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Direct speciation analysis of arsenic in sub-cellular compartments using micro-X-ray absorption spectroscopy
ARTICLE IN PRESS Environmental Research 110 (2010) 413–416
Contents lists available at ScienceDirect
Environmental Research journal homepage: www.elsevier.com/locate/envres
Direct speciation analysis of arsenic in sub-cellular compartments using micro-X-ray absorption spectroscopy$ Thomas Bacquart, Guillaume Deve s, Richard Ortega Cellular Chemical Imaging and Speciation Group, CNAB, CNRS, University of Bordeaux, 33175 Gradignan, France
a r t i c l e in fo
abstract
Article history: Received 25 March 2009 Received in revised form 8 September 2009 Accepted 14 September 2009 Available online 2 October 2009
Identification of arsenic chemical species at a sub-cellular level is a key to understanding the mechanisms involved in arsenic toxicology and antitumor pharmacology. When performed with a microbeam, X-ray absorption near-edge structure (m-XANES) enables the direct speciation analysis of arsenic in sub-cellular compartments avoiding cell fractionation and other preparation steps that might modify the chemical species. This methodology couples tracking of cellular organelles in a single cell by confocal or epifluorescence microscopy with local analysis of chemical species by m-XANES. Here we report the results obtained with a m-XANES experimental setup based on Kirkpatrick–Baez X-ray focusing optics that maintains high flux of incoming radiation (4 1011 ph/s) at micrometric spatial resolution (1.5 4.0 mm2). This original experimental setup enabled the direct speciation analysis of arsenic in sub-cellular organelles with a 10 15 g detection limit. m-XANES shows that inorganic arsenite, As(OH)3, is the main form of arsenic in the cytosol, nucleus, and mitochondrial network of cultured cancer cells exposed to As2O3. On the other hand, a predominance of As(III) species is observed in HepG2 cells exposed to As(OH)3 with, in some cases, oxidation to a pentavalent form in nuclear structures of HepG2 cells. The observation of intra-nuclear mixed redox states suggests an interindividual variability in a cell population that can only be evidenced with direct sub-cellular speciation analysis. & 2009 Elsevier Inc. All rights reserved.
Keywords: Arsenic Speciation Cell Synchrotron X-ray absorption spectroscopy
1. Introduction The precise identification of arsenic chemical species in human cells is a critical achievement to elucidate the mechanisms of arsenic toxicity and pharmacology. This is a challenging task because arsenic toxic effects occur at low concentration, and because arsenic can be present in numerous chemical species, with +3 or +5 valence, inorganic and organic forms. The aim of this paper is to present some recent advances in direct speciation analysis of chemical elements, such as arsenic, at the sub-cellular level using synchrotron radiation X-ray absorption spectroscopy (XAS). Usually chemical speciation analysis is performed by means of hyphenated techniques, which combine a purification/separation step with highly sensitive element detection. On the other hand, the biochemistry of sub-cellular compartments is traditionally investigated after cellular lysis and differential ultra-centrifugation. The purification and separation steps involved in the chromatographic and ultracentrifugation steps are prone to modify the native chemical species of the elements. The advantage of XAS is that it requires no sample
$ This work was funded in part by CNRS (Centre National de la Recherche Scientifique) and ESRF (European Synchrotron Radiation Facility). Corresponding author. Fax: +33 557 120 900. E-mail address: [email protected] (R. Ortega).
0013-9351/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2009.09.006
preparation or chromatographic separation, which enables to keep the sample in its native state. The main drawbacks are the lack of sensitivity of XAS compared to mass spectrometry for example; the typical detection limit for XAS being in the 10–100 mg/g range, and the lowest selectivity than chromatographic methods since XAS spectra of chemical element species are sometimes too similar to enable their identification (Lobinski et al., 2006). However, a second advantage of XAS is that it can now be applied with micrometric resolution to perform XAS at the sub-cellular level (Bacquart et al., 2007). XAS has proven to be a valuable tool to determine arsenic species in biological samples (Smith et al., 2005). Here we report the application of micro-XAS to sub-cellular speciation analysis of arsenic in cultured cells. Two cell lines have been investigated, human ovarian adenocarinoma cells (IGROV1) that were exposed to the antitumor agent As2O3, and human hepatocellular carcinoma cells (HepG2) a model cell line for As metabolism studies (Del Razo et al., 2001; Drobna et al., 2006) that were exposed to As(OH)3. 2. Material and methods 2.1. Cell culture and sample preparation IGROV1 and HepG2 cells were cultured onto 2 mm polycarbonate foils as adapted from previously described protocols (Carmona et al., 2008). IGROV1 cells
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were exposed to 9 mM As2O3 during 48 h. This concentration corresponds to the inhibition of 40% cell viability as measured by MTT assay. HepG2 cells were exposed to 50 mM As(OH)3 during 24 h, which corresponds to the inhibition of 50% cell viability. Mitochondria were marked with a fluorescent dye (Rhodamine123) and their intracellular location was determined either by epifluorescence microscopy or by confocal microscopy. Video images were recorded for further identification of cellular organelles. Cells were cryo-fixed in liquid nitrogen chilled isopentane and stored in liquid nitrogen until analysis in their frozen hydrated state at 150 K.
2.2. Synchrotron radiation X-ray absorption spectroscopy XANES (X-ray Absorption Near-Edge Spectroscopy) experiments were performed with a 1.5 4.0 mm2 spatial resolution using ESRF ID-22 hard X-ray microprobe with a Kirkpatrick–Baez focusing mirror as recently described (Bacquart et al., 2007). This experimental setup provided a photon flux of typically 1.5 1011 ph/s allowing X-ray absorption spectroscopy to be performed with micrometric resolution. The use of a liquid nitrogen cryo-stream enabled to irradiate the samples in their frozen hydrated state at 150 K. Low-temperature analysis was absolutely required to avoid any beam damage. The preservation of As chemical species during irradiation was checked by fast consecutive analyses of standards and samples, which show no spectral modifications (Bacquart et al., 2007). Prior to m-XANES, a quick synchrotron X-ray fluorescence mapping of arsenic and potassium distributions was performed in order to precisely identify the intracellular organelles. Arsenic chemical speciation was determined locally, by point analysis, into nucleus, mitochondria, and cytosol by scanning around As absorption K-edge from 11800 to 12000 eV with 0.5 eV energy
resolution. Arsenic chemical species were identified by the maximum energy of the white line, as reported by Smith et al. (2005); this method gives better results for the low-statistics m-XANES spectra than the energy position of the first infection point. This original experiment was applied for the direct speciation analysis of arsenic, at the sub-cellular level, with a 10 15 g detection limit.
2.3. Analysis of standard compounds Arsenic standard compounds have been prepared with high-purity ( 495%) chemicals: As(OH)3 (Fluka); AsO(OH)3 (Fluka); MMA(V), monomethylarsonic acid (Strem Chemical); DMA(V), dimethylarsinic acid (Chem Service); As2S2 (Strem Chemical); As2O5 (Strem Chemical); and As2O3 (Strem Chemical). XANES spectra were performed in fluorescence mode on powder standards mixed with cellulose and deposited on polycarbonate foils, as detailed above.
3. Results 3.1. Analysis of cancer cells exposed to As2O3 XANES spectra corresponding to the nucleus, cytosol, and mitochondrial network of individual IGROV1 human adenocarcinoma cells exposed to 9 mM As2O3 are presented in Fig. 1. Cellular
Fig. 1. Near-edge X-ray absorption spectra at arsenic K-edge obtained in the cytosol, mitochondrial network, and nucleus of IGROV1 cells exposed to 9 mM As2O3, and labelled with Rhodamine 123, a probe for mitochondrial network imaging (center image obtained by confocal microscopy). Each XANES spectrum is the sum of N = 5 energy scans of the same cellular area. Left and right spectra correspond, respectively, to the zones analysed on the left and right cells (white bars).
Fig. 2. (A) XANES spectra of reference compounds: As2S2, As2O3, DMA(V), and As2O5, and of the mitochondrial network, cytosol, and nucleus of IGROV1 cells exposed to 9 mM As2O3. The XANES spectra of sub-cellular compartments are the sum of 20 analyses. These analyses indicate that arsenic is mainly present in its + 3 oxidation state in all cellular compartments (dotted line). (B) XANES spectra of the reference compounds As(OH)3 and As2O3 showing a specific oscillation at 11,883 eV for As2O3 (arrows). This oscillation is not observed in cells, suggesting that arsenic trioxide is mainly transformed into As(OH)3 in IGROV1 cells.
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compartments were visualized using confocal microscopy images of Rhodamine 123 labeled cells prior to analysis. m-XANES was performed on frozen hydrated samples at 150 K. The absorption edge energy is the same for the three cellular compartments (11,871.370.3 eV). The comparison of this absorption edge energy with those of standard compounds (Fig. 2A) indicates that the main arsenic form in IGROV1 cells exposed to arsenic trioxide is As(OH)3 (11,871.570.5 eV). The detailed study of the XANES spectra after the absorption edge shows a difference in the region 11,882–11,883 eV between the spectrum from As2O3 powder and those from cellular organelles (Fig. 2A), which are similar to the As(OH)3 spectrum (Fig. 2B).
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57% As(OH)3, 8.5% of MMA(III), and 34.5% of DMA(III) (Del Razo et al., 2001). These m-XANES results and the results from Del Razo et al. (2001) are different from the recent XAS bulk analyses obtained on HepG2 freeze-dried pellet samples showing 98.2% As(GS)3 (Munro et al., 2008). This difference could result from the sample preparation because the process of freeze drying is known to affect the chemical state of major and trace elements (Hjorth, 2004), and especially of arsenic compounds (Huang and Ilgen, 2006). For the speciation analysis of arsenic it is strongly recommended to perform XANES on frozen hydrated cells in
3.2. Analysis of HepG2 cells exposed to As(OH)3
m-XANES has been directly performed in the nucleus, cytosol, and mitochondrial network (not shown) of HepG2 cells exposed to 50 mM As(OH)3 during 24 h (Fig. 3). The mean energies of arsenic absorption K-edge in the three cellular compartments are 11,870.7 eV in the nucleus (N=9), 11,871.2 eV in the cytosol (N= 3), and 11,871.2 eV in the mitochondrial network (N=2) (Fig. 3A). These energies correspond to the As(III) oxidation state. However, in one of the 9 analyzed cells a mixed valence state was observed within the nucleus in the presence of both As(III) and As(V), the oxidized form being preponderant (Fig. 3B). 4. Discussion 4.1. Analysis of cancer cells exposed to As2O3 Arsenic trioxide is a chemotherapeutic agent used to treat leukemia but whose mechanism of action is still elusive. As2O3 has been recently proposed as a potential agent to treat ovarian cancers in combination with other anticancer agents (Helm and States, 2009). m-XANES enabled determining the speciation of arsenic in the nucleus, cytosol, and mitochondrial network of IGROV1 human ovarian adenocarcinoma cells exposed to arsenic trioxide at IC40 concentration (Figs. 1 and 2). As(OH)3 is the principal chemical form observed in IGROV1 cellular compartments. This result is relevant with the cellular exposure conditions because As2O3 is dissolved in alkaline solutions giving arsenite before dilution in the culture medium and buffering. At neutral pH (pH= 7.4 in culture medium), As(OH)3 is known to be very stable (Ram´ırez-Sol´ıs et al., 2004). This result also indicates that arsenic is not metabolized in IGROV1 human ovarian adenocarcinoma cells. Therefore the cytotoxic effects of As2O3 are not due to metabolisation processes but rather to the presence of As(OH)3. 4.2. Analysis of HepG2 cells exposed to As(OH)3 We also performed m-XANES analysis of tumoral hepatocytes, HepG2 cells, a model cell line to study arsenic metabolization in vitro (Del Razo et al., 2001; Drobna et al., 2006). The mean energy of the maximum absorption edge is slightly different between the nucleus (11,870.7 eV) and the other two cellular areas, cytosol and mitochondrial network (11871.2 eV). The maximum energy of the absorption K-edge for trivalent arsenic reference compounds DMA(III), MMA(III), and As(OH)3 are, respectively, 11,869.0 70.5, 11,870.8 70.5, and 11,871.570.5 eV. From these data it can be concluded that the main oxidation state of arsenic in HepG2 cells is As(III), in all three cellular compartments (Fig. 3A). This is in good agreement with the reported speciation of arsenic in bulk samples of HepG2 cells exposed to 10 mM arsenite for 24 h giving
Fig. 3. Normalised XANES spectra at arsenic K-absorption edge in the cytosol and the nucleus of HepG2 cells exposed to 50 mM As(OH)3 during 24 h. Top spectra (A): sum of 9 local analyses of single cells showing that main arsenic oxidation state is As(III) both in cytosol and in nucleus. Bottom spectra (B): a mixture of trivalent and pentavalent arsenic is found in the nucleus, but not in the cytosol, of one of the 9 cells analysed. This observation suggests an inter-individual variability, which can only be pointed out by direct speciation analysis.
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order to preserve the native state of arsenic compounds (Bacquart et al., 2007). The second important result of HepG2 single cell analysis is the evidence of As(V) in one of the nucleus over the 9 cells analyzed (Fig. 3B). For pentavalent arsenic compounds DMA(V), MMA(V), and AsO(OH)3 the values of maximum absorption edge energies are, respectively, 11,873.070.5, 11,873.570.5, and 11,875.070.5 eV. The m-XANES spectra from this specific cell presents two maximum energy peaks: one at 11,871.2 eV, which corresponds to As(III), and another peak at 11,875.2 eV, which corresponds to As(V), and more specifically to AsO(OH)3. In the speciation experiment of bulk HepG2 samples (Del Razo et al., 2001), As(V) was not detected in cells. The m-XANES observation of mixed redox states of As(III) and As(V) in a single cell indicates that a minor fraction of arsenic could be present as As(V) although this fraction is difficult to evidence using bulk speciation analyses. The pentavalent As form has been detected in glioblastoma and bone marrow cells exposed to trivalent arsenic (Falnoga et al., 2007). The oxidation of As(III) to As(V) has been proposed as a mechanism of detoxification since As(V) compounds are less toxic than As(III) compounds (Aposhian et al., 2003). In addition, another very interesting observation is that As(V) is localized in a narrow region of the nucleus. One hypothesis could be that this zone corresponds to some micronuclei as arsenic is known to induce micronuclei in HepG2 cells (Gebel et al., 2002). However, this hypothesis remains to be verified. This singular result proves that the arsenic chemical species are not homogeneous in a cell population. It also points out the limitations of the m-XANES as this method requires long acquisition times, which limits the number of cells that can be analyzed during an allocated beam time on synchrotron radiation facilities.
5. Conclusion Thanks to the recent progress in m-XANES experimental devices, the speciation analysis of arsenic could be performed with high spatial resolution and with detection limits compatible with diluted concentrations. m-XANES is a powerful method to determine arsenic oxidation states at the sub-cellular level (Bacquart et al., 2007). It is probably the only method available for direct speciation analysis in cellular compartments. Direct speciation analysis allows the identification of chemical element species almost without sample preparation, keeping the cells and their chemical components very close to their native state. However it presents some limitations for the identification of arsenic chemical species when samples are constituted of a mixture of those species. It is therefore very important to compare
the experimental spectra with those of a large variety of reference compounds. Such pitfall could be overcome in the future if Extended X-ray Absorption Fine Structure (EXAFS) is performed at the sub-cellular level.
Acknowledgments The authors are grateful to Pr. Jean Rosenbaum, University of Bordeaux 2, for providing the HepG2 cells and to Dr. Jean Be nard, Institut Gustave Roussy, for the IGROV1 cells. We also acknowledge Dr. Sylvain Bohic and Dr. Re mi Tucoulou from ESRF for their valuable help during m-XANES experiments. References Aposhian, H.V., Zakharyan, R.A., Avram, M.D., Kopplin, M.J., Wollenberg, M.L., 2003. Oxidation and detoxification of trivalent arsenic species. Toxicol. Appl. Pharmacol. 193, 1–8. Bacquart, T., Deve s, G., Carmona, A., Tucoulou, R., Bohic, S., Ortega, R., 2007. Subcellular speciation analysis of trace element oxidation states using synchrotron radiation micro-X-ray absorption near edge structure. Anal. Chem. 79, 7353–7359. Carmona, A., Deve s, G., Ortega, R., 2008. Quantitative micro-analysis of metal ions in subcellular compartments of cultured dopaminergic cells by combination of three ion beam techniques. Anal. Bioanal. Chem. 390, 1585–1594. Del Razo, L.M., Styblo, M., Cullen, W.R., Thomas, D.J., 2001. Determination of trivalent methylated arsenicals in biological matrices. Toxicol. Appl. Pharmacol. 174, 282–293. ´ Drobna, Z., Xing, W., Thomas, D.J., Styblo, M., 2006. shRNA silencing of AS3MT expression minimizes arsenic methylation capacity of HepG2 cells. Chem. Res. Toxicol. 19, 894–988. Falnoga, I., Slejkovec, Z., Pucer, A., Podgornik, H., Tucek-Znidaric, M., 2007. Arsenic metabolism in multiple myeloma and astrocytoma cells. Biol. Trace Element Res. 116, 5–28. Gebel, T.W., Leister, M., Schumann, W., Hirsch-Ernst, K., 2002. Low-level selftolerance to arsenite in human HepG2 cells is associated with a depressed induction of micronuclei. Mutat. Res. 514, 245–255. Helm, C.W., States, J.C., 2009. Enhancing the efficacy of cisplatin in ovarian cancer treatment-could arsenic have a role. J. Ovarian Res. 14 (2), 2. Hjorth, T., 2004. Effects of freeze-drying on partitioning patterns of major elements and trace metals in lake sediments. Anal. Chim. Acta 526, 95–102. Huang, J.-H., Ilgen, G., 2006. Factors affecting arsenic speciation in environmental samples: sample drying and storage. Int. J. Environ. Anal. Chem. 86, 347–358. Lobinski, R., Moulin, C., Ortega, R., 2006. Imaging and speciation of trace elements in biological environment. Biochimie 88, 1591–1604. Munro, K.L., Mariana, A., Klavins, A.I., Foster, A.J., Lai, B., Vogt, S., Cai, Z., Harris, H.H., Dillon, C.T., 2008. Microprobe XRF mapping and XAS investigations of the intracellular metabolism of arsenic for understanding arsenic-induced toxicity. Chem. Res. Toxicol. 21, 1760–1769. Ram´ırez-Sol´ıs, A., Mukopadhyay, R., Rosen, B.P., Stemmler, T.L., 2004. Experimental and theoretical characterization of arsenite in water: insights into the coordination environment of As–O. Inorg. Chem. 43, 2954–2959. Smith, P.G., Koch, I., Gordon, R.A., Mandoli, D.F., Chapman, B.D., Reimer, K.J., 2005. X-ray absorption near-edge structure analysis of arsenic species for application to biological environmental samples. Environ. Sci. Technol. 39, 248–254.
Contents lists available at ScienceDirect
Environmental Research journal homepage: www.elsevier.com/locate/envres
Direct speciation analysis of arsenic in sub-cellular compartments using micro-X-ray absorption spectroscopy$ Thomas Bacquart, Guillaume Deve s, Richard Ortega Cellular Chemical Imaging and Speciation Group, CNAB, CNRS, University of Bordeaux, 33175 Gradignan, France
a r t i c l e in fo
abstract
Article history: Received 25 March 2009 Received in revised form 8 September 2009 Accepted 14 September 2009 Available online 2 October 2009
Identification of arsenic chemical species at a sub-cellular level is a key to understanding the mechanisms involved in arsenic toxicology and antitumor pharmacology. When performed with a microbeam, X-ray absorption near-edge structure (m-XANES) enables the direct speciation analysis of arsenic in sub-cellular compartments avoiding cell fractionation and other preparation steps that might modify the chemical species. This methodology couples tracking of cellular organelles in a single cell by confocal or epifluorescence microscopy with local analysis of chemical species by m-XANES. Here we report the results obtained with a m-XANES experimental setup based on Kirkpatrick–Baez X-ray focusing optics that maintains high flux of incoming radiation (4 1011 ph/s) at micrometric spatial resolution (1.5 4.0 mm2). This original experimental setup enabled the direct speciation analysis of arsenic in sub-cellular organelles with a 10 15 g detection limit. m-XANES shows that inorganic arsenite, As(OH)3, is the main form of arsenic in the cytosol, nucleus, and mitochondrial network of cultured cancer cells exposed to As2O3. On the other hand, a predominance of As(III) species is observed in HepG2 cells exposed to As(OH)3 with, in some cases, oxidation to a pentavalent form in nuclear structures of HepG2 cells. The observation of intra-nuclear mixed redox states suggests an interindividual variability in a cell population that can only be evidenced with direct sub-cellular speciation analysis. & 2009 Elsevier Inc. All rights reserved.
Keywords: Arsenic Speciation Cell Synchrotron X-ray absorption spectroscopy
1. Introduction The precise identification of arsenic chemical species in human cells is a critical achievement to elucidate the mechanisms of arsenic toxicity and pharmacology. This is a challenging task because arsenic toxic effects occur at low concentration, and because arsenic can be present in numerous chemical species, with +3 or +5 valence, inorganic and organic forms. The aim of this paper is to present some recent advances in direct speciation analysis of chemical elements, such as arsenic, at the sub-cellular level using synchrotron radiation X-ray absorption spectroscopy (XAS). Usually chemical speciation analysis is performed by means of hyphenated techniques, which combine a purification/separation step with highly sensitive element detection. On the other hand, the biochemistry of sub-cellular compartments is traditionally investigated after cellular lysis and differential ultra-centrifugation. The purification and separation steps involved in the chromatographic and ultracentrifugation steps are prone to modify the native chemical species of the elements. The advantage of XAS is that it requires no sample
$ This work was funded in part by CNRS (Centre National de la Recherche Scientifique) and ESRF (European Synchrotron Radiation Facility). Corresponding author. Fax: +33 557 120 900. E-mail address: [email protected] (R. Ortega).
0013-9351/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2009.09.006
preparation or chromatographic separation, which enables to keep the sample in its native state. The main drawbacks are the lack of sensitivity of XAS compared to mass spectrometry for example; the typical detection limit for XAS being in the 10–100 mg/g range, and the lowest selectivity than chromatographic methods since XAS spectra of chemical element species are sometimes too similar to enable their identification (Lobinski et al., 2006). However, a second advantage of XAS is that it can now be applied with micrometric resolution to perform XAS at the sub-cellular level (Bacquart et al., 2007). XAS has proven to be a valuable tool to determine arsenic species in biological samples (Smith et al., 2005). Here we report the application of micro-XAS to sub-cellular speciation analysis of arsenic in cultured cells. Two cell lines have been investigated, human ovarian adenocarinoma cells (IGROV1) that were exposed to the antitumor agent As2O3, and human hepatocellular carcinoma cells (HepG2) a model cell line for As metabolism studies (Del Razo et al., 2001; Drobna et al., 2006) that were exposed to As(OH)3. 2. Material and methods 2.1. Cell culture and sample preparation IGROV1 and HepG2 cells were cultured onto 2 mm polycarbonate foils as adapted from previously described protocols (Carmona et al., 2008). IGROV1 cells
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were exposed to 9 mM As2O3 during 48 h. This concentration corresponds to the inhibition of 40% cell viability as measured by MTT assay. HepG2 cells were exposed to 50 mM As(OH)3 during 24 h, which corresponds to the inhibition of 50% cell viability. Mitochondria were marked with a fluorescent dye (Rhodamine123) and their intracellular location was determined either by epifluorescence microscopy or by confocal microscopy. Video images were recorded for further identification of cellular organelles. Cells were cryo-fixed in liquid nitrogen chilled isopentane and stored in liquid nitrogen until analysis in their frozen hydrated state at 150 K.
2.2. Synchrotron radiation X-ray absorption spectroscopy XANES (X-ray Absorption Near-Edge Spectroscopy) experiments were performed with a 1.5 4.0 mm2 spatial resolution using ESRF ID-22 hard X-ray microprobe with a Kirkpatrick–Baez focusing mirror as recently described (Bacquart et al., 2007). This experimental setup provided a photon flux of typically 1.5 1011 ph/s allowing X-ray absorption spectroscopy to be performed with micrometric resolution. The use of a liquid nitrogen cryo-stream enabled to irradiate the samples in their frozen hydrated state at 150 K. Low-temperature analysis was absolutely required to avoid any beam damage. The preservation of As chemical species during irradiation was checked by fast consecutive analyses of standards and samples, which show no spectral modifications (Bacquart et al., 2007). Prior to m-XANES, a quick synchrotron X-ray fluorescence mapping of arsenic and potassium distributions was performed in order to precisely identify the intracellular organelles. Arsenic chemical speciation was determined locally, by point analysis, into nucleus, mitochondria, and cytosol by scanning around As absorption K-edge from 11800 to 12000 eV with 0.5 eV energy
resolution. Arsenic chemical species were identified by the maximum energy of the white line, as reported by Smith et al. (2005); this method gives better results for the low-statistics m-XANES spectra than the energy position of the first infection point. This original experiment was applied for the direct speciation analysis of arsenic, at the sub-cellular level, with a 10 15 g detection limit.
2.3. Analysis of standard compounds Arsenic standard compounds have been prepared with high-purity ( 495%) chemicals: As(OH)3 (Fluka); AsO(OH)3 (Fluka); MMA(V), monomethylarsonic acid (Strem Chemical); DMA(V), dimethylarsinic acid (Chem Service); As2S2 (Strem Chemical); As2O5 (Strem Chemical); and As2O3 (Strem Chemical). XANES spectra were performed in fluorescence mode on powder standards mixed with cellulose and deposited on polycarbonate foils, as detailed above.
3. Results 3.1. Analysis of cancer cells exposed to As2O3 XANES spectra corresponding to the nucleus, cytosol, and mitochondrial network of individual IGROV1 human adenocarcinoma cells exposed to 9 mM As2O3 are presented in Fig. 1. Cellular
Fig. 1. Near-edge X-ray absorption spectra at arsenic K-edge obtained in the cytosol, mitochondrial network, and nucleus of IGROV1 cells exposed to 9 mM As2O3, and labelled with Rhodamine 123, a probe for mitochondrial network imaging (center image obtained by confocal microscopy). Each XANES spectrum is the sum of N = 5 energy scans of the same cellular area. Left and right spectra correspond, respectively, to the zones analysed on the left and right cells (white bars).
Fig. 2. (A) XANES spectra of reference compounds: As2S2, As2O3, DMA(V), and As2O5, and of the mitochondrial network, cytosol, and nucleus of IGROV1 cells exposed to 9 mM As2O3. The XANES spectra of sub-cellular compartments are the sum of 20 analyses. These analyses indicate that arsenic is mainly present in its + 3 oxidation state in all cellular compartments (dotted line). (B) XANES spectra of the reference compounds As(OH)3 and As2O3 showing a specific oscillation at 11,883 eV for As2O3 (arrows). This oscillation is not observed in cells, suggesting that arsenic trioxide is mainly transformed into As(OH)3 in IGROV1 cells.
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compartments were visualized using confocal microscopy images of Rhodamine 123 labeled cells prior to analysis. m-XANES was performed on frozen hydrated samples at 150 K. The absorption edge energy is the same for the three cellular compartments (11,871.370.3 eV). The comparison of this absorption edge energy with those of standard compounds (Fig. 2A) indicates that the main arsenic form in IGROV1 cells exposed to arsenic trioxide is As(OH)3 (11,871.570.5 eV). The detailed study of the XANES spectra after the absorption edge shows a difference in the region 11,882–11,883 eV between the spectrum from As2O3 powder and those from cellular organelles (Fig. 2A), which are similar to the As(OH)3 spectrum (Fig. 2B).
415
57% As(OH)3, 8.5% of MMA(III), and 34.5% of DMA(III) (Del Razo et al., 2001). These m-XANES results and the results from Del Razo et al. (2001) are different from the recent XAS bulk analyses obtained on HepG2 freeze-dried pellet samples showing 98.2% As(GS)3 (Munro et al., 2008). This difference could result from the sample preparation because the process of freeze drying is known to affect the chemical state of major and trace elements (Hjorth, 2004), and especially of arsenic compounds (Huang and Ilgen, 2006). For the speciation analysis of arsenic it is strongly recommended to perform XANES on frozen hydrated cells in
3.2. Analysis of HepG2 cells exposed to As(OH)3
m-XANES has been directly performed in the nucleus, cytosol, and mitochondrial network (not shown) of HepG2 cells exposed to 50 mM As(OH)3 during 24 h (Fig. 3). The mean energies of arsenic absorption K-edge in the three cellular compartments are 11,870.7 eV in the nucleus (N=9), 11,871.2 eV in the cytosol (N= 3), and 11,871.2 eV in the mitochondrial network (N=2) (Fig. 3A). These energies correspond to the As(III) oxidation state. However, in one of the 9 analyzed cells a mixed valence state was observed within the nucleus in the presence of both As(III) and As(V), the oxidized form being preponderant (Fig. 3B). 4. Discussion 4.1. Analysis of cancer cells exposed to As2O3 Arsenic trioxide is a chemotherapeutic agent used to treat leukemia but whose mechanism of action is still elusive. As2O3 has been recently proposed as a potential agent to treat ovarian cancers in combination with other anticancer agents (Helm and States, 2009). m-XANES enabled determining the speciation of arsenic in the nucleus, cytosol, and mitochondrial network of IGROV1 human ovarian adenocarcinoma cells exposed to arsenic trioxide at IC40 concentration (Figs. 1 and 2). As(OH)3 is the principal chemical form observed in IGROV1 cellular compartments. This result is relevant with the cellular exposure conditions because As2O3 is dissolved in alkaline solutions giving arsenite before dilution in the culture medium and buffering. At neutral pH (pH= 7.4 in culture medium), As(OH)3 is known to be very stable (Ram´ırez-Sol´ıs et al., 2004). This result also indicates that arsenic is not metabolized in IGROV1 human ovarian adenocarcinoma cells. Therefore the cytotoxic effects of As2O3 are not due to metabolisation processes but rather to the presence of As(OH)3. 4.2. Analysis of HepG2 cells exposed to As(OH)3 We also performed m-XANES analysis of tumoral hepatocytes, HepG2 cells, a model cell line to study arsenic metabolization in vitro (Del Razo et al., 2001; Drobna et al., 2006). The mean energy of the maximum absorption edge is slightly different between the nucleus (11,870.7 eV) and the other two cellular areas, cytosol and mitochondrial network (11871.2 eV). The maximum energy of the absorption K-edge for trivalent arsenic reference compounds DMA(III), MMA(III), and As(OH)3 are, respectively, 11,869.0 70.5, 11,870.8 70.5, and 11,871.570.5 eV. From these data it can be concluded that the main oxidation state of arsenic in HepG2 cells is As(III), in all three cellular compartments (Fig. 3A). This is in good agreement with the reported speciation of arsenic in bulk samples of HepG2 cells exposed to 10 mM arsenite for 24 h giving
Fig. 3. Normalised XANES spectra at arsenic K-absorption edge in the cytosol and the nucleus of HepG2 cells exposed to 50 mM As(OH)3 during 24 h. Top spectra (A): sum of 9 local analyses of single cells showing that main arsenic oxidation state is As(III) both in cytosol and in nucleus. Bottom spectra (B): a mixture of trivalent and pentavalent arsenic is found in the nucleus, but not in the cytosol, of one of the 9 cells analysed. This observation suggests an inter-individual variability, which can only be pointed out by direct speciation analysis.
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order to preserve the native state of arsenic compounds (Bacquart et al., 2007). The second important result of HepG2 single cell analysis is the evidence of As(V) in one of the nucleus over the 9 cells analyzed (Fig. 3B). For pentavalent arsenic compounds DMA(V), MMA(V), and AsO(OH)3 the values of maximum absorption edge energies are, respectively, 11,873.070.5, 11,873.570.5, and 11,875.070.5 eV. The m-XANES spectra from this specific cell presents two maximum energy peaks: one at 11,871.2 eV, which corresponds to As(III), and another peak at 11,875.2 eV, which corresponds to As(V), and more specifically to AsO(OH)3. In the speciation experiment of bulk HepG2 samples (Del Razo et al., 2001), As(V) was not detected in cells. The m-XANES observation of mixed redox states of As(III) and As(V) in a single cell indicates that a minor fraction of arsenic could be present as As(V) although this fraction is difficult to evidence using bulk speciation analyses. The pentavalent As form has been detected in glioblastoma and bone marrow cells exposed to trivalent arsenic (Falnoga et al., 2007). The oxidation of As(III) to As(V) has been proposed as a mechanism of detoxification since As(V) compounds are less toxic than As(III) compounds (Aposhian et al., 2003). In addition, another very interesting observation is that As(V) is localized in a narrow region of the nucleus. One hypothesis could be that this zone corresponds to some micronuclei as arsenic is known to induce micronuclei in HepG2 cells (Gebel et al., 2002). However, this hypothesis remains to be verified. This singular result proves that the arsenic chemical species are not homogeneous in a cell population. It also points out the limitations of the m-XANES as this method requires long acquisition times, which limits the number of cells that can be analyzed during an allocated beam time on synchrotron radiation facilities.
5. Conclusion Thanks to the recent progress in m-XANES experimental devices, the speciation analysis of arsenic could be performed with high spatial resolution and with detection limits compatible with diluted concentrations. m-XANES is a powerful method to determine arsenic oxidation states at the sub-cellular level (Bacquart et al., 2007). It is probably the only method available for direct speciation analysis in cellular compartments. Direct speciation analysis allows the identification of chemical element species almost without sample preparation, keeping the cells and their chemical components very close to their native state. However it presents some limitations for the identification of arsenic chemical species when samples are constituted of a mixture of those species. It is therefore very important to compare
the experimental spectra with those of a large variety of reference compounds. Such pitfall could be overcome in the future if Extended X-ray Absorption Fine Structure (EXAFS) is performed at the sub-cellular level.
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