Uptake, Distribution, and Speciation of Selenoamino Acids by Human Cancer Cells: X-ray Absorption and Fluorescence Methods<xref rid="fn1"></xref>

Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies. X-ray absorption spectroscopy of bulk cell pellets treated with the selenoamino acids for 24 h showed that while selenium was found exclusively in carbon-bound forms in SeMet-treated cells, a diselenide component was identified in MeSeCys-treated cells in addition to the carbon-bound selenium species. X-ray fluorescence microscopy of single cells showed that selenium accumulated with sulfur in the perinuclear region of SeMettreated cells after 24 h, but microprobe selenium X-ray absorption near-edge spectroscopy in this region indicated that selenium was carbon-bound rather than sulfur-bound. X-ray absorption and X-ray fluorescence studies both showed that the selenium content of MeSeCys-treated cells was much lower than that of SeMet-treated cells. Selenium was distributed homogeneously throughout the MeSeCys-treated cells. Selenium is an essential element, which at supranutritional doses has been linked to reductions in cancer incidence and mortality. The seminal study in this field, The Nutritional Prevention of Cancer (NPC) Trial, showed that supplementation of 200 μg of Se/day as selenized yeast led to significant reductions in total cancer incidence and mortality and in the incidence of prostate cancer (1, 2). A more recent study, the Selenium and Vitamin E Cancer Prevention Trial (SELECT), investigated cancer risk associated with supplementation of 200 μg of Se/day as selenomethionine (SeMet), vitamin E, or a combination of both. The trial ceased 7 years into a planned 12-year study as there was no evidence of any benefit from either SeMet or vitamin E supplementation, but a small nonsignificant increase in the incidence of type 2 diabetes was observed in subjects taking only SeMet supplements (3). Selenized yeast tablets from the manufacturer of the NPC Trial tablets have been shown to have highly variable SeMet content (18-69%), with three other significant components remaining unidentified (4). The biological activity of Se is dependent on the chemical form of the Se compound, and it has been hypothesized that it is the methylated metabolites of Se supplements that are largely responsible for the anticancer properties of Se (5). As different Se compounds follow different metabolic pathways (6), the contradictory results of the NPC Trial and SELECT may be related to the use of different forms of Se supplementation. There is a clear need to identify themetabolites of different Se compounds in vivo to determine the most efficacious form of Se supplementation in cancer prevention. The selenoamino acids, selenomethionine (SeMet) and Semethylselenocysteine (MeSeCys), occur naturally in foods, are available as nutritional supplements, and have both been investigated for their anticancer properties (7). There are key differences between themetabolic pathways of these selenoamino acids and those of free selenide (formally HSe), the postulated cellular storage form of Se (8-10). SeMet can be adventitiously incorporated into proteins as SeMet or metabolized to selenocysteine (SeCys) and then lysed to HSe by β-lyase (11). SeMet may also be cleaved directly toMeSeH by γ-lyase (12). MeSeCys is not directly incorporated into proteins but is known to undergo cleavage by β-lyase to MeSeH, thereby largely avoiding metabolism to HSe (13). MeSeH is a putative generator of reactive oxygen species, and the ability of MeSeCys to produce MeSeH more efficiently than SeMet is thought to makeMeSeCys a more effective chemopreventative agent (9). This research was supported under the Australian Research Council’s Discovery Projects funding scheme (DP0984722 and DP0985807, QEII). We acknowledge travel funding provided by the International Synchrotron Access Program (ISAP) managed by the Australian Synchrotron. The ISAP is an initiative of the Australian Government being conducted as part of the National Collaborative Research Infrastructure Strategy. *To whom correspondence should be addressed. Telephone: þ61-88303-5060. Fax: þ61-8-8303-4358. E-mail: [email protected]. Abbreviations: NPC, Nutritional Prevention of Cancer; SELECT, Selenium and Vitamin E Cancer Trial; SeMet, selenomethionine; MeSeCys, Se-methylselenocysteine; HSe, hydrogen selenide; SeCys, selenocysteine; MeSeH, methylselenol; XAS, X-ray absorption spectroscopy; XANES, X-ray absorption near-edge structure; EXAFS, extended X-ray absorption fine structure; XRF, X-ray fluorescence; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SSRL, Stanford Synchrotron Radiation Lightsource; PCA, principal component analysis; APS, Advanced Photon Source; AS, Australian Synchrotron; CysSeSeCys, selenocystine; CysSSeCys, sulfoselenocystine. 1642 Biochemistry, Vol. 50, No. 10, 2011 Weekley et al. Studies of the speciation of Se in biological systems have relied largely on high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry, which is a method capable of accurately determining the concentration of major, minor, and trace components (6). However, this method requires extensive sample preparation that may itself alter the speciation of Se, thus confounding the results. Synchrotron X-ray absorption and fluorescence methods are ideal probes for investigating the chemical speciation and distribution of elements heavier than Si, such as Se, in biological systems with minimal sample preparation. X-ray absorption spectroscopy (XAS) and X-ray fluorescence (XRF) microscopy have previously been used to study the speciation and distribution of Cr in human lung cancer cells (14) and the metabolism of As in human hepatoma cells (15). While there are no previous reports of Se K-edge XAS conducted on mammalian systems, Pickering et al. (16) have shown that theX-ray absorption spectra of several biologically relevant Se compounds are sufficiently different to allow the identification of Se species within a biological matrix. Herein we investigate the speciation and distribution of Se in A549 human lung carcinoma cells treated with selenoamino acids, SeMet and MeSeCys, using X-ray absorption near edge structure (XANES) spectroscopy, extended X-ray absorption fine structure (EXAFS) spectroscopy, and synchrotron radiation X-ray fluorescence (XRF) microscopy. EXPERIMENTAL PROCEDURES Materials. Se-(methyl)selenocysteine hydrochloride (g95%) and seleno-L-methionine (g98%) were used as purchased from Sigma Aldrich. Solutions of the selenoamino acids in phosphatebuffered saline (PBS) (prepared using Milli-Q water) were prepared immediately before use. Cell Culture. A549 human lung adenocarcinoma epithelial cells, originally purchased from the American Tissue Culture Collection, were a gift from A. Levina (The University of Sydney). Cells were cultured as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (2%, v/v), L-glutamine (2 mM), an antibiotic/ antimicotic mixture (100 mg/mL penicillin and 100 units/mL streptomycin), and nonessential amino acids (100 units/mL) at 310 K in a 5% CO2-humidified incubator and were subcultured every 3-7 days. Cytotoxicity Assay. Cytotoxicity was assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (17). Briefly, cells were seeded at a density of 1 10 cells/well in a 96-well plate for 24 h at 310K in a 5%CO2humidified incubator. Solutions of the selenoamino acids prepared by serial dilution in PBS (10 μL) were added to full-serum DMEM (100 μL). After treatment for 72 h, the cells were incubated with an MTT solution (0.25 mg/mL in serum-free DMEM) for 3 h. The MTT solution was then replaced with dimethyl sulfoxide (100 μL), and the absorbance of the formazan solution was measured at a wavelength of 560 nm using a microplate spectrophotometer (BMG Lab Tech Fluostar Galaxy). Cell viability was reported as the percentage absorbance relative to the control as amean of three independent experiments (with eight replicates per experiment). IC50 values were determined by curve-fitting plots of cell viability versus the log of Se compound concentration. Sample Preparation. Bulk cell pellets from treated cultures were prepared for X-ray absorption spectroscopy. Cells were grown over 5 days to∼90%confluency in 75 cm culture flasks in completeDMEMandwere treated with SeMet (300 μL addition, yielding final concentrations of 100 and 200 μM), MeSeCys (300 μL addition, yielding final concentrations of 50 and 100 μM), or PBS (300 μL, as a vehicle-alone control) in fresh complete DMEM for 24 h. Cells were collected by gentle scraping and centrifugation. The supernatant was removed, and the cells were rinsed by resuspension in PBS (3 5 mL) and centrifugation before the pellet was collected and stored at 203 K and then vacuum-dried for 3 h. Cells used inXRF imaging were grown on 1.5mm 1.5mm 500 nm silicon nitride windows (Silson) in six-well plates as described previously (18). Briefly, the plates were seeded at a density of 1.8 10 cells/well in complete DMEM and were incubated at 310 K in a 5% CO2-humidified incubator for 24 h prior to treatment. Cells were treated with 50 μM SeMet or MeSeCys or PBS for 20min (1 h PBS treatment) and 10 or 50 μM SeMet or MeSeCys or PBS for 24 h (as a vehicle-alone control) before themediumwas removed and the cellswerewashed inPBS and fixed by being dipped in cold methanol (7 1 s). Samples were stored at 277 K. X-ray Absorption Spectroscopy and Data Analyses. Se K-edge X-ray absorption spectra of the bulk cell pellets were recorded at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 9-3. The X-ray beam was monochromated by reflection from a pair of Si(220) crystals. Harmonic rejection was achieved by setting the cutoff energy of a Rh-coated mirror to 15 keV.Cell pellets were compressed to approximately 3mm in diameter, secured betweenKapton tape, and cooled to∼10K in a flowing He cryostat. A spectrum of MeSeCys, used as a model compound for XANES fitting, was obtained from a frozen 5mM solution of MeSeCys in Milli-Q water. Spectra were recorded in fluorescence mode on a 30-element Ge detector array (Canberra) at 90 to the incident beam. The following energy ranges were used for XANES data collection: pre-edge region from 12425 to 12635 eV (10 eV steps), XANES region from 12635 to 12685 eV (0.25 eV steps), and postedge region from12685 to 12872 eV (0.05 Å steps in k-space). EXAFS spectra were recorded over the following energy ranges: pre-edge region from 12435 to 12635 eV (10 eV steps), XANES region from 12635 to 12685 eV (0.25 eV steps), and EXAFS region from 12685 to 13443 eV (0.05 Å steps in k-space to 14 Å). A hexagonal Se foil standard was used to calibrate the energy scale to the first peak of the first derivative of the Se edge (12658 eV). Data analysis, including calibration, averaging, and background subtraction of all spectra and principal component analysis (PCA), and target and linear regression analyses of XANES spectra were performed using EXAFSPAK (G. N. George, Stanford Synchrotron Radiation Laboratory, Stanford, CA). Spectra of model Se compounds for target and linear regression analyses were provided by G. N. George (University of Saskatchewan, Saskatoon, SK) except for that of MeSeCys, which was obtained during these experiments. X-ray Fluorescence Imaging, μ-XANES Spectra, and Data Analyses.XRF elemental distributionmaps of single cells were recorded on beamline 2-ID-E at the Advanced Photon Source (APS), Argonne National Laboratory, and on the X-ray fluorescence microprobe (XFM) beamline (19) at the Australian Synchrotron (AS).At theAPS, the beamwas tuned to an incident energy of 13.0 keV using a beam splitting Si(220) monochromator and was focused to a diameter of 1 μm using a “high-flux” zone plate. The tail of the scatter peak from the incident 13.0 keV Article Biochemistry, Vol. 50, No. 10, 2011 1643 beam does not significantly interfere with the Se KR peak at ∼11.2 keV (see Figure S1 of the Supporting Information). A single-element silicon drift energy dispersive detector (Vortex EX, SIINanotechnology, Northridge, CA), at 90 to the incident beam,was used to collect the fluorescence signal for 1 s per spatial point from samples under a He atmosphere. At the AS, a monochromatic 13.0 keV X-ray beam was focused (to a spot size of ∼1 μm ∼4 μm) using a zone plate, and the fluorescence signal was collected using a single-element silicon drift diode energy dispersive detector (Vortex EM, SII Nanotechnology), oriented at ∼73 degrees to the incident beam for 3 s per spatial point. Individual cells were located using images obtained from an optical microscope positioned in the beamline downstream from the sample. The fluorescence spectrum at each spatial point was fit to Gaussians, modified by the addition of a step function and a tailing function to describe mostly incomplete charge collection and other detector artifacts (20). For XRF images collected at the APS, two regions of interest corresponding to the whole cell (identified using optical images and the elemental distribution maps of P, S, Cl, K, and Zn) and nuclear regions (identified using optical images and regions of P and Zn colocalization) were selected. The integrated fluorescence spectra extracted from these regions were also fit with modified Gaussians to determine average elemental area densities (in units of micrograms per square centimeter). Quantification was performed by comparison to the corresponding measurements on the thin-film standards NBS-1832 and NBS-1833 from the National Bureau of Standards (Gaithersburg, MD). The analysis was performed using MAPS (21). At the AS, several regions of high Se concentration in the XRF maps of Se distribution were selected for μ-XANES analysis. Data were collected over the following energy ranges: pre-edge region from 12500 to 12640 eV (10 eV steps), XANES region from 12640 to 12690 eV (0.5 eV steps), and postedge region from 12690 to 12900 eV (10 eV steps) with a dwell time of 1 s per point. The specimen was repositioned through the μ-XANES scan to track the motion of the X-ray focus with energy. The stability of the analyzed region of each cell was characterized at ∼1 μm over the μ-XANES scan. A Se foil was used to calibrate the energy scale to the first peak of the first derivative of the elemental Se edge (12658 eV). Data analysis, including the calibration, averaging, and background subtraction of μ-XANES spectra, was performed using EXAFSPAK. Fitting of multiple model compounds using multiple linear regression analysis was not attempted because of the high noise level of the spectra.