability in

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the methylations of AsIII by hepatocytes from four donors 31, we have also observed inter-

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individual variability in the metabolic activity of our hepatocyte preparations for Rox.

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Hepatocytes from human donor 2 produced more AsV but less DMA, MMA, 3AHPAA, and

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NAHAA than HepG2 cells. The AsV concentration generated in the hepatocytes from human

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donor 2 was at least 15- and 5-times more than the ones from HepG2 cells when the cells were

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exposed to 20 µM and 100 µM Rox, respectively. DMA, MMA, 3AHPAA and NAHAA were

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non-detectable in hepatocytes from human donor 2 at both Rox concentration levels.

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Rox has relatively low bioavailability compared to other arsenic species, such as AsIII and

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AsB 32. Our study has demonstrated that the low percentage of the administrated Rox taken up

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by HepG2 cells or human primary hepatocytes undergoes metabolism. The sum concentration of

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metabolites only accounted for 9.7-10.9 % of the sum concentration of all arsenic species

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detected in HepG2, and the principal product of Rox biotransformation in HepG2 cells was

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3AHPAA. These results agree with that of Moody and Williams 33 who found that hens

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metabolized 18% Rox to 3AHPAA as the major metabolite.


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The metabolites that we have measured in HepG2 cells and human primary hepatocytes

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were in general agreement with the results of US FDA 34, showing the presence of AsV, DMA,

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MMA, 3AHPAA, and NAHAA in the livers of chickens fed Rox. In our study, several

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unidentified arsenic species were detected in HepG2 cells and hepatocytes after Rox exposure.

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Identifying these arsenic species can provide further information on illustrating the cellular

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metabolism of Rox in humans and understanding the toxicological significance of Rox

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metabolism.

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Although liver is the primary site for the metabolism of inorganic arsenic, it is not clear if

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liver is also the primary site for the metabolism of Rox. This is a limitation of using cultured

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hepatocytes as compared to whole-body models.

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Hepatocytes grown in a monolayer rather than in other configurations (e.g., sandwich

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cultured human hepatocytes or 3-D cultures) can have reduced biotransformation abilities. Thus,

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the metabolic conversion of Rox in the current study is potentially underestimated. Our results of

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the comparison between suspension culture and plate monolayer culture of hepatocytes provided

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supportive information. In the same batch of experiment where we tested the AsIII methylation in

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plate culture of the hepatocytes, we also tested the methylation of AsIII in suspension culture of

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the hepatocytes (SI Section 3). The plated hepatocytes exposed to 1 µM AsIII had AMR of 5 and

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6.5 pmol/h/106 cells for human 1 and 2, respectively. The AMR of suspended cells exposed to 1

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µM AsIII was 22.9 pmol/h/106 cells for human 1 and 34.3 pmol/h/106 cells for human 2. These

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results indicated that the AsIII methylation ability of hepatocytes in suspension culture was higher

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than that in plate culture.

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Hepatocytes have reductive intracellular environment with high concentrations of

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glutathione; this condition would favor the production of trivalent arsenicals. The HPLC-ICPMS



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method used here could separate MMAIII from other arsenicals but could not separate DMAIII

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from DMAV. We did not observe any MMAIII in our cell samples. MMAIII and DMAIII could

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easily be oxidized to the pentavalent MMAV and DMAV.35 The present study did not take any

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special measure to preserve the pentavalent methylarsenicals. Thus, the measured concentrations

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of monomethyl and dimethyl arsenic species represented both the trivalent and pentavalent forms

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of methylated arsenicals.

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Identification of arsenic species and a new arsenic metabolite. Figure 1c shows the

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presence of an arsenic species (Un) at a retention time of 25.7 min. The concentration of Un in

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the hepatocytes, estimated against the calibration of Rox, was at low µg/L levels. We first

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confirmed that the new arsenic species (Un) was present only in the hepatocyte samples (cell

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lysate and medium), but not in the culture medium control without incubation with the

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hepatocytes (SI Figure S3a). On a strong anion exchange column, the Un species eluted after

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Rox, and the retention time of Un did not match any of the common arsenic species that have

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standards available (SI Figure S3b). Separation of arsenic species on a reversed phase column

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(SI Figure S3c), collection of the Un fraction, and reanalysis of the collected fraction using anion

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exchange chromatography further confirmed the chromatographic behavior of the new arsenic

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species (Un) (SI Figure S3d).

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To identify the Un arsenic species, we developed a strategy that took advantages of the

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complementary information, such as high-resolution MS and MS/MS scans from quadrupole

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time-of-flight measurements, MRM from triple quadrupole linear ion trap analysis, and the

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HPLC retention time information, along with simultaneous detection of ICPMS and ESIMS. The

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details of the methodology are described in SI and the work flow is summarized in SI Figure S1.


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Briefly, from each HPLC analysis, we detected both precursor ions and fragment ions

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using a high-resolution quadrupole time-of-flight mass spectrometer, by acquiring both MS and

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MS/MS scans. Building on the previously reported mass spectral information (SI Table S2)

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about the common arsenic species, our analyses of the precursor ions, the fragment ions, and

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their inter-relations (SI Figures S4-S6) enabled us to narrow down the candidate list (SI Tables

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S2 and S3) and achieve a tentative identification. We supported our identification by performing

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repeated analyses in both the negative ionization mode (SI Figures S4-S6) and the positive

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ionization mode (SI Figures S7 and S8).

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We further confirmed the identification of Un as a thiolated Rox, using the authentic

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arsenic compound that we synthesized (SI Figures S9 and S10). Using the mass spectral

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information we obtained from the thiolated Rox (SI Figure S10) and Rox (SI Figure S11), we

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constructed an MRM method for the detection of Rox and its newly identified hepatic metabolite,

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thiolated Rox. Reversed phase HPLC separation with simultaneous detection by ICPMS (Figure

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2, top panel) and ESIMS detection (Figure 2, bottom panel) shows consistent results of Rox and

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the thiolated Rox in the human primary hepatocyte sample. We have also detected the thiolated

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Rox as a new metabolite of Rox in HepG2 cells.

290 291

The cytotoxicity of the thiolated Rox. The cytotoxicity of the thiolated Rox was then

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studied on two cell lines, urinary bladder cancer cell T24 and lung cancer cell A549. In the same

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batch of experiments, we evaluated the cytotoxicity of AsIII as a control. The 24-h IC50 values for

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AsIII were 42 ± 1 µM in A549 cells and 5.2 ± 0.2 µM in T24 cells, which were consistent with

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previous studies 30. The results of the toxicological experiments showed that the 24-h IC50 of

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thiolated Rox for A549 and T24 were 380 ± 80 µM and 42 ± 10 µM, respectively, whereas the



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24-h IC50 of Rox for A549 and T24 were 9300 ± 1600 µM and 6800 ± 740 µM, respectively.

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Therefore, the toxicity of the thiolated Rox was 23-161 fold higher than that of Rox. These

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results are consistent with previous cytotoxicity studies which suggested that the thiolated

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arsenic species are generally more toxic than their oxygenated analogues 36-42.

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We have considered whether the impurities Rox and in the synthesized thiolated Rox

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could contribute to the observed IC50 values. From the HPLC-ICPMS analysis of the synthesized

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thiolated Rox, we determined that the impurities were AsV and Rox, accounting for 0.9% and

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9.3% of total arsenic, respectively. The 24-h IC50 values of AsV was 1400 ± 130 µM for A549

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cells and 85 ± 6 µM for T24 cells (SI Table S4). When the apparent thiolated Rox was at its IC50

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value, e.g. about 400 µM for A549, the concentration of impurity as AsV (< 0.9%) was 4 µM

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which was much less than the IC50 of AsV. Thus the low concentration of AsV as an impurity

308

present in the thiolated Rox had little contribution to the observed cytotoxicity of thiolated Rox.

309 310

IMPLICATIONS AND OUTLOOK.

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Trace amounts of arsenic species, including Rox, have been previously detected in chicken fed

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Rox-containing food 12, 43. Rox is widely used as a poultry feed additive in many countries 44-47

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although it is no longer approved for this use in the United States 14, 48 and the European Union 15.

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A recent market-basket survey conducted in the United States showed that 46% of raw chicken

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meat samples purchased in the local food markets in the U.S. contained detectable Rox 3. The

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exact source(s) of the detected Rox in these chicken meat samples are not known. However,

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consumers of chicken meat are at risk of exposure to the residual Rox. Prior to this work, there is

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no study on the human metabolism of Rox. Our study, using both normal cells (human primary


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hepatocytes) and cancer cells (HepG2), demonstrates that human liver cells can metabolize Rox,

320

forming several arsenic species.

321

We determined the concentrations of arsenic species in the cell lysate after the filtration

322

through a 3kD cut-off membrane filter. This fraction accounted for (72 ± 10)% (mean ± SD) as

323

compared to the total concentration of arsenic in the whole cell digest (SI Section 1). These

324

results suggest that there was still arsenic remaining in the cell debris and/or in the high

325

molecular weight fraction (>3kD) in the filtrate. The remaining arsenic might be present in a

326

protein-bound form. Further studies could be carried out using size-exclusion chromatography to

327

determine both the protein-bound and free arsenicals in the cells.

328

One of the metabolites detected in the human liver cell samples is identified as a thiolated

329

Rox. This new arsenic species is more toxic than Rox. The thiolated Rox has IC50 values of 42

330

µM for T24 cells and 380 µM for A549 cells, representing 1-2 orders of magnitude lower than

331

the IC50 values for Rox. How the thiolated Rox exerts higher cytotoxicity than Rox to these cells

332

remains unclear. Higher cellular absorption could contribute to increased cytotoxicity 38.

333

Determination of cellular arsenic levels in cells exposed to Rox or thiolated Rox would be useful

334

in future studies.

335

There is no report on the quantitative amounts or estimations of human dietary intake of

336

Rox. Our findings of Rox metabolism in human hepatic cells and the discovery of the more toxic

337

arsenic metabolites warrant further research on human exposure, metabolism, and potential

338

health effects of these arsenic species.

339 340

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ACKNOWLEDGEMENTS

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This work was supported by the Natural Sciences and Engineering Research Council of Canada,

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Canadian Institutes of Health Research, the Canada Research Chairs Program, Alberta Innovates,

345

and Alberta Health.

346 347

SUPPORTING INFORMATION. Determination of recovery of arsenic from cell lysate

348

filtrates and recovery from the chromatographic column. Methylation of AsIII in suspension

349

culture of the hepatocytes. Determination of arsenic species using HPLC-ICPMS.

350

Characterization of arsenic species using ESIMS. Re-analyses of arsenic species along with

351

synthesized new arsenic species. The operating conditions for ICPMS and ESIMS. A list of

352

expected arsenic-containing fragment ions. A list of candidate precursor ions. The 24- and 48-

353

hour IC50 of arsenic species for A549 and T24 cells. Schematic of identification of new arsenic

354

species. Anion exchange HPLC-ICPMS chromatograms of HepG2, hepatocytes and cell medium.

355

Reversed-phase HPLC-ESI QqToFMS chromatograms of cell medium in negative and positive

356

ionization modes. Mass spectra of Rox and thiolated Rox on QqToFMS. Reversed-phase HPLC

357

with ICPMS and ESI-MRM chromatograms of cell samples.

358 359 360 361 362 363

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Table 1. The intracellular concentrations of arsenic species (µg/g protein) present in HepG2 cells

510

(n=3) and plated human primary hepatocytes from two individuals, designated Human 1 and

511

Human 2. Cells were incubated with 20 or 100 µM Rox for 24 h prior to the HPLC-ICPMS

512

analyses.

20 µM Rox (µg/g protein) 100 µM Rox (µg/g protein) HepG2 cells HepG2 cells Human 1 Human 2 Human 1 Human 2 (mean ± SD) (mean ± SD) DMA 0.008 ± 0.002 N.D.a N.D. N.D. N.D. 0.022 ± 0.001 N.D. N.D. N.D. N.D. MMA 0.005 ± 0.001 0.8 0.8 N.D. N.D. 0.010 ± 0.002 2 2 N.D. N.D. V As 0.08 ± 0.05 0.3 0.3 2 2 0.32 ± 0.06 0.7 0.6 2 2 a 3AHPAA 1.1 ± 0.2 N.D. N.D. N.D. N.D. 2.8 ± 0.5 2 1 N.D. N.D. NAHAA 0.03 ± 0.01 N.D. N.D. N.D. N.D. 0.05 ± 0.01 0.4 0.2 N.D. N.D. a 513 N.D.: non-detectable (below detection limit). SD: standard deviation.


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Figure 1. Chromatograms obtained from anion exchange HPLC-ICPMS analyses of (a) arsenic standards, (b) HepG2 cells exposed to 20 µM Rox, and (c) primary hepatocytes exposed to 20 µM Rox. The primary hepatocytes were from human donor 1. A new arsenic species to be identified is labelled as Un.



Figure 2. Chromatograms from reversed phase separation hyphenated with simultaneous (a) ICPMS detection and (b) ESI MS/MS detection of a hepatocyte sample. In ESI MS/MS, 5 transition ion pairs (278/95, 278/107, 278/111, 278/123, and 278/139) of thiolated Rox and 2 transition ion pairs (262/107 and 262/123) of Rox were monitored. The hepatocytes were prepared from a liver slice donated by human 1. Hepatocytes were incubated with 100 µM Rox for 24 h. Incubation medium from this culture was analyzed for arsenic species.


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Metabolism of a phenylarsenical in human hepatic ... - ACS Publications

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