Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing

JES-00950; No of Pages 12 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 6 ) XX X–XXX

Available online at www.sciencedirect.com

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Birget Moe1,2 , Hanyong Peng1 , Xiufen Lu1 , Baowei Chen1,3 , Lydia W.L. Chen1,4,5 , Stephan Gabos1 , Xing-Fang Li1 , X. Chris Le1,⁎

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Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing☆

1. Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2G3, Canada 2. Alberta Centre for Toxicology, Department of Physiology and Pharmacology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada 3. MOE Key Laboratory of Aquatic Product Safety, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510275, China 4. Department of Chemistry, Brock University, St. Catharines, Ontario L2S 3A1, Canada 5. Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4L8, Canada

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Article history:

The occurrence of a large number of arsenic species in the environment and in biological 21

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Received 10 October 2016

systems makes it important to compare the relative toxicity of the diverse arsenic species. 22

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Revised 11 October 2016

Toxicity of arsenic species has been examined in various cell lines using different assays, 23

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Accepted 11 October 2016

making comparison difficult. We report real-time cell sensing of two human cell lines to 24

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Available online xxxx

examine cytotoxicity of fourteen arsenic species: arsenite (AsIII), monomethylarsonous 25

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Keywords:

dimethylarsinic glutathione (DMAGIII), phenylarsine oxide (PAOIII), arsenate (AsV), 27

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Arsenic species

monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV), monomethyltrithioarsonate 28

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Thio-arsenicals

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Methylarsenicals

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Toxicity

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Real-time sensing

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Methylated and thiolated arsenic

the exposure time, giving rise to IC50 histograms as unique cell response profiles. Arsenic 33

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metabolites

accumulation and speciation were analyzed using inductively coupled plasma-mass spec- 34

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(MMTTAV), dimethylmonothioarsinate (DMMTAV), dimethyldithioarsinate (DMDTAV), 3-nitro- 29

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4-hydroxyphenylarsonic acid (Roxarsone, Rox) and 4-aminobenzenearsenic acid (p-arsanilic 30

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acid, p-ASA). Cellular responses were measured in real-time for 72 hr in human lung (A549) 31

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acid (MMAIII) originated from the oxide and iodide forms, dimethylarsinous acid (DMAIII), 26

and bladder (T24) cells. IC50 values for the arsenicals were determined continually over 32

trometry (ICP-MS). On the basis of the 24-hr IC50 values, the relative cytotoxicity of the tested 35 arsenicals was in the following decreasing order: PAOIII ≫ MMAIII ≥ DMAIII ≥ DMAGIII ≈ 36 DMMTAV > AsIII ≫ MMTTAV > DMDTAV > AsV ≫ DMAV > MMAV > Rox ≥ p-ASA. Step-wise 37 shapes of cell response profiles for DMAIII, DMAGIII, and DMMTAV coincided with the 38 conversion of these arsenicals to the less toxic, pentavalent DMAV. Dynamic monitoring of 39 the real-time cellular responses to arsenicals provided useful information for comparison of 40 41

the relative cytotoxicity of arsenicals.

© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 42 Published by Elsevier B.V. 43

☆ This manuscript honors Dr. William R. Cullen for his extraordinary contributions to the field of arsenic chemistry. ⁎ Corresponding author. E-mail: [email protected] (X. Chris Le).

http://dx.doi.org/10.1016/j.jes.2016.10.004 1001-0742/© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004

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Arsenic occurs naturally throughout the geosphere and is ubiquitous in the environment (Cullen and Reimer, 1989). Arsenic contamination of groundwater that serves as human drinking water sources is a serious public health concern (NRC, 1999). Chronic consumption of inorganic arsenic at elevated concentrations is a known cause of skin, bladder, and lung cancers (NRC, 2001; IARC, 2012; Cohen et al., 2016), and has also been associated with kidney, liver, and prostate cancers (IARC, 2012) as well as several non-carcinogenic ailments including diabetes, reproductive, cardiovascular, and neurological diseases (Schuhmacher-Wolz et al., 2009; Hughes et al., 2011; Maull et al., 2012; Naujokas et al., 2013). In humans, inorganic arsenic is enzymatically biotransformed to several methylated metabolites. This pathway of inorganic arsenic metabolism is generally accepted to follow: inorganic arsenate [AsV] → inorganic arsenite [AsIII] → monomethylarsonic acid [MMAV] → monomethylarsonous acid [MMAIII] → dimethylarsinic acid [DMAV] → dimethylarsinous acid [DMAIII] (Challenger, 1945; Cullen et al., 1989; Le et al., 2000; Styblo et al., 2002; Vahter, 2002; Thomas et al., 2001, 2004, 2007; Cullen, 2014). Alternative pathways postulating glutathione- or protein-conjugated intermediates have also been proposed (Hayakawa et al., 2005; Naranmandura et al., 2006; Dheeman et al., 2014), although chemical basis has been questioned (Cullen, 2014). Arsenic cytotoxicity is dependent on its oxidation state and chemical structure (speciation). In general, trivalent arsenicals are more cytotoxic than pentavalent species, and the methylated trivalent arsenicals, MMAIII and DMAIII, are more cytotoxic than the inorganic arsenicals, AsIII and AsV, which are more cytotoxic than the methylated pentavalent arsenicals, MMAV and DMAV (Styblo et al., 2000; Petrick et al., 2000; Dopp et al., 2004; Nascimento et al., 2008; Charoensuk et al., 2009; Naranmandura et al., 2011). Historically, the focus of arsenic toxicity studies has been the examination of the oxygenated metabolites of inorganic arsenic. However, as analytical techniques have improved to increase sensitivity and specificity, several thiolated arsenic metabolites have been identified (Hansen et al., 2004; Wang et al., 2015; Chen et al., 2016; Sun et al., 2016). A class of thiol-containing arsenicals that were first identified as metabolites in seaweed-fed sheep (Hansen et al., 2004), are the pentavalent sulfur-containing arsenic species, such as dimethylmonothioarsinate [DMMTAV], dimethyldithioarsinate [DMDTAV], and monomethylmonothioarsonate [MMMTAV]. These thio-arsenicals have been detected in human or animal urine as metabolites of inorganic arsenic (Hansen et al., 2004; Naranmandura et al., 2007b; Raml et al., 2007; Naranmandura et al., 2013; Chen et al., 2016), and are believed to be formed from reactions between oxygenated arsenicals and hydrogen sulfide (Wang et al., 2015). Monomethyltrithioarsonate [MMTTAV] is another thiol-containing pentavalent metabolite, but has only been found as a metabolite of anaerobic microbiota in vitro (Pinyayev et al., 2011). Recent cytotoxicity analysis of these newly identified thiolated pentavalent arsenicals suggests that thiol conjugation can modulate arsenic toxicity, prompting inclusion of thiolated metabolites in this study. DMMTAV has been found to be as toxic as the trivalent species, AsIII and DMAIII, in human cancer cell lines (Naranmandura et al.,

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2007a; Naranmandura et al., 2009). The trivalent glutathione conjugated arsenical, dimethylarsinic glutathione [DMAGIII], is suspected to play a key role in the transport of methylated arsenic species from the liver to the blood stream (Percy and Gailer, 2008). Reported IC50 values for DMAGIII are equal to or less than those of AsIII (Styblo et al., 2000; Vega et al., 2001). While inorganic arsenic and its metabolites are often considered the most important from a human health perspective, other organoarsenic species have become topics of recent research interest. Two pentavalent phenyl arsenic species used in poultry industry are 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, Rox) and 4-aminobenzenearsenic acid (p-arsanilic acid, p-ASA). As poultry feed additives, Rox and p-ASA not only improve feed efficiency, allowing for faster weight gain, but also help control intestinal bacteria and parasites (Jones, 2007; Chen and Huang, 2012; Nachman et al., 2013). Both arsenicals have been phased out of use in the European Union and the United States; however, they are still uses in many other countries (Kazi et al., 2013; Yao et al., 2013; Mafla et al., 2015; Mangalgiri et al., 2015; Wang and Cheng, 2015). Although several studies have shown that these arsenicals can accumulate in the tissue and organs of livestock (Aschbacher and Feil, 1991; Desheng and Niya, 2006; Nachman et al., 2013; Peng et al., 2014; Liu et al., 2015; Liu et al., 2016), little is known about the cytotoxicity of these pentavalent arsenic species to human cells. Another arsenic species that is used in laboratory research as a known inhibitor in various biochemical reactions to elucidate toxicity mechanisms is phenylarsine oxide [PAOIII]. This trivalent organoarsenic species is not naturally-occurring, but it is found in the environment at sites contaminated with chemical warfare agents, as it is a degradation product of the chemical warfare agent, diphenylarsine dichloride (also known as Pfiffikus) (Leermakers et al., 2006). Studies have shown PAOIII to be a potent cytotoxicant (Charoensuk et al., 2009). Extensive data are available surrounding the cytotoxicity of individual arsenicals. However, these data have been obtained using various assays on different cell lines. The species-dependent cytotoxicity and variations in different assays make it difficult to compare the relative cytotoxicity of different arsenic species. In addition, some arsenicals have therapeutic uses, as with the treatment of acute promyelocytic leukemia with arsenic trioxide (As2O3) (Shen et al., 1997; Wang et al., 2004; Chen et al., 2015) and refractory solid tumors with DMAGIII (alternate names: S-dimethylarsino-glutathione, ZIO-101, and darinaparsin). Understanding the relative cytotoxicity of various arsenic species may direct its exploitation for further therapeutic investigation. Real-time cell-electronic sensing analysis is an impedancebased detection technique that can simultaneously perform 96 in vitro tests of cytotoxicity. Because the real-time cell sensing technique is label-free and dye-free, it is less invasive than traditional colorimetric cytotoxicity assays. It provides continuous monitoring, revealing more dynamic and complete cytotoxic response information, and it has been demonstrated in chemical cytotoxicity testing (Solly et al., 2004; Xing et al., 2005; Boyd et al., 2008; Moe et al., 2016). It is also one of the cell-based in vitro assay technologies implemented in the United States Environmental Protection Agency ToxCast program to prioritize the vast number of environmental chemicals, many of which are already under heavy commercial use, for

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Introduction

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Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004

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1. Experimental

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1.1. Cell cultures

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Two cell lines, A549 and T24, were used in this study. The human lung carcinoma cell line, A549 (CCL-185; American Type Culture Collection (ATCC), Manassas, VA), was cultured in RPMI 1640 media (Gibco/Invitrogen, Waltham, MA). The human bladder carcinoma cell line, T24 (HTB-4; ATCC) was cultured in McCoy's 5A modified media (ATCC). Both media were supplemented with 10% fetal bovine serum (Sigma-Aldrich, Oakville, ON, Canada) and 1% penicillin–streptomycin (Invitrogen). The incubation conditions were maintained at 37°C, 5% CO2, and 90% humidity. During the study, cells were sub-cultured twice weekly into standard 10 cm × 20 mm cell culture dishes (Corning Incorporated, Corning, NY, USA) containing fresh media, using 0.05% trypsin–EDTA (Invitrogen) for cell detachment.

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Solutions of AsIII and AsV in deionized (DI) water were prepared from commercially available sodium arsenite and sodium arsenate (Sigma/Aldrich, St. Louis, MO, USA). Solutions

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of MMAIII in DI water were prepared from synthesized standards of methyldiiodoarsine (Millar et al., 1960; Zingaro, 1996; Cullen et al., 2016) and methylarsine oxide [MAOIII] (Cullen et al., 1989, 2016), and the solution of DMAIII in DI water was prepared from a synthesized standard of dimethyliodoarsine (Millar, 1960; Cullen et al., 2016). Solutions of DMAGIII, DMMTAV, MMTTAV, and DMDTAV in DI water were also prepared from synthesized standards prepared in our laboratory (Cullen et al., 2016). DMAGIII stock solutions also contained 2% methanol (Fisher Scientific, Nepean, ON, Canada). Solutions of PAOIII, 3-nitro-4-hydroxyphenylarsonic acid [Rox], and 4-aminobenzenearsenic acid [p-ASA] in DI water were also prepared from commercially available standards (Sigma/Aldrich). PAOIII stock solutions contained 2% methanol, and p-ASA stock solutions contained up to 10% methanol (Fisher Scientific). Table S1 in Appendix A presents a list of the prepared arsenic species with their chemical structure. Solutions were sterilized via filtration (0.22 μm) and the final concentration of arsenic in each solution was calibrated using an Agilent 7500cs inductively coupled plasmamass spectrometry (ICP-MS) system (Agilent Technologies, Japan). AsIII, AsV, and PAOIII stock solutions were stored at 4°C until use. Rox and p-ASA solutions re-precipitate over time in DI water at 4°C and had to be used within a week of preparation. All remaining arsenic solutions are unstable at 4°C and had to be prepared fresh the day of treatment.

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further toxicological testing through the development of predictive in vitro assays (Xia et al., 2008; Judson et al., 2010). The features of high-throughput and real-time cytotoxicity monitoring make real-time cell sensing a desirable tool for prioritizing chemicals for surveillance and regulatory consideration. The objective of this study is to examine the relative cytotoxicity and cellular responses of fourteen arsenic species in two human cell lines using a real-time cell sensing technique. We choose two cell lines, A549 and T24, for this study. A549 cells, derived from a human alveolar adenocarcinoma, have been shown to express arsenic (+3 oxidation state) methyltransferase (As3MT) (Sumi et al., 2011). T24 cells, derived from a human urinary bladder carcinoma, lack the ability to methylate inorganic AsIII (Drobna et al., 2005). The enzyme responsible for methylation is known to be As3MT, which catalyzes the transfer of a methyl group from the donor molecule, S-adenosylmethionine (Lin et al., 2002). Therefore, these two cell lines represent cells that are either able or unable to methylate arsenic. The species under investigation include the inorganic arsenicals [AsIII, AsV], their trivalent methylarsenical metabolites [MMAIII, DMAIII], pentavalent methylarsenical metabolites [MMAV, DMAV], and thiolated metabolites [DMAGIII, MMTTAV, DMMTAV, DMDTAV]. The study also included three pheylarsenicals [PAOIII, Rox, p-ASA]. We choose the cell-electronic sensing technique because it is able to provide real-time monitoring of cytotoxic response of the test cells to the arsenicals. The dynamic monitoring provides information necessary for generating quantitative IC50 histograms, revealing important cytotoxic responses that are dependent on cell type, arsenic species, concentration, and exposure time. In addition, the determination of cytotoxic response using a single assay will allow for comparison of the relative cytotoxicity of the arsenic species under investigation. Hence, the results of this study will provide information that can be used to better inform the human health risk assessment and pharmaceutical application of arsenicals.

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The principles of the impedance-based real-time-cellelectronic sensing (RTCES) system (ACEA Biosciences, San Diego, CA, USA) have been described previously (Xing et al., 2005; Boyd et al., 2008; Moe et al., 2016). The measured impedance is automatically converted to cell index (CI). Increases in CI result from an increase in the number of cells adhered to the microelectrodes (via cell proliferation), an increase in cell adhesion, or an increase in cell spreading. Decreases in CI occurs when the number of cells decreases due to cell death, detachment from the microelectrodes, and morphological changes. Therefore, changes in CI can represent multiple cytological responses to the testing compound. Cells were seeded into 96-well or 16-well E-plates of the 96×- or 16×-RTCA systems (ACEA Biosciences, San Diego, CA) at pre-calibrated concentrations that allowed for a CI of 1 to be reached between 18 and 24 hr after seeding to standardize the treatment time of replicate experiments. A549 cells were seeded at 4000–4500 cells per well and T24 cells at 3500–4000 cells per well. When a CI of 1 was reached, the arsenic solutions described above were serially diluted in the respective media of the A549 and T24 cell lines to achieve the proper dose range for quantitative analysis. Two hundred microliters of each treatment concentration was added to triplicate wells. Negative controls (untreated media) and solvent controls (methanol) were added at a volume of 200 μL to triplicate wells. After treatment, CI was measured at hourly intervals for at least 70 hr post-exposure. A minimum of three separate experimental runs with all corresponding negative and solvent controls were performed for each arsenic species on each cell line (n ≥ 3). CI scales were normalized using multiplication factors for easier visual display of results.

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Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004

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volume was 30 μL. An Agilent 7500cs ICP-MS system was used 346 as the detector. 347

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The determination of IC50 values over time and the statistical analysis of data (two-way analysis of variance with Bonferroni post-tests, t-test) were performed using Prism 5.0 (Graph Pad Software Inc., San Diego, CA, USA). IC50 values were defined as the concentration of arsenical that resulted in a 50% reduction in normalized CI as compared to the normalized CI of non-treated control cells at a given time point.

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2. Results and discussion

1.5. Determination of arsenic species in media of treated A549 cells A549 cells were seeded into standard 96-well culture dishes (Corning, NY, USA) at a density of 4.5 × 104 cells/mL. When the cells reached 50%–60% confluency, the AsIII, MMAIII, DMAIII, DMAGIII, AsV, MMTTAV, DMMTAV, and DMDTAV solutions were serially diluted in media to produce the desired treatment concentrations based on the 24 hr IC50 values for A549 cells determined using RTCA. Due to the available quantity of DMDTAV standard, a treatment concentration of only ¼ IC50 (1.05 mmol/L) was prepared. Each treatment concentration was added to 24 wells, and 150 μL of media from 3 replicate wells was collected at each of 8 post-exposure time points. The selected time points for analysis were at the time of treatment (0 hr) and at 3, 6, 9, 12, 24, 36, 48, and 56 hr post-exposure. High-performance liquid chromatography (HPLC) (Agilent 1100 series; Agilent Technologies) separation of arsenic species was performed on a Prodigy™ ODS-3 column (3 μm particle size, 100 Å, 100 × 4.6 mm; Phenomenex, CA, USA). The mobile phase was prepared as follows: 5 mmol/L tetrabutylammonium hydroxide, 200 mmol/L malonic acid, and 5% methanol, with the pH adjusted to 5.85. The flow rate was maintained at 1.2 mL/min for the entire 6 min. The column temperature was maintained at 50°C. The injection

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To illustrate the cell-electronic sensing method for cytotoxicity assessment of the fourteen arsenicals, we first tested inorganic arsenicals, AsIII and AsV. Fig. 1 shows the real-time sensing of cellular response of A549 and T24 cells to AsIII and AsV. The top two graphs in Fig. 1 show the normalized cell index (CI) over time after the specified concentration of arsenic species was added to the cell medium. They represent typical real-time cytotoxicity response profiles of AsIII and AsV in A549 (Fig. 1a) and T24 (Fig. 1b) cells. The concentration-dependent cytotoxicity was observed for A549 cells (Fig. 1a, top panel) between 40 and 250 μmol/L AsIII and for T24 cells (Fig. 1b, top panel) between 1 and 75 μmol/L AsIII. Likewise, the dose–response for AsV in A549 cells (Fig. 1a, middle panel) was in the mmol/L range (1–5 mmol/L) and in the μmol/L range (20–500 μmol/L) for T24 cells (Fig. 1b, middle panel). These results indicate that T24 cells are more sensitive than A549 cells to both inorganic arsenicals. The cell-dependent and speciation-dependent arsenic cytotoxicity is further demonstrated by the quantitative IC50 histograms (in μmol/L, mean ± SEM), as shown in the bottom graphs of Fig. 1. The IC50 histograms were obtained from the corresponding real-time dose–response profiles (Fig. 1 top two graphs) and represent the concentration of arsenical that results in a 50% reduction in CI compared to the untreated control cells at a given time point. The data at 24-hr exposure on Fig. 1 is indicated with a dotted line. The display of IC50 histograms is a unique feature of the real-time cell sensing, which is difficult to obtain using conventional endpoint cytotoxicity assays. These histograms show that AsIII has significantly lower IC50 values throughout the exposure period, supporting that AsIII is more cytotoxic than AsV in both cell lines. These results are consistent with previous reports comparing cytotoxicity of AsIII and AsV (Styblo et al., 2000; Vega et al., 2001; Hirano et al., 2003; Charoensuk et al., 2009).

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Cells were seeded into standard 6-well culture dishes (Greiner BioOne, Oakville, ON, Canada) at concentrations that had previously been determined to allow the cells to reach 50%– 60% confluency after 18–24 hr growth. A549 cells were seeded at a density of 4.5 × 104 cells/mL and T24 cells at 4.0 × 104 cells/mL. When the cells had reached the proper confluency, the AsIII, MMAIII, and DMAIII solutions described above were serially diluted in media to produce concentrations equivalent to the 24 hr IC50 value and ½ IC50 value, as determined using RTCA. The old media in each well was aspirated and replaced with the treated media, with triplicate wells prepared for each concentration. The control wells (without arsenic treatment) were also prepared in triplicate. Cells were incubated for 24 hr. After 24 hr exposure, cells were washed twice with phosphate buffered saline (PBS; Gibco), detached using 0.05% trypsin–EDTA, and suspended in fresh media. Cells were pelleted by centrifugation at 1700 r/min for 3 min and washed with ice-cold PBS. The cells were then counted using a hemocytometer. After pelleting again at 1000 r/min for 10 min, the PBS was carefully aspirated to avoid disturbing the pellet. The cell pellets were then resuspended in 2% HNO3 (Fisher Scientific) and sonicated in a water sonicator (Fisher Scientific) for 30 min for lysis. After pelleting the now lysed cells at 2500 r/min for 30 min, the supernatant (containing the intracellular components) was carefully collected to avoid disturbing the pellet and placed into a fresh 0.5 mL microcentrifuge tube (Fisher Scientific). Because these samples were prepared for total arsenic analysis, samples were parafilmed and stored at 4°C until analysis by ICP-MS with either an ELAN 6000 ICP-MS system (PerkinElmer, Waltham, MA, USA) or an Agilent 7500cs ICP-MS system.

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Fig. 2 presents the cytotoxicity response profiles of A549 and 396 T24 cells exposed to the several methylated and thiolated 397 arsenicals, MMAIII, DMAIII, DMAGIII, MMTTAV, DMMTAV, and 398

Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004

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IC50 ( mol/L)

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DMDTAV. These cytotoxicity response profiles demonstrate the concentration-, cell-, and species-dependent nature of arsenical cytotoxicity. For the testing of toxicity of MMAIII, both the methyldiiodoarsine and MAOIII forms were used. They both form the same MMAIII species in solution. Therefore, it is expected to observe similar cytotoxicity of these species. Indeed, our results (Fig. 2a and c) show that both response profiles are similar when both starting forms of MMAIII were tested. The response profiles of DMAIII (Fig. 2b) show a step-wise shape in the concentration range of 7.5–10 μmol/L for the A549 cells and 5–7 μmol/L for the T24 cells. The profile shapes of the thiolated trivalent arsenic species, DMAGIII (Fig. 2d), in both cell lines are similar to those of DMAIII (Fig. 2b). The step-wise profile shape is visible in the concentrations of DMAGIII between 10 and 25 μmol/L for A549 cells and 2.5– 5 μmol/L for T24 cells. The step-wise response may be due to the conversion of DMAIII and DMAGIII in solution to DMAV over the testing period, as this will be discussed later. The cytotoxicity response profiles for the pentavalent thiolated arsenic metabolites, MMTTAV (Fig. 2e), DMMTAV (Fig. 2f), and DMDTAV (Fig. 2g), also show distinct speciesdependent responses in both A549 and T24 cells. DMMTAV (Fig. 2f) shows step-wise profile shapes that are similar to those of DMAIII (Fig. 2b) and DMAGIII (Fig. 2d). In A549 cells, this step-wise profile shape is seen with the concentrations between 25 and 30 μmol/L DMMTAV (Fig. 2f), while in T24 cells, it is seen with concentrations between 5 and 7 μmol/L DMMTAV (Fig. 2f). The CI response shapes of

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Fig. 1 – Normalized cell index (CI) and IC50 values (μmol/L) obtained from continuous, real-time cell-electronic sensing of A549 and T24 cells exposed to AsIII and AsV. Qualitative and quantitative cellular response characterization of AsIII and AsV exposure in (a) A549 and (b) T24 cells. The hourly dose–response data from the cytotoxicity profiles of CI over time are used to generate temporal IC50 histograms of the hourly IC50 values (μmol/L mean ± SEM) over the exposure period. The data collected at 24 hr is indicated with a dotted line.

MMTTAV (Fig. 2e) and DMDTAV (Fig. 2g) are characterized by initial increase of CI values follow by a rapid decrease. Although the reasons for the distinct profiles are not known, possibility of different biological effects and the involvement of arsenic species conversion during cell incubation cannot be ruled out. Real-time cell sensing of A549 and T24 cells incubated with PAOIII (Fig. 3a), Rox (Fig. 3b), and p-ASA (Fig. 3c) shows that the trivalent PAOIII is much more cytotoxic than the two pentavalent phenylarsenicals. PAOIII is highly cytotoxic with a dose–response in the nmol/L range, consistent with previous report (Charoensuk et al., 2009). Rox and p-ASA exhibit a dose– response in the mmol/L range, five to six orders of magnitudes higher than PAOIII. Our results of Rox and p-ASA are consistent with previous cytotoxicity studies, which reported p-ASA dose–response ranges of 0.05–50 mmol/L in rat primary hepatocytes (Yuan et al., 2006) and 1–25 mmol/L in mouse embryonic stem cells (Kang et al., 2014), both determined using the tetrazolium (MTT) colorimetric assay. To quantitatively compare the relative cytotoxicity of the organoarsenicals, we generated temporal IC50 histograms from the concentration–response profiles for each arsenical tested in both the A549 and T24 cell lines. As an example to illustrate the range of IC50 values that were determined amongst the tested arsenicals, Fig. 4 shows the IC50 values over time for the most cytotoxic PAOIII, in comparison to inorganic AsIII and the much less cytotoxic Rox, in both the A549 and T24 cells. These results demonstrate the strong influence of arsenic speciation on cytotoxicity, as the IC50

Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456

6

J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 6 ) XXX –XXX

A549

20

T24

30

40

50

60

70

0

10

20

30

40

50

60

70

60

70

0

10

20

30

40

50

60

70

Time (hr)

50

60

70

0

10

20

30

40

50

60

70

10

30

40

50

60

0

f. DMMTAV T24

30

40

50

60

70 0

10

20

30

40

50

60

70

10 9 8 7 6 5 4 3 2 1 0 0

A549

10

20

20

30

40

50

60

70

0

10

20

Time (hr)

40

50

60

70

Control 5 / 2.5 mol/L 25 / 5 mol/L 30 / 7 mol/L 40 / 10 mol/L 50 / 12.5 mol/L 60 / 15 mol/L 100 / 30 mol/L 30

40

50

60

70

D

Time (hr)

A549

T24

10

20

30

40

Time (hr)

50

60

70

0

10

20

30

40

50

60

70

Control 100 mol/L / --125 / 5 mol/L 250 / 25 mol/L 500 / 50 mol/L 1,000 / 100 mol/L 2,500 / 250 mol/L 7.5 / 1 mmol/L 15 / 5 mmol/L

Time (hr)

C

T

E

Normalized Cell Index

g. DMDTAV 10 9 8 7 6 5 4 3 2 1 0 0

30

T24

Time (hr)

Time (hr)

10

Time (hr)

Time (hr)

Control 25 / 1 mol/L 200 / 10 mol/L 300 / 20 mol/L 400 / 30 mol/L 500 / 40 mol/L 1,000 / 50 mol/L 6,000 / 250 mol/L

Control 5 / 2.5 mol/L 10 / 4 mol/L 20 / 5 mol/L 22.5 / 6 mol/L 25 / 7 mol/L 30 / 9 mol/L 50 / 15 mol/L

T24

20

Time (hr)

A549

20

A549

F

40

10 9 8 7 6 5 4 3 2 1 0 0

R O

Normalized Cell Index Normalized Cell Index

50

O

30

e. MMTTAV

10

40

P

20

Control 1 / 0.25 mol/L 10 / 1.5 mol/L 11 / 2 mol/L 13.5 / 2.5 mol/L 15 / 3 mol/L 17.5 / 3.5 mol/L 20 / 5 mol/L

T24

Time (hr)

10 9 8 7 6 5 4 3 2 1 0 0

30

20

d. DMAGIII

A549

10

10

Control 1 / 1 mol/L 7.5 / 5 mol/L 10 / 6 mol/L 15 / 6.5 mol/L 20 / 7 mol/L 25 / 8 mol/L 30 / 15 mol/L

T24

A549

Time (hr)

c. MAOIII 10 9 8 7 6 5 4 3 2 1 0 0

10 9 8 7 6 5 4 3 2 1 0 0

Time (hr)

Time (hr)

Normalized Cell Index

10

Control 1 / 0.25 mol/L 8 / 1 mol/L 10 / 2 mol/L 12 / 2.5 mol/L 15 / 3 mol/L 18 / 4 mol/L 30 / 5 mol/L

Normalized Cell Index

Normalized Cell Index

10 9 8 7 6 5 4 3 2 1 0 0

Normalized Cell Index

b. DMAIII

a. MMAIII

R

R

E

Fig. 2 – Cytotoxicity response profiles of the normalized cell index (CI) over time for A549 and T24 cells exposed to (a) MMAIII, (b) DMAIII, (c) MAOIII, (d) DMAGIII, (e) MMTTAV, (f) DMMTAV, and (g) DMDTAV. The tested concentration range for each arsenical is included for both A549 (left) and T24 (right) cell lines. Untreated control cells are indicated in red in each cytotoxicity profile. MMAIII: monomethylarsonous acid; DMAIII: dimethylarsinous acid; MAOIII: methylarsine oxide; DMAGIII: dimethylarsinic glutathione; MMTTAV: monomethyltrithioarsonate; DMMTAV: dimethylmonothioarsinate.

463

2.3. Accumulation of arsenic in A549 and T24 cells

464

To explore whether the cellular uptake and accumulation of arsenic contribute to the observed differences in the cytotoxicity between A549 and T24 cells, we first determined the intracellular concentrations of total arsenic in A549 and T24 cells after 24 h exposure to AsIII, MMAIII, and DMAIII. Table 1 shows the average amounts of arsenic in A549 and T24 cells after the cells were incubated with arsenic species at concentrations equivalent to the respective 24-hr IC50 values or half the IC50 values. The average number of 4.35 × 108 arsenic atoms/cell in T24 cells treated with 6.9 μmol/L AsIII was almost twice the amount (2.55 × 108 As atoms/cell) found in A549 cells treated with 76.6 μmol/L AsIII. These results suggest that T24 cells are capable of more efficient uptake

461

465 466 467 468 469 470 471 472 473 474 475 476

C

460

N

459

U

458

O

462

values range from nmol/L for PAOIII to μmol/L for AsIII and to mmol/L for Rox. IC50 histograms of other tested arsenic species are shown in Appendix A Fig. S1. From continuous real-time cell sensing, these results provide information on the toxicity of each arsenic species to the cells over the entire 70-hr incubation period.

457

and/or retention of AsIII than A549 cells. The concentrations of intracellular arsenic in cells treated with the 24-hr IC50 value concentrations of MMAIII were similar in both cell lines at around 0.40 × 108 arsenic atoms/cell. Because the concentration of MMAIII present in the cell culture medium of the treated T24 cells (1.9 μmol/L) was nearly seven times lower than in the medium of the treated A549 cells (13.6 μmol/L), the similar amounts in the cells suggest that T24 cells are also capable of more efficient uptake and/or retention of arsenic than A549 cells exposed to MMAIII. In the case of incubation with DMAIII, T24 cells contained 0.44 × 108 arsenic atoms/cell after 24-hr incubation with 6.4 μmol/L μM DMAIII, while A549 cells contained 0.80 × 108 arsenic atoms/cell after 24-hr incubation with 14.1 μmol/L DMAIII (Table 1). Another reason for the observed differences in the cytotoxicity between A549 and T24 cells may be related to the differences in the ability of these cells to methylate arsenic. The enzymatic methylation of inorganic arsenic is an important step in arsenic metabolism to promote elimination. The enzyme responsible for methylation is known to be arsenic (+3 oxidation state)-methyltransferase (As3MT), which catalyzes the transfer of a methyl group from the

Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004

477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498

7

J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 6 ) XX X–XXX

10 9 8 7 6 5 4 3 2 1 0 0

A549

10

20

30

40

50

60

70

Control 0.1 µ mol/L 0.4 µ mol/L 0.6 µ mol/L 0.7 µ mol/L 0.8 µ mol/L 0.85 µ mol/L 0.9 µ mol/L 0.95 µ mol/L 1 µ mol/L 1.6 µ mol/L

Normalized Cell Index

Normalized Cell Index

a. PAOIII 10 9 8 7 6 5 4 3 2 1 0 0

T24

10

20

Time (hr)

30

40

50

60

70

Control 0.01 µ mol/L 0.05 µ mol/L 0.075 µ mol/L 0.1 µ mol/L 0.125 µ mol/L 0.15 µ mol/L 0.175 µ mol/L 0.225 µ mol/L 0.5 µ mol/L

Time (hr)

10

20

30

40

50

60

70

T24

10

20

Time (hr)

30

40

50

60

70

40

50

60

70

Control 2 mmol/L 4 mmol/L 6 mmol/L 7 mmol/L 8 mmol/L 9 mmol/L 10 mmol/L 12 mmol/L

10

20

30

40

50

60

70

Time (hr)

T

Time (hr)

T2 4

P

Normalized Cell Index

10 9 8 7 6 5 4 3 2 1 0 0

D

20

Control 3 mmol/L 11 mmol/L 11.5 mmol/L 12 mmol/L 13 mmol/L 14 mmol/L

E

Normalized Cell Index

A5 4 9

10

30

Time (hr)

c. p-ASA 10 9 8 7 6 5 4 3 2 1 0 0

Control 1 mmol/L 3 mmol/L 4 mmol/L 4.5 mmol/L 5.25 mmol/L 5.75 mmol/L 6 mmol/L 6.25 mmol/L 7 mmol/L 8 mmol/L

F

10 9 8 7 6 5 4 3 2 1 0 0

O

Control 8 mmol/L 10 mmol/L 10.5 mmol/L 11 mmol/L 11.5 mmol/L 12 mmol/L 13 mmol/L 14 mmol/L

A549

R O

10 9 8 7 6 5 4 3 2 1 0 0

Normalized Cell Index

Normalized Cell Index

b. Rox

E

C

Fig. 3 – Cytotoxicity response profiles of the normalized cell index (CI) over time for A549 and T24 cells exposed to (a) PAOIII, (b) Rox, and (c) p-ASA. PAOIII: phenylarsine oxide; Rox: Roxarsone; p-ASA: p-arsanilic acid.

400 300 200 100 4 2 0 0

R 10

20

30

40

50

60

Rox III

As PAOIII

70

Time (hr)

b. T24 9500 8000 6500 5000 90 50 10 8 6 4 2 0 0

U

IC50 (µ mol/L)

R

15000 12000 9000

N C O

IC50 (µ mol/L)

a. A549

Rox III

As PAOIII

10

20

30

40

50

60

70

Time (hr) Fig. 4 – Temporal IC50 histograms of the hourly IC50 values (μmol/L mean ± SEM) for (a) A549 and (b) T24 cells exposed to PAOIII, inorganic AsIII, and Rox. PAOIII: phenylarsine oxide; AsIII: arsenite; Rox: Roxarsone.

donor molecule, S-adenosylmethionine (AdoMet) (Lin et al., 2002). In the pathway of oxidative methylation, As3MT catalyzes the conversion of AsIII to MMAV and MMAIII to DMAV. A549 cells, derived from a human alveolar adenocarcinoma, have been shown to express As3MT (Sumi et al., 2011). However, T24 cells, derived from a human urinary bladder carcinoma, lack the ability to methylate inorganic AsIII (Drobna et al., 2005). Hence, the observed difference in cytotoxicity and accumulation of the arsenicals in T24 and A549 cells is likely influenced by the inability of T24 cells to methylate AsIII. The higher cytotoxicity observed in T24 cells exposed to AsIII or MMAIII is consistent with the fact that T24 cells lack the enzyme to catalyze the conversion of AsIII to the less toxic MMAV, or the conversion of MMAIII to the less toxic DMAV. It is also known that methylation affects clearance of arsenic from cells and tissues. Previous reports have shown higher accumulation of total arsenic in non-methylating versus methylating cell lines (Styblo et al., 2000; Dopp et al., 2005, 2008, 2010). Retention of arsenic species in tissues of As3MT knockout mice was also higher than in the wild-type mice (Drobna et al., 2009; Chen et al., 2011; Naranmandura et al., 2013; Currier et al., 2016). Thus, it is reasonable that T24 cells would have higher accumulation of arsenic after exposure to AsIII or MMAIII than A549 cells. Our results of higher accumulation of arsenic in T24 cells than A549 cells exposed to AsIII or MMAIII are consistent with the differences in the ability of these cells to methylate arsenic.

Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004

499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525

8

Table 1 – Concentrations of total arsenic in A549 and T24 cells after 24-hr exposure to AsIII, MMAIII, and DMAIII at their respective IC50 and half IC50 concentrations.

t1:3

t1:4

A549 As in medium ⁎

t1:5

As in cells

MMAIII DMAIII

– 76.6 μmol/L 38.3 μmol/L 13.6 μmol/L 6.8 μmol/L 14.1 μmol/L 7.05 μmol/L

IC50 ½ IC50 IC50 ½ IC50 IC50 ½ IC50

U

2500 2250 1750

50

P

15

300 150 0

MMTTA V

5000 3750 2500

0 0

20 30 40 Time (hr)

50

20 30 40 Time (hr)

Concentration ( µ mol/L)

16 8 0

DMAGIII

50 40 30

10 0

50

16

8 4 0 75

DMMTA V

60 45 30

10

20 30 40 Time (hr)

50

10

30 20 40 Time (hr)

50

h. 1050 µmol/L DMDTAV DMA V DMMTA V DMA III DMDTA V

12

0 0

DMAV DMAGIII

20 10

15 10

IC50 (µ mol/L)

DMAIII

25

g. 20.5 µmol/L DMMTAV Species 1 Species 2 Species 3

450

32 24

60

20

5 0

50

1250 20 30 40 Time (hr)

0

6250

AsV

1250 10

D

T

20 40 30 Time (hr)

600

C

2000

N

Concentration ( µ mol/L)

AsV

3000

0

10

5

30

f. 600 µmol/L MMTTAV

4000

1000

R

5 0

50

MMAIII

10

10

O

20 40 30 Time (hr)

Concentration ( µ mol/L)

10

3250 IC50 (µ mol/L)

15 10

e. 1400 µmol/L As V

750 0

20

DMA V DMA III

15

Concentration ( µ mol/L)

85 70

25

R

AsIII

IC50 (µ mol/L)

100

55 0

0 30

150

IC50 (µ mol/L)

IC50 (µ mol/L)

350

5

20

DMDTA V

1600 1200 800 400 0

15000 10000

IC50 (µ mol/L)

0

10

Concentration ( µ mol/L)

20

MMAV MMAIII

15

IC50 (µ mol/L)

40

20

C

60

d. 23 µmol/L DMAGIII

c. 14.1 µmol/L DMAIII

E

AsV AsIII

Concentration ( µ mol/L)

Concentration ( µ mol/L)

80

R O

O

b. 13.6 µmol/L MMAIII

a. 74.2 µmol/L As III

media of A549 cells incubated with AsIII, MMAIII, DMAIII, DMAGIII, AsV, MMTTAV, DMMTAV, and DMDTAV. The initial concentrations of these arsenic species were equivalent to the respective 24-hr IC50 values of these arsenic species, and the concentrations of arsenic species in the cell culture were determined using HPLC–ICP-MS (e.g., Fig. S2 in Appendix A). These results show that the inorganic arsenicals AsIII (Fig. 5a)

E

Because the trivalent methylarsenicals are not stable at room temperature and can be oxidized to the pentavalent methylarsenicals (Gong et al., 2001), we determined arsenic speciation in the cell culture medium over time. Fig. 5 shows the concentrations of main arsenic species in the culture

531

1.5 ± 0.6 435 ± 43 114 ± 9.3 40 ± 5 5.3 ± 0.4 44 ± 15 22 ± 6

AsIII: arsenite; MMAIII: monomethylarsonous acid; DMAIII: dimethylarsinous acid. As atoms/cell (×106) values were mean ± SEM (n = 3). ⁎ As in medium represents the treatment concentrations of arsenic species that corresponded to IC50 and ½ IC50 values.

527

530

– 6.9 μmol/L 3.45 μmol/L 1.9 μmol/L 0.95 μmol/L 6.4 μmol/L 3.2 μmol/L

2.2 ± 0.7 255 ± 9 59 ± 21 36 ± 3 6.8 ± 0.6 80 ± 7 23 ± 4

2.4. Arsenic speciation in culture media over time

529

As atoms/cell (×106)

F

Control AsIII

526

528

As in cells

As atoms/cell (× 10 )

Concentration ( µ mol/L)

t1:14 t1:15 t1:16 t1:18 t1:17

As in medium 6

t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13

T24

IC50 (µ mol/L)

t1:1 t1:2

J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 6 ) XXX –XXX

DMDTA V

7500 5000 2500 0 0

10

20 30 40 Time (hr)

50

Fig. 5 – Concentrations of arsenic species present in the culture media of A549 cells (top graphs) and IC50 histograms (bottom graphs) over the same incubation period and incubation temperature (37°C). Parallel experiments were carried out by incubating A549 cells with the arsenic compound at a concentration equivalent to its respective 24-hr IC50 value. The media was collected from A549 cells treated with (a) 74.2 μmol/L AsIII, (b) 13.6 μmol/L MMAIII, (c) 14.1 μmol/L DMAIII, (d) 23 μmol/L DMAGIII, (e) 1400 μmol/L AsV, (f) 600 μmol/L MMTTAV, (g) 20.5 μmol/L DMMTAV, and (h) 1050 μmol/L DMDTAV. Arsenic species were determined using HPLC–ICP-MS. AsIII: arsenite; MMAIII: monomethylarsonous acid; DMAIII: dimethylarsinous acid; DMAGIII: dimethylarsinic glutathione; AsV: arsenate; MMTTAV: monomethyltrithioarsonate; DMMTAV: dimethylmonothioarsinate. Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004

532 533 534 535 536 537 538

9

J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 6 ) XX X–XXX

550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573

t2:1

2.5. Cytotoxicity ranking of arsenic species

584

The 24-hr and 48-hr IC50 values for the fourteen arsenicals examined in this study are summarized in Table 2. Our IC50 values determined using the real-time cell sensing technique were consistent with results from other arsenic studies using traditional end-point cytotoxicity assays. The 24-hr IC50 value of 74 ± 4 μmol/L for AsIII in the A549 cell line was in good agreement with previously reported values of around 100 μmol/L for the same cell line, determined using MTT and cell counting assays (Talbot et al., 2008; Chiang and Tsou, 2009). The 24-hr IC50 values for MMAIII, DMAIII, and AsIII in T24 cells determined using the real-time cell sensing method were also consistent with results of MTT tests, where the potency of these arsenicals was MMAIII (2.5 μmol/L) > DMAIII, AsIII (>10 μmol/L) (Styblo et al., 2002). Our quantitative results were also consistent with Bartel et al. (2011), who reported IC70 values in A549 cells after 24 h exposure to arsenicals using cell counting methods. They found DMAIII (5.1 μmol/L) ≈ MMAIII (5.6 μmol/L) > DMMTAV (12.1 μmol/L) > AsIII (57.2 μmol/L) (Bartel et al., 2011), in comparison to our IC50 values: MMAIII (14 μmol/L) ≈ DMAIII (14 μmol/L) > DMMTAV (20 μmol/L) > AsIII (74 μmol/L). A second study from the Schwerdtle group determined 24-hr IC70 values in A549 cells using a cellular

585

F

549

O

548

R O

547

P

546

574

D

545

increase in IC50 values over time for DMMTAV in comparison to MMAIII, DMAIII, and DMAGIII is consistent with the partial conversion exhibited by the DMMTAV. These results show that the unique IC50 histograms for MMAIII (Fig. 5b), DMAIII (Fig. 5c), and DMAGIII (Fig. 5d) coincide with the conversion of the more toxic trivalent methylarsenicals to the less toxic pentavalent methylarsenicals. These results are understandable because the cells were actually exposed to the pentavalent arsenicals of lower toxicity during the later incubation time.

E

544

T

543

C

542

E

541

R

540

and AsV (Fig. 5e) were relatively stable over the entire incubation period at 37°C. The pentavalent thiolated methylarsenicals MMTTAV, DMMTAV, and DMDTAV also appeared to be stable, although MMTTAV and DMMTAV contained mixtures of arsenic species. Importantly, the three trivalent methylarsenicals, MMAIII (Fig. 5b), DMAIII (Fig. 5c), and DMAGIII (Fig. 5d), were oxidized over time. MMAIII was converted to the less toxic pentavalent MMAV, with complete conversion by 56 h exposure. DMAIII and DMAGIII were both converted to the less toxic DMAV over time. A complete conversion of DMAIII to DMAV was observed by 9 hr post-exposure, while the conversion of DMAGIII to DMAV was complete after 36 hr. Therefore, in the tests of toxicity of MMAIII, DMAIII, and DMAGIII, the actual concentrations of these highly toxic arsenic species experienced by the cells decreased over time. During the incubation period, the cells were actually exposed to a mixture of both the more toxic trivalent methylarsenicals and the less toxic pentavalent methylarsenicals. It is possible that the conversion of the more toxic arsenic species to the less toxic arsenic species in the culture medium could contribute, at least in part, to the observed unique profiles of cell index (e.g., Fig. 2). For further comparison, the IC50 histograms (bottom graphs in Fig. 5) are shown along with the HPLC–ICP-MS results of the arsenic species in the cell culture (top graphs in Fig. 5). The arsenicals that did not undergo conversion over time (AsIII, AsV, MMTTAV, DMDTAV) had decreasing CI values over time (Figs. 1 and 2) and correspondingly, decreasing IC50 values over time (Fig. 5). With no observed conversion, these arsenicals maintained their potency over the exposure period. However, the arsenic species (MMAIII, DMAIII, DMAGIII, DMMTAV) that exhibited conversion to less toxic arsenicals over time showed increasing CI values over time for mid-dose range treatments (Fig. 2), which resulted in increasing IC50 values over time after 20 h exposure (Fig. 5). The more gradual

Table 2 – The 24-hr and 48-hr IC50 values (μmol/L mean ± SEM) for fourteen arsenicals in A549 and T24 cells.

R

539

t2:4 t2:5

N C O

t2:3 t2:2

24-hr

III

a

A549

T24 48-hr q

24-hr

48-hr j

0.11 ± 0.01z,⁎ 1.1 ± 0.2aa 2.2 ± 0.2bb 5 ± 1cc 5 ± 1cc 8 ± 2cc 5 ± 1cc 29 ± 5dd 80 ± 8ee 63 ± 13ee,⁎ 560 ± 30ff,⁎ 2600 ± 240gg 6400 ± 620x 7200 ± 1900

PAO MAOIII MMAIII DMAIII DMAGIII DMMTAV AsIII MMTTAV AsV DMDTAV DMAV MMAV Rox p-ASA

t2:20 t2:21 t2:22 t2:24 t2:23 t2:25 t2:27 t2:26 t2:29 t2:28 t2:30

PAOIII: phenylarsine oxide; MAOIII: methylarsine oxide; MMAIII: monomethylarsonous acid; DMAIII: dimethylarsinous acid; DMAGIII: dimethylarsinic glutathione; DMMTAV: dimethylmonothioarsinate; AsIII: arsenite; MMTTAV: monomethyltrithioarsonate; AsV: arsenate; DMMTAV: dimethylmonothioarsinate; DMAV: dimethylarsinic acid; MMAV: monomethylarsonic acid; Rox: Roxarsone; p-ASA: p-arsanilic acid. a–gg : Denotes significant difference (p < 0.05), two-way analysis of variance of arsenical and cell line IC50 values at each time point with Bonferroni post-tests. ⁎: Denotes significant difference (p < 0.05), unpaired t-test of 24-hr and 48-hr IC50 values. 1 : Value not included in analysis (variable standard deviation for 24-hr IC50 values).

U

t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19

0.7 ± 0.1 12 ± 2b 14 ± 2b,c 14 ± 2b,c 23 ± 1c 20 ± 4b,c 74 ± 4d 600 ± 79e 1400 ± 130f 4300 ± 3000a 6030 ± 390g 28000 ± 1020i 9300 ± 1600g,h Undetermined

0.9 ± 0.1 14 ± 2r 17 ± 3r,s 17 ± 3r,s 29 ± 3s 21 ± 7r,s 67 ± 3t 280 ± 22u,⁎ 1100 ± 93v 230 ± 74u 3300 ± 170w,⁎ 25000 ± 280y 9300 ± 1200x Undetermined

0.08 ± 0.01 1.2 ± 0.4k 1.9 ± 0.1k 5 ± 1l 5 ± 1l 6 ± 1l 6.9 ± 0.5l 25 ± 2m 85 ± 6n 342 ± 95a 1500 ± 210o 2600 ± 350p 6800 ± 740h 6300 ± 3200

Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004

575 576 577 578 579 580 581 582 583

586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 Q5 605 606

10

J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 6 ) XXX –XXX

621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642

647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664

668 667

This study was supported by Alberta Health, Alberta Innovates, the Canada Research Chairs Program, the Canadian Institutes of Health Research (CIHR), and the Natural Sciences and Engineering Research Council (NSERC) of Canada. The authors acknowledge Dr. Hongsui Sun for synthesizing many of the arsenic compounds used in this study.

669

F

Acknowledgments

O

620

C

619

E

618

R

617

R

616

O

615

C

614

N

613

U

612

Appendix A. Supplementary data

670 671 672 Q6 673 674

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The cytotoxicity of more than a dozen arsenic compounds is dependent on the cell type, the arsenic species, and the concentration and exposure time of the arsenic species. The two selected cell lines represent those expressing As3MT arsenic methyltransferase (A549) or lack of this enzyme (T24) for arsenic methylation. The real-time cell sensing method enables continuous monitoring of the dynamic changes in cell index, providing qualitative and quantitative information for the assessment of arsenical cytotoxicity in real-time. The IC50 histograms show the relative cytotoxicity of the fourteen arsenic compounds in the test cell lines over the entire incubation period. The continuous monitoring over the entire cell incubation period reveals unique profiles that may suggest different processes/mechanisms involved in the cellular toxicity. Although the mechanisms responsible for the observed differences in temporal profiles are not fully understood, the results coincide with the conversion of the more toxic trivalent MMAIII, DMAIII, and DMAGIII to the less toxic pentavalent MMAV and DMAV. Further studies are

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dehydrogenase assays and found DMAGIII (5.8 μmol/L) > DMMTAV (20.5 μmol/L) > AsIII (97.0 μmol/L) (Leffers et al., 2013), which compare to our 24-hr IC50 values of DMAGIII (23 μmol/L) ≈ DMMTAV (20 μmol/L) > AsIII (74 μmol/L). Our IC50 values for DMAGIII were consistent with previous reports that found the cytotoxicity of DMAGIII was similar to that of AsIII in most of the tested cell lines (Styblo et al., 2000). Statistical analysis of the IC50 values at each individual time point (24 and 48 hr) quantitatively demonstrated celland arsenical-dependent cytotoxicity, as the IC50 values were determined to be statistically different (p < 0.05) across both the arsenical and cell line tested (two-way analysis of variance with Bonferroni post-tests). With the exception of Rox, IC50 values of T24 cells were significantly lower than IC50 values of A549 cells for all arsenicals at both time points (Table 2). On the basis of the 24-hr IC50 values in Table 2, the relative toxicities of arsenicals to A549 cells are: PAOIII > MMAIII ≈ DMAIII ≥ DMMTAV ≥ DMAGIII > AsIII > MMTTAV > AsV > DMDTAV > Rox > p-ASA. In T24 cells, the relative toxicities are: PAOIII > MMAIII > DMAIII ≈ DMAGIII ≈ DMMTAV ≈ AsIII > MMTTAV > AsV > DMDTAV > Rox ≈ p-ASA. In general, we found that the trivalent arsenicals were more cytotoxic than most pentavalent species, supportive of the trend reported in the literature (Styblo et al., 1997, 2000; Lin et al., 1999). DMMTAV, a pentavalent thioarsenical, was more cytotoxic than AsIII in the A549 cell line, and was equally cytotoxic as AsIII in the T24 cell line. The results of high toxicity of DMMTAV are consistent with other studies of DMMTAV (Naranmandura et al., 2009, 2011). Naranmandura et al. (2011) reported that DMMTAV was more cytotoxic than AsIII to EJ-1 cells. The authors also found that DMMTAV generated intracellular reactive oxygen species. Of the pentavalent thio-arsenicals examined in this study, DMMTAV was also the most cytotoxic in both cell lines, with a 24-hr IC50 value either one or two orders of magnitude lower than those of the other tested thio-arsenicals, MMTTAV and DMDTAV.

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Please cite this article as: Moe, B., et al., Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing, J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.10.004