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Jul 3, 2008 - Sydney, NSW 2006, Australia, X-Ray Science DiVision, Argonne National ... (XRF) and X-ray absorption spectroscopy (XAS) analyses were ...
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Microprobe XRF Mapping and XAS Investigations of the Intracellular Metabolism of Arsenic for Understanding Arsenic-Induced Toxicity Kristie L. Munro,† Anna Mariana,† Andrejs I. Klavins,† Amalanie J. Foster,‡ Barry Lai,§ Stefan Vogt,§ ZhongHou Cai,§ Hugh H. Harris,# and Carolyn T. Dillon†,* School of Chemistry, UniVersity of Wollongong, NSW 2522, Australia, School of Chemistry, UniVersity of Sydney, NSW 2006, Australia, X-Ray Science DiVision, Argonne National Laboratory, Argonne, Illinois 60439, School of Chemistry and Physics, UniVersity of Adelaide, SA 5005, Australia ReceiVed April 7, 2008

Arsenic (As) is responsible for mass-poisonings worldwide following the ingestion of As-contaminated drinking water. Importantly, however, As toxicity is exploited in the antileukemia drug, Trisenox (As2O3), which successfully cures 65-80% of patients suffering relapsed acute promyelocytic leukemia. In an effort to determine the intracellular organelle and biomolecular targets of As, microprobe X-ray fluorescence (XRF) and X-ray absorption spectroscopy (XAS) analyses were performed on HepG2 cells following their exposure to high doses of arsenite (1 mM) or arsenate (20 mM). Microprobe XRF elemental mapping of thin-sectioned cells showed As accumulation in the euchromatin region of the cell nucleus (following arsenite exposure) synonymous with As targeting of DNA or proteins involved in DNA transcription. X-ray absorption near edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS) analysis of arsenite-treated cells, however, showed the predominance of an As tris-sulfur species, providing increased credence to As interactions with nuclear proteins as a key factor in As-induced toxicity. Introduction Chronic arsenic (As) poisoning, arsenicosis, is associated with drinking As-contaminated water with toxicity observed following intake as low as 0.05 mg As/L for a period of 6 months (1–5). It is a problem predominantly affecting third-world countries where groundwater, particularly from tube wells, is the major source of drinking water, e.g., Argentina, Chile, China, Mexico, and India (1). In Bangladesh and West Bengal alone, more than 70 million people are at risk of drinking Ascontaminated water, and it is estimated that more than 4 million people currently suffer the effects of arsenicosis, namely, cancers of the skin (keratosis), kidney, bladder, lung, and liver, and black foot disease (gangrene) (1, 6). While the potential incidence of As-induced toxicity is clearly phenomenal, the mechanism of action of As is poorly understood (1). Paradoxically, the toxicity of As is exploited in Trisenox, an FDA1-approved treatment for relapsed and refractory acute promyelocytic leukemia (APL) (7). Intravenous administration of the arsenic trioxide-containing therapeutic results in complete remission in 60-85% of patients who have previously relapsed following treatment with the conventional therapy, all-transretinoic acid (7, 8). In addition, the use of As2O3 and other As compounds is being explored for the treatment of other leukemias and malignancies (9, 10). Clearly, the need to determine the mechanisms of As-induced toxicity is two-fold: (i) to improve our understanding of As-induced toxicities for the prevention of As-induced cancers and illnesses and (ii) to * To whom correspondence should be addressed. Tel: (612) 4221 4930. Fax: (612) 4221 4287. E-mail: [email protected]. † University of Wollongong. ‡ University of Sydney. § Argonne National Laboratory. # University of Adelaide.

assist in the design of future As-containing anticancer drugs (4). Currently, the proposed mechanisms of As-induced toxicity are numerous and include factors such as protein binding (e.g., tubulin-binding leading to the disruption of the mitotic spindle, adenine nucleotide translocator (ANT)- binding leading to mitochondrial induced apoptosis, and chronic stimulation of growth factors), interference with DNA repair processes and DNA methylation, and/or oxidative stress (involving radical damage) (8, 11–14). No single mode of action, if indeed one dominates, has been widely accepted for As-induced toxicities, and it is likely that the mode of action varies depending on the As species involved. Consequently, it is important to (i) determine the predominant intracellular targets of As, including both organelle and biomolecular targets and (ii) identify the chemical structure of As found at these targets to gauge the biomolecular interactions that lead to the observed toxicities. The determination of organelle targets of As should be readily achievable by 1 Abbreviations: ANBF, Australian National Beamline Facility; ANOVA, analysis of variance; ANT, adenine nucleotide translocator; APL, acute promyelocytic leukemia; APS, Advanced Photon Source; CHO-K1, Chinese hamster ovary (cell line); Cyt, cytoplasmic; DMA(III), dimethylarsinous acid; DMA(V), dimethylarsinic acid; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; ESI-MS, electrospray ionization-mass spectrometry; EXAFS, extended X-ray absorption fine structure; FDA, Food and Drug Administration; GM, growth medium (containing DMEM, fetal bovine serum, and penicillin/streptomycin); GSH, glutathione; GS, glutathiol; HepG2, human hepatoma (cell-line); HG-AAS, hydride generationatomic absorption spectroscopy; HPLC-ICP-MS, high performance liquid chromatography-inductively coupled plasma-mass spectrometry; MMA(III), monomethylarsonous acid; MMA(V), monomethylarsonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); Nuc, nuclear; PBS, phosphate-buffered saline; SH, sulfhydryl; SR, synchrotron radiation; SRIXE, synchrotron radiation-induced X-ray emission (spectroscopy); UVB, ultraviolet B (radiation); XANES, X-ray absorption near edge spectroscopy; XAS, X-ray absorption spectroscopy; XRF, X-ray fluorescence (spectroscopy).

10.1021/tx800128d CCC: $40.75  2008 American Chemical Society Published on Web 07/03/2008

Micro-XRF and XAS Studies of Intracellular Arsenic Scheme 1. Classical Metabolic Pathway for As (21–23)

Scheme 2. Metabolic Pathway of As Proposed by Hayakawa (24)

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(III) (see Scheme 2), on reverse-phase C18 columns during HPLC-ICP-MS analysis (29). In contrast, XAS is highly sensitive to the oxidation state, coordination geometry, and coordination sphere of As. It has been successfully employed for the direct analysis of As species in water samples (30) and biological samples, such as plants (31, 32) and earthworms (33), following minimal sample preparation procedures. In addition, the technique has recently been combined with microprobe XRF (our studies and those of Ortega (34)), establishing that As(III) is the ultimate product in cells and mammalian tissue. Unfortunately, however, the sensitivity of microprobe XAS failed to provide high quality postedge structure voiding discernment of the intracellular As species beyond identification of the oxidation state (34). In this work, attention was focused on the biological effects of inorganic As compounds, namely, arsenite and arsenate, since they represent a large proportion of the As species ingested by humans in As-contaminated drinking water and since arsenite is the active ingredient of Trisenox at physiological pH. The high sensitivity and spatial resolution of microprobe SR-XRF have been exploited to investigate the intracellular organelle targets of As. In addition, low temperature (10 K) XAS analysis of bulk samples has been used to improve the resultant XAS spectra in an effort to resolve the dominant biotransformation products and consequently biomolecular targets of arsenite in HepG2 cells. Implications for the mechanisms of toxicity are discussed on the basis of the data presented.

Experimental Procedures

microprobe SR-XRF (synchrotron radiation-X-ray fluorescence, also referred to as SRIXE (synchrotron-induced X-ray emission)) (15–17), which provides spatial detection of As to 0.2 µm resolution within mammalian cells without the need for fluorescent tagging, immuno- or radioactive- labeling (18–20). A knowledge of the biotransformation products of As, and consequently the species responsible for cellular damage, has been the subject of interest for some time. The ground-breaking work of Challenger (21) over 60 years ago and that of subsequent workers (5, 22, 23) showed that arsenite (the soluble form of arsenic trioxide) undergoes significant biotransformation reactions including sequential methylation and oxidation/reduction cycles resulting in a number of As metabolites in ViVo (Scheme 1) (5, 21–23). In 2005, Hayakawa et al. (24) highlighted the importance of [As(GS)3] and glutathiol-bound methylated As intermediates in the metabolic pathway (Scheme 2) based on the identification of [As(GS)3] as a major constituent in bile (25). A key criticism (26, 27) of previous analyses for determining the above-reported As metabolites has been limitations associated with the commonly used analytical procedures, such as HG-AAS (hydride generation-atomic absorption spectroscopy) or HPLC-ICP-MS (high performance liquid chromatographyinductively coupled plasma-mass spectrometry) (28). The main drawback of HG-AAS is the fact that the extensive preparative procedures and nature of the analytical technique used exclude the possibility of directly determining the original As oxidation states. Of concern is the finding that these techniques may also favor more stable species that withstand the preparation conditions (26, 27). For instance, important intermediates, such as [As(GS)3] and [MMAIII(GS)2], are known to degrade to the equivalent nonglutathiol-coordinated species, iAs(III) and MMA-

Caution: Arsenic compounds are carcinogenic and toxic. Due care should be taken to aVoid inhalation of the compounds and to aVoid contact with skin. Chemicals. All chemicals were of AR grade or better unless otherwise stated and were used as obtained. The As compounds administered in the cell culture assays were obtained from Sigma Aldrich: sodium arsenite (NaAsO2, 99%) and disodium hydrogenarsenate heptahydrate (Na2HAsO4.7H2O, >98%). For brevity, sodium arsenite and disodium hydrogenarsenate heptahydrate will be referred to as arsenite and arsenate, respectively, throughout the latter sections of the article. Standards for XAS analysis, chosen on the basis of metabolic mechanisms proposed in Schemes 1 and 2, were obtained directly from the Japanese distributor WAKO: sodium arsenite (99%), disodium hydrogenarsenate heptahydrate (99%), cacodylic acid (DMA(V), 98%), and monomethylarsonic acid (MMA(V), 95%). XAS standards for arsenic trisglutathione ([As(GS)3]) and the methylated As(III) complexes, [MMAIII(GS)2] and [DMAIII(GS)], were prepared using glutathione (GSH, >99%, Sigma) as described below. Cell Line. HepG2 human hepatoma cells were chosen for cell studies since the liver is the main site of metabolism where As methylation occurs. The cells were obtained from Dr. A. Ammit (University of Sydney) and were grown in growth medium (GM) containing Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, 11885-084) supplemented with 10% fetal bovine serum (Thermo Electron Corporation) and penicillin/streptomycin (100 IU/mL and 100 µg/mL, respectively, Thermo Electron Corporation). They were grown in a 37 °C, humidified, 5% CO2 atmosphere incubator (Revco Ultima, USA), and the cells were subcultured weekly to prevent overcrowding and cell death. Cytotoxicity. Cytotoxicity was assessed using a modified method of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay described by Mosmann (35) and Huxham (36). Briefly, a cell solution (1 × 105 cells/100 µL) was dispensed into each well of a 96-well plate (Becton Dickinson) and incubated for 24 h at 37 °C in the 5% CO2 incubator. The next day, fresh solutions of the specified As compounds were prepared in DMEM. To each well, 100 µL of As/DMEM solution was added, and the cells were

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incubated for 4 h. The treatment solution was then removed by aspiration and the cells were washed twice with phosphate buffered saline (PBS, 200 µL, pH 7.4, 0.172 M, Oxoid). Fresh GM (100 µL) was added, and the cells were left to recover in the incubator for 24 h. After the recovery period, filtered-MTT solution (50 µL, 2 mg/mL in PBS) was added to each well, and the plate was incubated for 4 h. The wells were carefully aspirated to remove the MTT solution and then washed twice with PBS. The remaining formazan was dissolved by the addition of DMSO (200 µL) followed by gentle agitation of the 96-well plate for 1 h. The absorbance was recorded at 570 nm using a Spectromax250 microplate spectrophotometer (Molecular Devices Corporation) with accompanying Softmax PRO 4.0 control software. Data was reported as the percentage obtained from the absorbance intensity ratio of the As-treated cells to the control cells (eq 1) for six replicate values.

Cell Viability (%) ) (A570As-treated cells/A570control cells) × 100% (1) The calculated cell viability (as determined by mitochondrial dehydrogenase activity) was then plotted against the As concentrations and, the IC50 value of each compound was determined directly from the graph as the concentration of As that inhibits 50% activity. Microprobe SR-XRF Mapping of Thin-Sectioned Cells. Preparation of Thin-Sectioned Cells. HepG2 cells were treated with arsenite (100 µL, 1 mM final concentration), arsenate (100 µL, 20 mM final concentration), or milli Q water (100 µL) in DMEM for 4 h at 37 °C. They were then washed, fixed, dehydrated, and thin-sectioned from Spurrs resin to produce 1 µm sections as described previously by Dillon (18–20) and detailed in the Supporting Information. XRF Data Collection and Analysis of As-Treated Cells. Prior to analysis, the samples were inspected and imaged using a Leica DMXRE Epi-fluorescence/visual microscope (Leica, Germany) at 200× magnification. Individual cells were then positioned in the beam by comparing the X-ray transmission image with the micrographs, using the markers on the grid. Cells were chosen for analysis on the basis of their healthy appearance and location away from any particulate material. Because of the high X-ray energy needed to excite the As K-edge and consequent leakage of unfocused X-rays through the central stop of the zone plate optics, comparatively high background scattering was detected. Consequently, the effects of scattering were reduced by analyzing cells that were located away from the gold grid. Hard X-ray microprobe experiments were performed on XOR beamline 2ID-D at the Advanced Photon Source (APS), Argonne National Laboratory (Chicago, USA). All measurements were conducted using a monochromatic 11.9 keV X-ray beam that was focused to submicron resolution using a stacked pair of zone plates. The sample was mounted on a high precision x,y,z motorized stage and was housed in a He atmosphere. Accurate location of the cells in the sections was achieved by collecting low resolution X-ray fluorescence maps at 2.5 µm steps over a scan area of 30 × 30 µm2. High resolution maps were collected at 0.3 µm steps, and the emitted X-rays were detected for either 1.5 or 3 s per point using a Vortex silicon drift detector (SII Nanotechnology, USA). Elements analyzed included P, Cl, S, K, Ca, Fe, Cu, Zn, and As. Elemental area densities (micrograms per square micron) were determined using the MAPS v1.6.0 software, by fitting the full fluorescence spectrum at every point to modified Gaussians and comparing with measurements on the thin film standards, NBS-1832 and NBS-1833 obtained from the National Bureau of Standards (Gaithersburg, MD, USA) (37, 38). Statistical analysis of the data was performed using the ANOVA Tukey-Kramer method whereby a value of p < 0.05 (p ) probability) was considered significant (39). XAS of As Standards and As-Treated Cell Pellets. Preparation of Standards. XAS spectra were recorded for As standards in aqueous solution (chosen on the basis of proposed metabolic Schemes 1 and 2) to be used as models for determining the intracellular As speciation. Arsenite (pH 5.5, 7.4, and 13), arsenate

Munro et al. (pH 4, 7.4, and 9), MMA(V), and DMA(V) solutions were freshly prepared in milli Q-water, to which glycerol (final concentration 30% v/v) was added as a glassing agent to produce final As concentrations of 10 mM. [As(GS)3] was prepared by the reaction of arsenite (300 µL, 30 mM) with GSH (300 µL, 150 mM) in Milli-Q water for 1 h. The resultant solution was mixed with glycerol (300 µL) for analysis. [MMAIII(GS)2] was produced by reaction of MMA(V) (300 µL, 30 mM) with GSH (300 µL, 150 mM) at 60 °C for 5 h. At the conclusion of the reaction, glycerol (300 µL) was added and the solution was mixed thoroughly. [DMAIII(GS)] was produced by heating DMA(V) (300 µL, 30 mM) with GSH (300 µL, 150 mM) at 37 °C for 5 h. After the reaction period, glycerol (300 µL) was added, and the solution was mixed thoroughly. All final solutions (500 µL) were injected into a polycarbonate solution cell and rapidly frozen in liquid nitrogen prior to insertion into the cryostat for XAS analysis. Characterization of the above-mentioned solutions of [As(GS)3], [MMAIII(GS)2], and [DMAIII(GS)] was also performed using ESI-MS to confirm that the desired product was the predominant As species (Supporting Information, Figures 1S-3S). Bulk cell preparation for XANES analysis was performed by treatment of 106 HepG2 cells in 60-mm2 dishes with arsenite solution (100 µM in DMEM, 4 h) to try to increase the probability of detecting any methylated intracellular As species. EXAFS analysis, however, was performed on cells treated with the IC50 value of arsenite (700 µM in DMEM, 4 h) to improve the signalto-noise ratio over an extended energy range. After the treatment period, the As/DMEM solution was removed, and the cells were washed twice with PBS to remove any extracellular As or detached cells. The cells were then harvested (using trypsin), and the cell pellet was washed twice with repeated centrifugation and PBS replacement. Following the final centrifugation, PBS was removed, and the cell pellet was rapidly frozen in liquid nitrogen. The cell pellet was stored in liquid nitrogen until immediately prior to traveling, at which point it was freeze-dried for 24-48 h and stored in a desiccator until analysis (40). Neat freeze-dried cell samples were pressed into 0.5-mm pellets supported in an Al spacer between two 63.5-µm kapton tape windows. Data Collection. Arsenic K-edge X-ray absorption spectra were collected for As frozen solution standards and bulk cell samples on the Australian National Beamline Facility (ANBF, beamline 20B) at the Photon Factory, Tsukuba, Japan. The beam energy was 2.5 GeV, and the beam current was 300-400 mA. A Si[111] channel-cut monochromator was detuned by 50%. All spectra were recorded in fluorescence mode at a temperature of ∼10 K (maintained with a closed-cycle He CryoIndustries REF-1577-D22 cryostat) using a 36-element Ge-array detector (Eurisys/Canberra Industries) and a 1 mm beam-defining vertical slit. An arsenate standard was simultaneously analyzed in transmission mode downstream of the sample for energy calibration purposes, and an average of 2-3 scans were performed for each of the data analyses. The energy of the first peak of the first derivative in the spectrum of the solid arsenate standard was assumed to be 11871.7 eV. Data Processing. Processing of XAS data was performed with EXAFSPAK (31, 41). Multiple linear regression analyses of XANES spectra were performed using the DATFIT module of EXAFSPAK. When fitting the As model spectra to the As-treated cells, all small components ( 0.05). While this might be clarified by analysis of a greater sample size, the current data indicates that As uptake into cells is associated with significant localization into the nucleus following acute exposure (4 h, 1 mM). This is also confirmed by analysis of the Nuc:Cyt As concentration ratios: control cells ) 1.2; arsenate cells ) 1.9; arsenite-treated cells ) 1.9. The distribution of Ca in the nucleus and cytoplasm across the range of cell treatments is shown in the bottom panel in Figure 4. A small, significant increase in the Ca concentration was observed in the nucleus versus the cytoplasm of the control cells ([Ca] Nuc:[Ca] Cyt ) 1.5; p < 0.05). The increase in Ca concentration is more pronounced in the nucleus versus that in the cytoplasm of the As-treated cells: arsenate-treated cells [Ca] Nuc:[Ca] Cyt ) 1.9, p < 0.001; arsenite-treated [Ca] Nuc:[Ca] Cyt ) 2.2, p < 0.001. In addition, the Ca concentration was higher in the nucleus of the arsenite and arsenate-treated cells versus the nucleus of the control cells (p < 0.01 in both instances). These results clearly show a trend toward higher Ca in the nucleus of As-treated cells. Importantly, no significant changes in the Ca concentrations were observed in the cytoplasm of the control and As-treated cells. Bulk Cell XAS Analysis. Figure 5 shows the K-edge X-ray absorption spectra of the As(III) and As(V) solution standards: (A) arsenite (pH 5.5) and (B) arsenate (pH 4); the relevant As(III) standards of the biotransformation products, (C) [As(GS)3], (D) [MMAIII(GS)2], and (E) [DMAIII(GS)], and (F) a HepG2 cell pellet obtained following treatment with arsenite (100 µM, 4 h). As expected, the As(III) species gave rise to edge energies and white line peaks approximately 3.5 eV lower than the As(V) species (white line energies: arsenite, 11870.6 eV; arsenate, 11874.1 eV). Coordination of As by three GS ligands results in a shift of the edge energy to lower eV (white line energy: 11869.81 eV) compared to that of arsenite. Close inspection of the near postedge structure reveals a significant depression in the absorption intensity at approximately 11875 eV for [As(GS)3]. Reduction of MMA(V) and DMA(V) to [MMAIII(GS)2] and [DMAIII(GS)], respectively, by GSH resulted in expected shifts to lower edge energies of the As(III) complexes. The white line energies are [MMAIII(GS)2], 11870.0 eV; MMA(V), 11873.4 eV; [DMAIII(GS)], 11870.7 eV; DMA(V), 11873.2 eV. Furthermore, the shoulder in the post edge region (at 11875 eV) becomes more evident as the number of coordinated GS ligands decreases. Close examination of the spectra also reveals that the edge increases slightly as the number of methyl groups increases, and the number of GS ligands consequently decreases, suggesting that the overall bonding

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Figure 2. Microprobe SR-XRF maps of P, S, Cl, K, Ca, Zn, Cu, and As obtained from a thin-sectioned HepG2 cell that had been treated with (A) milli-Q water (100 µL in 5 mL DMEM); scan dimensions, 16 × 18 µm2; stepsize, 0.3 µm; dwell-time, 1.5 s/pt; (B) arsenite (1 mM, 4 h); scan dimensions, 20 × 20 µm2; stepsize, 0.3 µm; dwell time, 3 s/pt; or (C) arsenate (20 mM, 4 h); scan dimensions, 16 × 13 µm2; stepsize, 0.3 µm; dwell time, 1.5 s/pt.

Micro-XRF and XAS Studies of Intracellular Arsenic

Figure 3. (A) Correlative light micrograph of a toluidine-blue stained thin section of HepG2 cells that have been exposed to arsenite (1 mM, 4 h) and (B) the corresponding microprobe SR-XRF maps of P, Zn, As, and the colocalization of the elements shown in Figure 2B.

change results in a more positively charged As in (E) compared that in (C). The spectrum of [MMAIII(GS)2] shifts to higher energy relative to [As(GS)3], although the shift is not as large as that observed by [DMAIII(GS)]. The lower symmetry of the methylated species produces a broader peak such that any shift to higher energy is masked under the low resolution conditions of the experiment. Figure 5F shows the spectrum obtained from HepG2 cells that had been exposed to arsenite (100 µM) for 4 h. The edge energy and the white line peak energy (11869.9 eV) are indicative of an As(III) species, and the shoulder at 11875 eV is much more pronounced than that expected for arsenite. Figure 6 shows the result of a multiple linear regression fit of the XANES spectra of the model compounds from Figure 5 to the XANES spectrum of the arsenite-treated HepG2 cells. While all potential metabolites were initially fitted to the HepG2 XANES spectrum, species such as arsenite (pH 9), arsenate (pH 4, 7.4 and 13), MMA(V), DMA(V), [MMAIII(GS)2], and [DMAIII(GS)] showed no significant contributions with fractions ranging from 10-7-10-13. It is clear from Figure 5 that the best fit to the models was obtained when the dominant species was [As(GS)3] (97%) with a small contribution (3%) from arsenite (pH 5.5) to give a residual of 35.4610 cm2 g-1. Importantly, the next best fit was obtained following sole contribution from [As(GS)3] (101%) (residual ) 36.2375 cm2 g-1). Figure 7 shows the best single-scattering fit to the EXAFS data recorded for HepG2 cells treated for 4 h with 700 µM arsenite. The parameters for this fit with three sulfur scatterers are shown in Table 1, along with an alternative and poorer fit

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Figure 4. Quantifications of As and Ca in the cytoplasmic (Cyt) and nucleus (Nuc) regions of the cells. All data were collected using a 0.3 µm stepsize and a 1.5 s dwell time. Columns represent the average mass of the specified element in a defined area (per µm2) where n ) 2-4, and the error bars represent the standard deviation from the mean. (Significant differences from the Control Cyt are represented by * p < 0.05, ** p < 0.01, *** p < 0.001, and differences from the Control Nuc are represented by # p < 0.05, ## p < 0.01, and ### p < 0.001.) In addition, significant differences (p < 0.001) were obtained for [Ca] between the cytoplasm and nucleus in arsenite- and arsenate-treated cells.

with only two sulfur scatterers. Attempts at fitting models with either carbon or oxygen scatterers, at ∼1.95 or 1.7 Å, respectively, diverged from physically realistic solutions.

Discussion The finding that there is negligible As in the control cells validates the use of microprobe SR-XRF as a technique for determining subcellular targets of As without the need of fluorescent tags or other labels that may alter the uptake and biotransformation of As. The microprobe SR-XRF elemental distribution maps obtained from the thin-sections of arsenitetreated HepG2 cells clearly indicate that As is capable of entering the cell nucleus where it can potentially interact with DNA or proteins involved in DNA replication. High dominance of P in areas morphologically identified as heterochromatin regions by the light microscopy image (Figure 3A and B) is consistent with reports by Quintana (47) that also showed a strong correlation between P and dense chromatin of mammalian cell nuclei. The exclusion of As from the regions of heterochromatin suggests that As penetration through the densely

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Figure 5. Arsenic K near-edge spectra of aqueous solutions of standards and arsenite-treated HepG2 cells: arsenite pH 5.5 (A), arsenate pH 4 (B), [As(GS)3] (C), [MMAIII(GS)2] (D), [DMAIII(GS)] (E), HepG2 cells treated with arsenite (100 µM, 4 h) (F).

Figure 6. Quantitative analysis of the intracellular HepG2 As by fitting the sum of aqueous As standard spectra. The spectra include the acquired data from the arsenite-treated HepG2 cells (A), the best fit (B), 2.9% aqueous arsenite at pH 5.5 (C), 98.2% aqueous [As(GS)3] (D), and the residual obtained from the fit (E).

packed chromatin does not occur. Furthermore, the localization of As in euchromatin is highly significant since the euchromatin region is an area of high DNA transcription, unlike the heterochromatin region, which generally consists of dormant (or conserved) DNA (46). This places As in the vicinity of active DNA and gives credence to the suggestion that it is causing damage either by interacting directly with DNA or by interacting with DNA binding proteins. On the basis of the review of Kitchin and Wallace (13), however, there is more evidence to suggest that As binds to proteins over DNA.

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The observed increase in nuclear Ca localization (Figures 2B and 4) is consistent with studies performed by Yee-Chien and Haimei (48) that showed that arsenite treatment of CHO-K1 cells triggered Ca accumulation in the nucleus. The importance of the redistribution of intracellular Ca is multifaceted. First, a loss of Ca2+ homeostatic control, including subtle changes in Ca2+ distribution within the cells, is an indicator of apoptosis (49). Furthermore, cytotoxic agents that cause oxidative stress and depletion of GSH (as arsenicals have been reported to do) were found to cause a lethal influx of Ca2+ or perturb intracellular Ca distribution (49). It is also known that As interacts with the SH groups of the adenine nucleotide translocator (ANT) (50) opening the mitochondrial permeability transition pore. Mitochondria play an important role in Ca2+ storage and are vital for Ca2+ homeostasis (51) such that mitochondrial-induced apoptosis is associated with changes in intracellular Ca. While Cai et al. (52) showed that high dose As exposure was associated with the collapse of the mitochondrial transmembrane potential believed to occur via interactions with the SH groups of ANT, it is unclear from the present work whether the increases in nuclear Ca are related to As-induced mitochondrial damage. Microprobe SR-XRF analysis of thin-sectioned cells resulting from arsenite treatment indicated that the dominant As species was intracellular rather than membrane-bound As. This is not only consistent with the findings of Dopp et al. (53) following uptake studies of arsenite in HepG2 cells but also indicates that the subsequent bulk cell XAS analyses provided information regarding intracellular As. While microprobe XRF/XAS studies by us and Ortega (34) failed to provide high quality postedge structure in the X-ray microprobe XAS spectra, XAS analysis of bulk cells provided adequate information to determine the atomic coordination to As. Multiple linear regression analysis of the XANES of the cellular As indicated that the best fit corresponded to As bound by three S ligands (97%) and a small amount (3%) being oxygen bound, suggesting that the administered arsenite may undergo incomplete biotransformation during the 4 h exposure period. Importantly, however, it is clear that As does undergo significant biotransformation to a thiolbound species. It should also be noted that the fit of the arsenitetreated cells with one standard, [As(GS)3], resulted in only a slightly worse fit, which would not be considered significantly different. Single-scattering analysis of the EXAFS recorded from a pellet of cells treated with arsenite for 4 h indicated that intracellular As was bound by three sulfur atoms and showed no evidence for the presence of lighter atoms bound to As. However, this does not preclude the existence of oxygen-bound As as a minor (∼1%) component in the cells, as the stronger backscattering amplitude of S would mask the small O backscattering signal. The predominantly tris-sulfur binding is consistent with previously reported XAS results obtained from As species analyzed in fur and feathers (54), and the chlorogogenous tissue of earthworms (55), which have attributed the species to proteinbound As. Similarly, the dominance of the As-thiol species in the work presented here (Figures 6 and 7) gives strong credence to the interactions between As and proteins as the likely basis for its toxicity since there is little evidence for DNA-bound As. While there is an obvious discrepancy between the XAS spectrum of the HepG2 cells and [As(GS)3], this is potentially accounted for by the protein constraining the geometry around the As to one distinct from that of free [As(GS)3]. Although, the existence of a further significant component cannot be ruled out, there are no biologically relevant ligands that are more

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Figure 7. EXAFS analysis (left) and corresponding Fourier Transforms (right) of a freeze-dried pellet of HepG2 cells treated with 700 µM arsenite for 4 h showing experimental (s) and calculated (---) data. Fit parameters are shown in Table 1.

Table 1. Summary of EXAFS Fitting Results for Arsenite-Treated HepG2 Cellsa No. of S interatomic Debye-Waller scatterers (N) distance (R, Å) factor (σ2, Å2) -∆E0 (eV)b Fit-Errorc 3 2

2.244(4) 2.241(4)

0.0027(2) 0.0007(2)

6.6(8) 7(1)

0.536 0.564

a Freeze-dried bulk cell pellet of cells treated with 700 µM arsenite for 4 h. The k-range was 1-12.5 Å-1 for all fits. A scale factor (S02) of 1 was used for all fits. The values in parentheses are the estimated standard deviations (precisions) obtained from the diagonal elements of the covariance matrix. Note that the accuracies will always be somewhat higher than the precisions, typically ( 0.02 Å for R and ( 20% for N and σ2. The coordination number was not a variable in these fits. b ∆E 0 ) E0 - 11885 (eV), where E0 is the threshold energy. c The Fit-Error is defined as [Σk6(χexptl - χcalcd)2/Σk6χexptl2]1/2.

electron donating than GS. In addition, the EXAFS analysis that indicates a greater probability for three S scatterers over two S scatterers is also consistent with a recent report by Kitchin and Wallace (13) that shows a much longer lifetime of tridentate coordination of protein cysteine to As (1-2 h) in comparison to bidentate (1-2 min) and monodentate (0.01-1 s) protein cysteine coordination. The absence of significant contributions from methylated As metabolites in the XAS spectrum of the HepG2 cells is consistent with findings by Chen et al. (56). For instance, it was reported that the proportion of methylated As decreased following an increase in the arsenite administration (0.1 µM arsenite administration yielded inorganic As, 28%; MMA, 11%; and DMA, 61%; while 1 µM arsenite administration yielded inorganic As, 72%; MMA, 18%; and DMA, 10%). Since the studies reported here used 100 µM arsenite and XAS spectra represent an average of all contributing species, it is not surprising that there were no significant contributions from the methylated As species. These results are also consistent with pharmacokinetic studies of As metabolites in the urine by Wang et al. (57) that showed that increased As administration (As2O3intravenous injection of APL patients) inhibited the methylation of arsenic. In conclusion, microprobe SR-XRF analysis of arsenite- and arsenate-treated cells showed a clear accumulation of As in the nucleus following acute exposure (1 mM As(III) or 20 mM As(V) for 4 h). In arsenite-treated cells, the accumulation was associated with euchromatin regions implying that toxicity was associated with As binding to DNA or DNA transcription proteins. XAS analysis of cells treated with 100 µM arsenite showed clear evidence that

the ultimate As species was coordinated through three S-atoms, implicating As-protein binding, which was consistent with EXAFS studies of HepG2 cells treated with 700 µM arsenite. While it has been stipulated that reduced GSH levels associated with As exposure may cause toxicity through oxidative stress, it was not apparent from the XAS that As-GSH was the predominant species. Instead a more constrained symmetry, possibly associated with protein binding was predicted from the XAS spectra assuming one predominant species. Ca2+ increases in the nucleus were consistent with studies by other workers and indicated evidence of cell toxicity. While these may be associated with mitochondrial-induced apoptosis, the current spatial resolution of the microprobe XRF technique excludes the possibility of exploring As localization in mitochondria. Consequently, the results of this study point most strongly to damage induced by the ability of As to enter and accumulate in the nucleus where it is likely to interact with pertinent proteins. Finally, in the current microprobe XRF study of thin-sectioned cells, the As doses were chosen to reflect the IC50 values and toxicity of arsenite toward the cells and to ensure detectability of intracellular As. We are currently conducting similar assays at 10 and 100 µM doses and longer exposure times to approach plasma concentrations observed following administration of Trisenox. Acknowledgment. We are grateful for financial support from the University of Wollongong, Small Grants Scheme and to the Australian Synchrotron Research Program (ASRP) for access to the APS and ANBF facilities (C.T.D., H.H.H., K.L.M., A.M., and A.I.K.). ASRP is funded by the Commonwealth of Australia under the Major National Research Facilities Program. The use of APS facilities was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract no. DE-AC0206CH11357. We thank Dr. Garry Foran and Dr. Bernt Johannessen (ANBF) for assistance with XAS. We also acknowledge the facilities at the Australian Microscopy and Microanalysis Facility at the Electron Microscope Unit, The University of Sydney, in particular, Emine Korkmaz and Tony Romeo for their suggestions and helpfulness during the thin-sectioning procedures. K.L.M. is grateful to the Australian Rotary Health Fund and Ian and Jean Simpson for her Ph.D. scholarship. Supporting Information Available: Preparation of thinsectioned cells for SR-XRF mapping and the ESI-MS of

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[As(GS)3], [MMAIII(GS)2], and [DMAIII(GS)]. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) WHO (2001) EnVironmental Health Criteria 224. Arsenic and Arsenic Compounds. 2nd ed.,World Health Organisation, Geneva. (2) Wang, J. P., Qi, L., Moore, M. R., and Ng, J. C. (2002) A review of animal models for the study of the arsenic carcinogenesis. Toxicol. Lett. 133, 17–31. (3) Schoen, A., Beck, B., Sharma, R., and Dube, E. (2004) Arsenic toxicity at low doses: epidemiological and mode of action considerations. Toxicol. Appl. Pharmacol. 198, 253–267. (4) Abernathy, C. O., Liu, Y.-P., Longfellow, D., Aposhian, H. V., Beck, B., Fowler, B., Goyer, R., Menzer, R., Rossman, T., Thompson, C., and Waalkes, M. (1999) Arsenic: health effects, mechanisms of action, and research issues. EnViron. Health Perspect. 107, 593–597. (5) Cullen, W. R., and Reimer, K. J. (1989) Arsenic speciation in the environment. Chem. ReV. 36, 315–361. (6) Smith, A. H., Lingas, E. O., and Rahman, M. (2000) Contamination of drinking-water by arsenic in Bangladesh: a public health emergency. Bull. World Health Org. 78, 1093–1103. (7) Cohen, M. H., Hirschfeld, S., Honig, S. F., Ibrahim, A., Johnson, J. R., O’Leary, J. J., White, R. M., Williams, G. A., and Pazdur, R. (2001) Drug approval summaries: arsenic trioxide, tamoxifen citrate, anastrazole, paclitaxel, bexarotene. The Oncologist 6, 4–11. (8) Bachleitner-Hofmann, T., Kees, M., and Gisslinger, H. (2002) Arsenic trioxide: acute promyolocytic leukemia and beyond. Leuk. Lymphoma 43, 1535–1540. (9) Lu, D.-P., Qiu, J.-Y., Jiang, B., Wang, Q., Liu, K.-Y., Liu, Y.-R., and Chen, S.-S. (2002) Tetra-arsenic tetra-sulfide for the treatment of acute promyelocytic leukemia: a pilot report. Blood 99, 3136–3143. (10) Don, A. S., Kisker, O., Dilda, P., Donoghue, N., Zhao, X., Decollogne, S., Creighton, B., Flynn, E., Folkman, J., and Hogg, P. J. (2003) A peptide trivalent arsenical inhibits tumor angiogenesis by perturbing mitochondrial function in angiogenic endothelial cells. Cancer Cell 3, 497–509. (11) Rojewski, M. T., Korper, S., and Schrezenmeier, H. (2004) Arsenic trioxide therapy in acute promyelocytic leukemia and beyond: from bench to bedside. Leuk. Lymphoma 45, 2387–2401. (12) Chou, W.-C., and Dang, C. V. (2004) Acute promyelocytic leukemia: recent advances in therapy and molecular basis of response to arsenic therapies. Curr. Opin. Hematol. 12, 1–6. (13) Kitchin, K. T., and Wallace, K. (2008) The role of protein binding of trivalent arsenicals in arsenic carcinogenesis and toxicity. J. Inorg. Biochem. 102, 532–539. (14) Don, A. S., and Hogg, P. J. (2004) Mitochondria as cancer drug targets. Trends Mol. Med. 10, 372–378. (15) Cai, Z., Lai, B., Yun, W., McNulty, I., Khounsary, A., Maser, J., Ilinski, P., Legnini, D., Trakhtenberg, E., Xu, S., Tieman, B., Wiemerslage, G., and Gluskin, E. (2000) Performance of a high-resolution X-ray microprobe at the Advanced Photon Source. Am. Inst. Phys. Conf. Proc. 521, 31–34. (16) Bohic, S., Simionovici, A., Ortega, R., Heymann, D., Schroer, C., and Snigirev, A. (2001) Synchrotron-induced X-ray microfluorescence on single cells. Nucl. Instr. Meth. Phys. Res. B 181, 728–733. (17) Jacobsen, C. (1999) Soft X-ray microscopy. Trends Cell Biol. 9, 44– 47. (18) Dillon, C. T., Lay, P. A., Kennedy, B. J., Stampfl, A. P. J., Cai, Z., Ilinski, P., Rodrigues, W., Legnini, D. G., Lai, B., and Maser, J. (2002) Hard X-ray microprobe studies of chromium-treated V79 Chinese hamster lung cells: further insights into chromium carcinogenesis. J. Biol. Inorg. Chem. 7, 640–645. (19) Hall, M. D., Dillon, C. T., Zhang, M., Beale, P., Cai, Z., Lai, B., Stampfl, A. P. J., and Hambley, T. W. (2003) The cellular distribution and oxidation state of platinum(II) and platinum(IV) antitumour complexes in cancer cells. J. Biol. Inorg. Chem. 8, 726–732. (20) Waern, J. B., Harris, H. H., Lai, B., Cai, Z., Harding, M. M., and Dillon, C. T. (2005) Intracellular mapping of the distribution of metals derived from the antitumor metallocenes. J. Biol. Inorg. Chem. 10, 443–453. (21) Challenger, F. (1945) Biological methylation. Chem. ReV. 36, 315– 361. (22) Kitchin, K. T. (2001) Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Pharmacol. 172, 249–261. (23) Thomas, D. J., Waters, S. B., and Styblo, M. (2004) Elucidating the pathway for arsenic methylation. Toxicol. Appl. Pharmacol. 198, 319– 326.

Munro et al. (24) Hayakawa, T., Kobayashi, Y., Cui, X., and Hirano, S. (2005) A new metabolic pathway of arsenite: arsenic-glutathione complexes are substrates for human arsenic methyltransferase Cyt19. Arch. Toxicol. 79, 183–191. (25) Kala, S. V., Kala, G., Prater, C. I., Sartorelli, A. C., and Lieberman, M. W. (2004) Formation and urinary excretion of arsenic triglutathione and methylarsenic diglutathione. Chem. Res. Toxicol. 17, 243–249. (26) Styblo, M., Del Razo, L. M., Vega, L., Germolec, D. R., LeCluyse, E. L., Hamilton, G. A., Reed, W., Wang, C., Cullen, W. R., and Thomas, D. J. (2000) Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Arch. Toxicol. 74, 289–299. (27) Telolahy, P., Morel, G., Cluet, J. L., Yang, H. M., Thieffry, N., and de Ceaurriz, J. (1995) An attempt to explain interindividual variability in 24-h urinary excretion of inorganic arsenic metabolites by C57 BL/ 6J mice. Toxicology 103, 105–112. (28) Brima, E. I., Jenkins, R. O., and Haris, P. I. (2006) Understanding arsenic metabolism through spectroscopic determination of arsenic in human urine. Spectroscopy 20, 125–151. (29) Cui, X., Kobayashi, Y., Hayakawa, T., and Hirano, S. (2004) Arsenic speciation in bile and urine following oral and intravenous exposure to inorganic and organic arsenics in rats. Toxicol. Sci. 82, 478–487. (30) Melitas, N., Wang, J., Conklin, M., O’Day, P., and Farrell, J. (2002) Understanding soluble arsenate removal kinetics by zerovalent iron media. EnViron. Sci. Technol. 36 2074–2081. (31) Hansel, C. M., La Force, M. J., Fendorf, S., and Sutton, S. (2002) Spatial and temporal association of As and Fe species on aquatic plant roots. EnViron. Sci. Technol. 36 1988–1994. (32) Pickering, I. J., Gumaelius, L., Harris, H. H., Prince, R. C., Hirsch, G., Banks, J. A., Salt, D. E., and George, G. N. (2006) Localizing the biochemical transformations of arsenate in a hyperaccumulating fern. EnViron. Sci. Technol. 40, 5010–5014. (33) Langdon, C. J., Meharg, A. A., Feldmann, J., Balgar, T., Charnock, J., Farquhar, M., Piearce, T. G., Semple, K. T., and Cotter-Howells, J. (2002) Arsenic-speciation in arsenate-resistant and non-resistant populations of earthworm, Lumbricus rubellus. J. EnViron. Monit. 4, 603–608. (34) Bacquart, T., Deves, G., Carmona, A., Tucoulou, R., Bohic, S., and Ortega, R. (2007) Subcellular speciation analysis of trace element oxidation states using synchrotron radiation micro-X-ray absorption near-edge structure. Anal. Chem. 79, 7375–7359. (35) Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. (36) Huxham, L. A., Cheu, E. L. S., Patrick, B. O., and James, B. R. (2003) The synthesis, structural characterization, and in Vitro anti-cancer activity of chloro(p-cymene) complexes of ruthenium(II) containing a disulfoxide ligand. Inorg. Chim. Acta 352, 238–246. (37) Vogt, S. (2003) MAPS: A set of software tools for analysis and visualisation of 3D X-ray fluorescence data sets. J. Physique IV 104, 635–638. (38) Van Espen, P. (2002) In Handbook of X-ray Spectrometry: ReVised and Expanded (Van Grieken, R., and Markowicz, A., Eds.), Dekker, New York. (39) Graphpad Instat (1998) Version 3.01, GraphPad Software, Inc., La Jolla, CA. (40) Jones, R. A., Smail, A., and Wilson, M. R. (2002) Detecting mitochondrial permeability transition by confocal imaging of intact cells pinocytically loaded with calcein. Eur. J. Biochem. 269, 3990– 3997. (41) George, G. N., and Pickering, I. J. (2001) EXAFSPAK. A Suite of Computer Programs for Analysis of X-ray Absorption Spectra, Stanford Synchrotron Research Laboratory, Stanford, CA. http:// ssrl.slac.stanford.edu/exafspak.html. (42) Weng, T.-C., Waldo, G. S., and Penner-Hahn, J. E. (2005) A method for normalization of X-ray absorption spectra. J. Synch. Rad. 12, 506– 510. (43) Ankudinov, A. L., Ravel, B., Rehr, J. J., and Conradson, S. D. (1998) Real space multiple scattering calculation and interpretation of X-ray absorption near edge structure. Phys ReV B. 58, 7565–7576. (44) Ankudinov, A. L., Bouldin, C., Rehr, J. J., Sims, J., and Hung, H. (2002) Parallel calculation of electron multiple scattering using Ianczos algorithms. Phys ReV B. 65, 104–107. (45) Branden, C., and Tooze, J. (1999) Introduction to Protein Structure, Garland Publishing Inc., New York. (46) Bozzola, J. J., and Russell, L. D. (1998) Electron Microscopy. Principles and Techniques for Biologists, 2nd ed., Jones and Bartlett, Sudbury, MA. (47) Quintana, C. (1991) X-ray microanalysis of cell nuclei. J. Electron Microsc. Tech. 18, 411–423. (48) Yee-Chien, L., and Haimei, H. (1996) Lowering extracellular calcium content protects cells from arsenite-induced killing and micronuclei formation. Mutagenesis 11, 75–78.

Micro-XRF and XAS Studies of Intracellular Arsenic (49) Orrenius, S., Zhivotovsky, B., and Nicotera, P. (2003) Regulation of cell death: the calcium-apoptosis link. Nature ReV. Mol. Cell. Biol. 4, 552–565. (50) Belzacq, A.-S., and Brenner, C. (2003) The adenine nucleotide translocator: a new potential chemotherapeutic target. Curr. Drug Targets 4, 517–524. (51) Butow, R. A., and Avadhani, N. G. (2004) Mitochondrial signalling: the retrograde response. Mol. Cell 14, 1–15. (52) Cai, X., Shen, Y. L., Zhu, Q., Jia, P. M., Yu, Y., Zhou, L., Huang, Y., Zhang, J. W., Xiong, S. M., Chen, S. J., Wang, Z. Y., Chen, Z., and Chen, G. Q. (2000) Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia. Leukemia 14, 262–270. (53) Dopp, E., Hartmann, L. M., von Recklinghausen, U., Florea, A. M., Rabieh, S., Zimmermann, U., Shoukouhi, B., Yadav, S., Hirner, A. V., and Rettenmeier, A. W. (2005) Forced uptake of trivalent and pentavalent methylated and inorganic arsenic and its cyto-/genotoxicity in fibroblasts and hepatoma cells. Toxicol. Sci. 87, 46–56.

Chem. Res. Toxicol., Vol. 21, No. 9, 2008 1769 (54) Smith, P. G., Koch, I., and Reimer, K. J. (2008) An investigation of arsenic compounds in fur and feathers using X-ray absorption spectroscopy speciation and imaging. Sci. Total EnViron. 390, 198– 204. (55) Langdon, C. J., Winters, C., Sturzenbaum, S. R., Morgan, A. J., Charnock, J. M., Meharg, A. A., Piearce, T. G., Lee, P. H., and Semple, K. T. (2005) Ligand arsenic complexation and immunoperoxidase detection of metallothionein in the earthworm Lumbricus rubellus inhabiting arsenic rich soil. EnViron. Sci. Technol. 39, 2042–2048. (56) Chen, G.-Q., Zhou, L., Styblo, M., Walton, F., Jing, Y., Weinberg, R., Chen, Z., and Waxman, S. (2003) Methylated metabolites of arsenic trioxide are more potent than arsenic trioxide as apoptotic but not differentiation inducers in leukemia and lymphoma cells. Cancer Res. 63, 1853–1859. (57) Wang, Z., Zhou, J., Lu, X., Gong, Z., and Le, X. C. (2004) Arsenic speciation in urine from acute promyelocytic leukemia patients undergoing arsenic trioxide treatment. Chem. Res. Toxicol. 17, 95–103.

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