extfieldsofstudy:
Medicine
Materials Science

s2fieldsofstudy: Biology Engineering

text:
Differential stress reaction of human colon cells to oleic-acid-stabilized and unstabilized ultrasmall iron oxide nanoparticles

Therapeutic engineered nanoparticles (NPs), including ultrasmall superparamagnetic iron oxide (USPIO) NPs, may accumulate in the lower digestive tract following ingestion or injection. In order to evaluate the reaction of human colon cells to USPIO NPs, the effects of non-stabilized USPIO NPs (NS-USPIO NPs), oleic-acid-stabilized USPIO NPs (OA-USPIO NPs), and free oleic acid (OA) were compared in human HT29 and CaCo2 colon epithelial cancer cells. First the biophysical characteristics of NS-USPIO NPs and OA-USPIO NPs in water, in cell culture medium supplemented with fetal calf serum, and in cell culture medium preconditioned by HT29 and CaCo2 cells were determined. Then, stress responses of the cells were evaluated following exposure to NS-USPIO NPs, OA-USPIO NPs, and free OA. No modification of the cytoskeletal actin network was observed. Cell response to stress, including markers of apoptosis and DNA repair, oxidative stress and degradative/autophagic stress, induction of heat shock protein, or lipid metabolism was determined in cells exposed to the two NPs. Induction of an autophagic response was observed in the two cell lines for both NPs but not free OA, while the other stress responses were cell- and NP-specific. The formation of lipid vacuoles/droplets was demonstrated in HT29 and CaCo2 cells exposed to OA-USPIO NPs but not to NS-USPIO NPs, and to a much lower level in cells exposed to equimolar concentrations of free OA. Therefore, the induction of lipid vacuoles in colon cells exposed to OA utilized as a stabilizer for USPIO NPs is higly amplified compared to free OA, and is not observed in the absence of this lipid in NS-USPIO NPs.

Introduction
Nanotechnology and the extended use of nanomaterials in nanomedicine is a rapidly developing field. [1][2][3] Nanomaterials are materials with one, two, or three dimensions in the nanoscale, while nanoparticles (NPs) are usually defined as systems for which all three dimensions are roughly 1 to 100 nm. In the field of nanomedicine, the acceptance of this definition extends to particles or nanotherapeutics with dimensions up to 1,000 nm. Since the initial development of therapeutic NPs, the field of nanotechnology has gained a lot of interest due to their huge potential for applications in industry and medicine. The primary goals of nanoparticulate systems in nanomedicine are to target specific tissues or cells, to deliver drugs in a controlled manner, thus lowering the necessary dose of chemotherapeutics for therapeutic efficacy together with decreasing the toxicity of the corresponding treatment to alleviate the side effects of chemotherapeutics. However, the biocompatibility of therapeutic NPs must be ensured and their intrinsic toxicity must be controlled so that it does not overtake the benefits of decreasing the toxicity of the drugs or treatments. Therefore, a deep understanding of their interactions with living tissues and knowledge of their possible effects in the human body are necessary for the safe use of nanoparticulate formulations. However, the knowledge about the possible interactions of engineered therapeutic NPs with living cells and tissues and their metabolic fate presently are not complete or well-defined.
After ingestion or intravenous injection, therapeutic NPs may reach the lower digestive tract. The effects of the interaction of NPs, in particular of metal-based solid-core NPs, with the colon epithelia, the potential toxicity profile of NPs, and the evaluation of the effects of NPs' exposure on the functional response of colon cells are currently limited. It is generally accepted that not only the size, but also the chemical composition, surface chemistry, charge, and shape are relevant for considering the behavior of NPs in the biological environment.
Ultrasmall superparamagnetic iron oxide (USPIO) NPs were initially developed as therapeutic and diagnostic agents in nanomedicine for magnetic resonance imaging, for perfusion imaging, and for angiography and tumor vascular imaging. Their uses have been recently extended to new applications. [4][5][6][7] We have previously shown that USPIO NPs are taken up by human cells, even when functionalized with a fluorophore 8,9 or with therapeutic anticancer agents, 10,11 but that the biochemical characteristics of the surface functionalization of the NPs were important for their cellular trafficking and the reaction of the cells to their uptake. In particular, we had observed that the intracellular localization of cationic USPIO NPs functionalized with hydrophilic or hydrophobic drugs was dependent on the lipophilicity of the drugs. 10,11 We have also previously shown that monolayers and spheroids of human colon cells can internalize cationic USPIO NPs, but cannot release or transport them across models of the human colon epithelial barrier. 12 In the present study, in order to further evaluate the effects of the biophysical characteritics of USPIO NPs in human colon cells, we compared the reaction of human HT29 and CaCo 2 colon epithelial cells to deeply characterized non-stabilized (bare) and oleic-acid-stabilized USPIO NPs, demonstrating that the main colon cell stress response was the induction of high amounts of large lipid vacuoles dependent on the oleic acid stabilizer of the USPIO NPs, but not on free oleic acid or the iron oxide core.

Materials and methods NPs
Non-stabilized USPIO NPs (Fe 3 O 4 ; NS-USPIO NPs) were purchased from PlasmaChem (PlasmaChem GmbH, Adlershof, Germany) as a ∼3% nanosuspension in water with a nominal NP size of 8±3 nm (as determined by dynamic light scattering [DLS], value provided by the provider) and 18 mg iron/mL by quantitative Prussian Blue reaction. Oleicacid-stabilized USPIO NPs (Fe 3 O 4 , 3% oleic acid coating; OA-USPIO NPs) were obtained from PlasmaChem as a ∼7% nanosuspension in water with a nominal particle iron oxide core size of 8±3 nm, a hydrodynamic size of 14-15 nm (both values determined by DLS by the provider), and 206 mg iron/mL by quantitative Prussian Blue reaction.

Physicochemical characterization of the UsPIO NPs
The nominal size of both USPIO-NPs was measured by transmission electron microscopy (TEM) using asreceived material and Holey carbon 300 mesh copper grids (Polysciences, Warrington, PA, USA) to confirm the supplier data provided. NP properties and stability characteristics were assessed at Trinity College Dublin; NPs were placed in ultrapure deionized water for 24 hours, then the NPs were incubated in 10% heat-inactivated fetal calf serum (FCS) in phosphate-buffered saline (PBS), in Dulbecco's Modified Eagle's Medium (DMEM) culture medium, in DMEM containing 10% FCS, or in cell culture medium preconditioned for 72 hours by the CaCo 2 and HT29 cells (CaCo 2 -CM and HT29-CM). Stock solutions of NS-USPIO NPs and OA-USPIO NPs were vortexed for 5 seconds to disperse the particles evenly and then diluted in the relevant solutions. NS-USPIO NP and OA-USPIO NP characterization was carried out for the identification of nominal and hydrodynamic radii, zeta potentials, colloidal and aggregation stabilities by DLS (Malvern Zetasizer; Malvern Instruments, Malvern, UK), and NP tracking analysis (NTA; Nanosight NS500; Malvern Instruments) of the two NPs dispersed in the different biological matrices after vortexing for 5 seconds. Three USPIO NP concentrations were used (eg, 25, 50, and 100 µg/mL). All measurements were carried out three times at physiologically relevant pH (pH =7) and averages and standard deviations (SDs) are reported. For both techniques, hydrodynamic radius and polydispersity index (PDI) were measured at room temperature using single depth position, recording six videos of 90 seconds each. Quality assurance over the measurements carried out was guaranteed by the adoption of Quality Nano (QNano, FP7 project) standard operating procedures, which have been developed as part of large inter-laboratory comparative study focused on NP physicochemical characterization. 13 International Journal of Nanomedicine 2014:9 submit your manuscript | www.dovepress.com Dovepress Dovepress 3483 stress reaction of human colon cells to iron oxide nanoparticles cell models and culture conditions Human HT29 and CaCo 2 colon cancer cells were obtained from the American Tissue Culture Collection (Manassas, VA, USA). Cells were grown in DMEM containing 4.5 g/L glucose, 10% heat-inactivated FCS, and penicillin/streptomycin (all cell culture reagents from Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). To obtain cell-conditioned medium (cell-CM), HT29 and CaCo 2 cells were grown to 75% confluence, the medium was changed to fresh complete medium and cell-CM was harvested after 3 days, centrifuged, and stored in aliquots at -20°C.

cell uptake of UsPIO NPs
Cells were grown in 48-well plates (Costar; Corning Incorporated, Corning, NY, USA) until 75% confluent, and exposed to NPs diluted in DMEM with 10% FCS without phenol red (Invitrogen) for 24 hours at the concentrations and for the time indicated. Cell-associated iron was quantified using the Prussian Blue reaction essentially as previously described. 12 Briefly, the cell layers were dissolved at room temperature for 1 hour in 6 M HCl (125 µL/well), then 125 µL of a 5% weight/volume (w/v) solution of K 4 [Fe(CN) 6 ]⋅3H 2 O (EMD Millipore, Billerica, MA, USA) in water was added for 10 minutes and the absorbance was read at 690 nm in a multi-well plate reader (iEMS Reader; Labsystems, BioConcept Ltd., Allschwil, Switzerland). A standard curve of iron chloride in 6 M HCl treated under the same conditions was used to quantify the amount of cell-bound iron. All experiments were performed in triplicate wells, and repeated twice. Means ± SDs were calculated.

Determination of cytotoxicity
Cells were exposed to the NPs or to oleic acid (EMD Millipore) at the concentrations and for the time indicated. Following exposure, cell viability was evaluated using the 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) assay (200 µg/mL final concentration; Sigma-Aldrich Co., St Louis, MO, USA), essentially as previously described. 12 Absorbance at 540 nm was measured in a multi-well plate reader (iEMS Reader; BioConcept Ltd.), and the absorbance values of treated cells were compared with the absorbance values of untreated cells. The interference of the NPs with the assay was controlled as described in more detail elsewhere and no interference was observed in the conditions of our assay. 14 Experiments were conducted in triplicate wells and repeated twice. Means ± SDs were calculated. Statistical significance was ascertained using a Student's t-test.

histochemical determination of iron and lipids
For histological iron determination, after exposure to USPIO NPs or to oleic acid, the cell layers were washed with saline and incubated for 20 minutes at room temperature with a 1:1 solution of 10% HCl and 10% K 4 [Fe(CN) 6 ] . 3H 2 O in H 2 O, washed with distilled water, counterstained with Nuclear Fast Red (EMD Millipore), dehydrated in graded ethanol to xylol, and mounted. Slides were photographed under a Nikon digital camera (DXM 1200; Nikon Corporation, Tokyo, Japan). For histological lipid determination, after exposure to the NPs or to oleic acid, the cell layers were washed with PBS, fixed in 4% buffered formaldehyde at 4°C for 30 minutes and the histological Prussian Blue reaction was performed first, washed once with distilled water, then stained with Oil-O-Red (saturated solution in isopropanol, diluted 3:2 in distilled water; EMD Millipore), counterstained with hematoxylin (Sigma-Aldrich Co.) and mounted in Pertex (Leica Biosystems, Wetzlar, Germany). Slides were photographed under a Nikon digital camera (DXM 1200; Nikon Corporation).

TeM for cell imaging
Cells were grown in 9 cm diameter Petri dishes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and exposed to the NPs or oleic acid for the appropriate times. Cells were washed twice with PBS, fixed in 2% glutaraldehyde buffered in 0.1 M cacodylate for 1 hour at 4°C, then washed in 0.2 M cacodylate at 4°C (three times for 10 minutes), gently scraped in 0.2 M cacodylate and harvested by centrifugation (5 minutes at 1,000 rpm). The pellets were coated with agarose (type III low gelling temperature; Sigma-Aldrich Co.) and cut in small pieces. The samples were postfixed in 1%-2% buffered osmium tetroxide for 1 hour at room temperature, washed in 0.2 M cacodylate (twice for 5 minutes), dehydrated in graded ethanol (twice for 10 minutes in 70% ethanol, twice for 10 minutes in 90% ethanol, and three times for 20 minutes in 100% ethanol), dehydrated again in propylene oxide (twice for 30 minutes), embedded in equal volumes of propylene oxide and Epon (50% Epon 812 substitute, 26% dodecenylsuccinic anhydride, 23% methyl nadic anhydride, 1% 2,4,6-tris[dimethylaminomethyl]phenol); all dilutions w/v, all chemicals from Fluka; Sigma-Aldrich Co.) for 1 hour, and then embedded for 16 hours in Epon. Specimens were embedded in Epon blocks that were cured for 48 hours at 60°C, and then trimmed and cut into 100 nm sections and mounted on slides for post-visualization by methylene blue/azure blue staining. Thin sections (50 nm) were cut from the cured blocks using an ultramicrotome (Ultracut E; Reichert-Jung Optische Werke AG, Wien, Austria) and mounted on 3 mm 200-mesh copper grids. Grids were heavy-metal stained using a standard two-step uranyl acetate/lead citrate technique (Leica EM Stain; Leica Microsystems, Wetzlar, Germany) and then examined and photographed at 80 kV with a Philips CM12 transmission electron microscope (Philips International B.V., Amsterdam, the Netherlands) combined with a MegaView III, Soft Imaging System (Gloor Instruments AG, Kloten, Switzerland). The quantification of the number per cell and diameter of the lipid vacuoles were determined on TEM images obtained at 2 µm resolution of single cells without prior selection (n20 per treatment) using the ImageJ software (imagej.net; National Institutes of Health, Bethesda, Maryland, USA) and were then represented in boxplot figures. Normality of the data was tested using the Shapiro-Wilk test, and statistical significance was ascertained using the Wilcoxon-Mann-Whitney test.

acetate incorporation
Radioactive acetate incorporation was performed according to the protocol published by Beckers et al. 15 The cells were seeded and grown in 6-well plates (Costar; Corning Incorporated) for 48 hours, then exposed to the NPs or to oleic acid for 24 hours. Six hours prior to the end of the treatment, etomoxir (10 µg/mL, an inhibitor of fatty acid oxidation; Sigma-Aldrich Co.) and [ 14 C]sodium acetate (10 nCi/mL final concentration; American Radiolabeled Chemicals, Inc., St Louis, MO, USA) were added to the cells for 24 hours. The cell layers were precipitated with 10% trichloroacetic acid, then dissolved in 0.1% sodium dodecyl sulfate in 0.1 N NaOH and 4.5 mL scintillation cocktail (Optiphase HI-Safe; PerkinElmer Inc., Waltham, MA, USA). Radioactivity was counted in a β-counter (WinSpectra, Wallac, Finland). The radioactivity counts of treated cells were compared to the radioactivity counts of untreated cells. Cellular protein concentration was determined using the bicinchoninic acid protein assay, according to the provider's instructions (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific) using bovine serum albumin (BSA) as the standard. Measurements were performed in triplicate and repeated three times. Means ± SDs were calculated. Statistical significance was ascertained using a Student's t-test.

Western blotting experiments
Cells were grown in 9 cm diameter Petri dishes (Becton, Dickinson and Company) and exposed to the NPs or oleic acid for the appropriate times. Following treatments, the cell layers were washed once with cold PBS and lysed in 250 µL of lysis solution (17.6 mM Tris-HCl, 120.56 mM NaCl, 8.8% volume/volume glycerol, 0.88% volume/volume NP40, 0.88% w/v CHAPS, 2 mM vanadate, 50 mM NaF, pH 7.85) and 10 µL of proteinase inhibitor cocktail (Sigma-Aldrich Co.), and extracted by four cycles of freeze/thawing. Supernatants were submitted to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Whatman; GE Healthcare Europe GmbH, Freiburg, Germany). The membranes were blocked with 5% fat-free milk in PBS, 5% BSA in PBS, washed in 0.05% Tween-20 (Sigma-Aldrich Co.) in PBS, and incubated overnight at 4°C with the primary antibodies diluted in 1% fat-free milk or 1% BSA in 0.05% Tween-20 in PBS. Membranes were then exposed for 1 hour at room temperature to peroxidase-conjugated secondary antibodies and visualized using chemoluminescence (ECL; GE Healthcare UK Ltd, Little Chalfont, UK). The primary and secondary antibodies, buffers, and dilutions used are summarized in Supplementary material, Table S1. To control for loading, the membranes were stripped by successive incubation in 0.1 M glycine pH 2.3, 1 M NaCl in PBS, and 0.05% Tween 20 in PBS, blocked for 1 hour with 5% fat-free milk in PBS, and exposed to a polyclonal anti-human β-actin rabbit antibody (diluted 1:5,000; Sigma-Aldrich Co.) for 1 hour at room temperature and treated as washed and visualized. For densitometric analysis, the protein band areas were evaluated using ImageJ software (imagej. net; National Institutes of Health), then the ratio of the band area of each protein to the area of the actin band was determined.

glutathione-s-transferase (gsT) activity
The activity of GST in cell extracts was determined using 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as previously described. 16 Briefly, cells were grown for 24 hours in 9 cm diameter Petri dishes (Becton, Dickinson and Company) and exposed to the NPs or oleic acid for 72 hours. Then, the cell layers were scraped in cold pH 6.5 PBS, extracted by 4 cycles of freezing/thawing, and centrifuged at 10,000 rpm at 4°C for 10 minutes and the supernatants were collected. The GST activity in the supernatants was determined by measuring the increase in absorbance at 340 nm in a multi-well plate reader (iEMS Reader; BioConcept Ltd.) for 30 minutes at 37°C after addition of CDNB and GSH (1 mM final concentrations; Sigma-Aldrich Co.). GST activity was expressed as nM CDNB-GSH conjugates/minute/mg Measurements were performed in triplicate and repeated three times. Means ± SDs were calculated. Statistical significance was ascertained using a Student's t-test.
staining of cellular actin with fluorescent phalloidin Cells were grown for 48 hours in 4-chamber glass slides (Becton, Dickinson and Company) and exposed to the NPs or oleic acid for 24 hours. At the end of the treatment, the cell layers were washed in PBS, fixed in 4% buffered formaldehyde, washed with PBS, permeabilized for 5 minutes in 0.1% Triton X-100 in PBS, and washed. Then, 200 µL of a 1% BSA (Sigma-Aldrich Co.) and 2.5% Oregon Green 488 Phalloidin (6.6 µM stock solution in methanol; Thermo Fisher Scientific) solution in PBS were added. After 20 minutes incubation at room temperature, the cell layers were washed with PBS and 2 mL of 1 µg/mL 4′,6′-diamidino-2-phenylindole (DAPI) (Hoffman-La Roche Ltd., Basel, Switzerland) in PBS was added for 20 minutes. Slides were washed with PBS and mounted in 20% glycerol in PBS. Fluorescence images were taken with a Zeiss Axioplan 2 Imaging microscope (Carl Zeiss Meditec AG, Jena, Germany) at 400× magnification and λ ex /λ em =365/420 nm (DAPI) or λ ex /λ em = 450-490/515-565 nm (Oregon Green 488 Phalloidin).

characterization of the Ns-UsPIO NPs and Oa-UsPIO NPs
The determination of the nominal size of NS-USPIO NPs and OA-USPIO NPs was assessed using TEM to confirm  Figure 1) highlighted the spherical shape of the particles, their crystalline structures and wide size distribution, and tendency to aggregate under TEM conditions for both samples. No major differences were evident between the NS-USPIO NPs and OA-USPIO NPs from TEM analysis. The average diameter size for both USPIO NPs was measured to be 9.3±3.5 nm. Higher magnification TEM images also confirmed that the particles were synthesized by physical vapor deposition due to the organized crystalline structure. NS-USPIO NP and OA-USPIO NP characterization was carried out using either DLS to measure NS-USPIO NPs' and OA-USPIO NPs' nominal and hydrodynamic particle size, zeta potential, PDI, and mobility, or NTA (Nanosight NS500; Malvern Instruments) to measure particle size, PDI, and colloidal and aggregation stabilities at physiologically relevant conditions (Table 1, Table 2, Table S2, Table S3, and Figure 2). The NPs were analyzed first in water, then after incubation for 24 hours into relevant biological matrixes: 10% FCS in PBS (FCS), DMEM culture medium (DMEM), DMEM culture medium containing 10% FCS (DMEM + FCS) ( Table 1 and Figure 2), or culture medium preconditioned for 72 hours by the CaCo 2 and HT29 cells (HT29-CM and CaCo 2 -CM) ( Table 2 and Figure 2). Results were consistent between both DLS and NTA, and between the three NP concentrations analyzed; thus, only NTA results at a NP concentration of 50 µg/mL are shown in Figure 2. From the analysis of the results obtained, it is   Table 2 and also shown by the NTA batch analysis particle size plots reported in Figure 2. The zeta potentials of both USPIO NPs could not be determined in the biological matrices, since the NPs agglomerate. The zeta potentials are provided only for the nanosized NPs contained in the agglomerates in Table S2A and Table S2B; these values are in parentheses to underline this uncertainty.

Uptake and cytotoxicity of the Ns-UsPIO NPs and Oa-UsPIO NPs
In cells exposed to NS-USPIO NPs, the NPs were mostly cell-associated after 24 hours of exposure ( Figure 3A) as determined by the quantitative Prussian Blue reaction of iron, whereas the level of cell-associated iron was much lower in cells exposed to OA-USPIO NPs, close to the detection limit of the Prussian Blue reaction, but still detectable ( Figure 3B). None of the USPIO NPs were cytotoxic for the colon cells ( Figure 4A). The cytotoxicity of oleic acid for the human colon cells was also evaluated. In a 50 µg iron/mL OA-USPIO NPs solution, the concentration of oleic acid was 23 µM, which represents the maximal oleic acid concentration achieved in our experiments; thus, we evaluated the cytotoxicity of multiples of this initial concentration. While a ten-fold-concentration of oleic acid had no effect on the viability of the human colon cells, only the 100-and 1,000-fold-concentrations dramatically decreased cell survival ( Figure 4B). Ethanol, the solvent of oleic acid, at the highest concentration achieved in our experiments, had no effect on cell survival. Thus, neither the oleic acid stabilizer of the OA-USPIO NPs nor the two USPIO NPs were cytotoxic for the cells.     Added iron (µg/mL)

Iron/well (µg) Iron/well (µg)
Added iron (µg/mL) A B Figure 4 cytotoxicity of Ns-UsPIO NPs, Oa-UsPIO NPs, and free oleic acid for human colon cells. Notes: (A) cytotoxicity of Ns-UsPIO NPs and Oa-UsPIO NPs. caco 2 and hT29 cells were exposed for 24 hours to increasing amounts of Ns-UsPIO NPs or Oa-UsPIO NPs, then the cell metabolic activity was determined using an MTT assay. cytotoxicity is expressed as the percentage of cell survival of exposed cells compared to unexposed cells. Means ± sD were calculated. : caco 2 cells; ♦: hT29 cells. (B) cytotoxicity of free oleic acid. human hT29 and caco 2 cells were exposed for 72 hours to increasing concentrations of oleic acid or to the highest concentration (1.75%) of ethanol used to dissolve oleic acid, then an MTT test was performed. results are expressed as the percentage of cell survival of exposed cells compared to unexposed cells and are the means ± sD of triplicates of two independent experiments. Statistical significance was assessed using a student's t-test. Abbreviations: MTT, 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide; NPs, nanoparticles; Ns-UsPIO NPs, non-stabilized UsPIO NPs; Oa-UsPIO NPs, oleicacid-stabilized UsPIO NPs; sD, standard deviation; UsPIO, ultrasmall superparamagnetic iron oxide. involved in other cellular responses was assessed using Western blotting experiments ( Figure 6A and Figure 6B). The expression and/or activation state of markers of apoptosis (cleaved poly [ADP-ribose] polymerase [PARP]), double-strand DNA damage (phosphorylated histone H2AX [γH2AX]), oxidative stress (the GST-π), as well as the enzymatic activity of GST (results not shown), or hypoxia (hypoxia-inducible factor 1-α [HIF-1α]) were not affected by the exposure of colon cells to NS-USPIO NPs, OA-USPIO NPs, or free oleic acid. A weak increase of the expression of the lysosomal protease procathepsin D and cathepsin D was observed in CaCo 2 cells treated with NS-USPIO NPs, and a clear increase in the expression of the autophagic marker LC3I/ II was shown in CaCo 2 cells exposed to both NS-USPIO NPs and OA-USPIO NPs, but not in cells exposed to free oleic acid. The transferrin receptor-1/CD71, an iron transporter regulated by intracellular iron, was only expressed by HT29 cells and its expression slightly decreased when the cells were exposed to NS-USPIO NPs, which are internalized at high levels by the cells. Of the three heat shock protein (HSP) chaperones evaluated, only the expression of HSP90 significantly increased in CaCo 2 cells, but not in HT29 cells, exposed to NS-USPIO NPs and increased at an even higher level in those cells exposed to OA-USPIO NPs. Thus cell-selective and NP-selective effects of USPIO NPs in the lysosomal, iron transport, and autophagic responses of colon cells, and a stress response mediated by the chaperone HSP90 were demonstrated.

Oa-UsPIO-NPs-induced formation of lipid vacuoles by colon cells
Then we evaluated the effect of oleic acid, either free or USPIO-NPs-associated, on the metabolism and handling of lipids in the human colon cells. Both NS-USPIO NPs and OA-USPIO NPs, but not free oleic acid, decreased the incorporation of acetate in CaCo 2 cells, but not in HT29 cells (Figure 7). OA-USPIO NPs decreased acetate incorporation significantly more than NS-USPIO NPs, suggesting a selective interference with lipid metabolism in these cells. Therefore, we examined the effects of NS-USPIO NPs, OA-USPIO NPs, and free oleic acid on the aspect of cells when examined under light microscopy ( Figure 8). In cells exposed to NS-USPIO NPs, high amounts of the iron oxide cores of NPs could be seen inside cells in the TEM images (Figure 8, black arrows), while only low amounts of the iron oxide cores could be detected by TEM in cells exposed to OA-USPIO NPs, which are internalized at much lower levels than NS-USPIO NPs. However, TEM images demonstrated the presence of a high amount of vacuoles in cells exposed to OA-USPIO NPs, and of a much lower amount of vacuoles in cells exposed to free oleic acid (Figure 8, white arrows). The lipidic nature of these vacuoles was demonstrated by histological staining with Oil-Red-O, a fat-soluble red-colored dye which allows visualizing and evaluating lipids in cells (Figure 9). In cells exposed to OA-USPIO NPs, many red-colored vacuoles were observed, while in cells exposed to an equimolar concentration of free oleic acid achieved in OA-USPIO NPs, only rare lipid vacuoles were observed. Thus, the Oil-Red-O histological staining strongly supported a lipidic nature for the vacuoles observed in TEM images. Thus the OA-USPIO NPs, despite their poor cellular uptake, were inducing a cell reaction suggesting an effect of the oleic acid stabilizer in the cell metabolism.
As shown by TEM, the appearance of the lipid in cells exposed to OA-USPIO NPs was time-dependent ( Figure 10A, white arrows). In order to validate the TEM observations, the diameter and the number of lipid droplets/vacuoles per cell were determined on TEM images of at least 20 different cells per treatment randomly selected from cultures of HT29 and CaCo 2 cells exposed to OA-USPIO NPs or free oleic acid, Notes: hT29 and caco 2 cells were exposed for 24 hours to either 23 µM free Oa, Ns-UsPIO NPs (50 µg iron/ml), or Oa-UsPIO NPs (50 µg iron/ml), then the expression of proteins involved in DNa repair, apoptosis, oxidative stress response, lysosomal and autophagic activation (degradative response), iron uptake, or heat shock chaperone induction was analyzed by Western blotting (A), then the ratio of the band intensities of each protein to the band intensity of actin was evaluated (B). Abbreviations: cD71, transferrin receptor; cParP, cleaved poly(aDP-ribose) polymerase; gsT-π, glutathione s-transferase-π; γh2aX, phospho-histone h2aX; hIF-1α, hypoxia-inducible factor 1-α; hsP, heat shock protein; NPs, nanoparticles; Ns-NPs, Ns-UsPIO NPs; Ns-UsPIO NPs, non-stabilized UsPIO NPs; NT, non-treated; Oa, oleic acid; Oa-NPs, Oa-UsPIO NPs; Oa-UsPIO NPs, oleic-acid-stabilized UsPIO NPs; UsPIO, ultrasmall superparamagnetic iron oxide.

Figure 8
Induction of vacuoles in human colon cancer cells exposed to Oa-UsPIO NPs or free Oa. Notes: human hT29 or caco 2 colon cancer cells were either unexposed (control) or exposed to Ns-UsPIO NPs or Oa-UsPIO NPs (50 µg iron/ml), or free Oa (23 µM) for 24 hours, then TeM images were acquired. Black arrows: iron oxide cores; white arrows: vacuoles. Abbreviations: NPs, nanoparticles; Ns-UsPIO NPs, non-stabilized UsPIO NPs; Oa, oleic acid; Oa-UsPIO NPs, oleic-acid-stabilized UsPIO NPs; UsPIO, ultrasmall superparamagnetic iron oxide; TeM, transmission electron microscopy.  Figure 10B). In both colon cancer cell lines, the number and the diameter of the vacuoles significantly increased after exposure to OA-USPIO NPs in comparison to cells exposed to free oleic acid or in control cells.

Discussion
Magnetic USPIO NPs are in clinical use or under evaluation as diagnostic, therapeutic, and theranostic agents, which include contrast agents for magnetic resonance imaging, targeted drug delivery, cell sorting and tracking, or hyperthermia. With these expanding applications, the potential toxicity/cytotoxicity of magnetic NPs is of concern and is under experimental evaluation. Therefore, in the present manuscript we compared the responses of two human colon epithelial cancer cells (HT29 and CaCo 2 cells) to exposure to two USPIO NPs with the same core nominal size and morphology, but either NS-USPIO NPs or OA-USPIO NPs, as well as the free oleic acid which was used as the stabilizing acid of the USPIO NPs. First, the two USPIO NPs were characterized in detail for their biophysical characteristics in several relevant biomedical cell culture media, including cell culture media unconditioned or preconditioned by the cells studied (HT29-CM or CaCo 2 -CM). The determination by TEM of the shape, size distribution, and average diameter of the iron oxide core were similar for both USPIO NPs. The detailed physicochemical properties of NS-USPIO NPs and OA-USPIO NPs were also assessed using DLS and NTA in the different biological media. The incremental characterization from water to FCS, to DMEM only, then to DMEM + FCS, and finally to cell-CM was carried out to systematically dissect each single aspect of the NP suspension in biological media to understand how the components making the media induced aggregation. The physicochemical characterization of the NS-USPIO NPs showed very different behavior than that of the OA-USPIO NPs. OA-USPIO NPs displayed a narrower size distribution in water and FCS than NS-USPIO NPs. In the presence of FCS, NS-USPIO NPs were initially highly aggregating but tended to better stabilize, showing less aggregation in DMEM + FCS over the long-term than OA-USPIO NPs. In both HT29-CM and CaCo 2 -CM, OA-USPIO NPs showed less aggregation than NS-USPIO NPs, but aggregation recovery after incubation in the cell-CM. The surface characteristics, size, agglomeration state, and chemical composition are important factors to be considered for the reaction of cells to the stress of being exposed to NPs.
Thus, after incubation in the cell culture medium conditioned by the CaCo 2 and HT29 cells, OA-USPIO NPs underwent a stabilization response, suggesting a different mechanical interaction between the particles and the cells in their culture medium. Different surface properties of NPs attract specific molecules from the surrounding medium, especially proteins, thus forming different protein corona. 17 Thus, we can hypothesize that the protein corona of USPIO NPs in cell-CM is responsible for their improved dispersibility.
The factors to be considered for the reaction of cells to the stress of being exposed to NPs are multiple. The state of aggregation/agglomeration of NPs is an important feature that influences the cell responses to NPs. For example, cytotoxicity and genotoxicity of TiO 2 NPs were dependent on the state of the NPs aggregation. 18 Unstabilized NPs tend to agglomerate, while stabilized NPs are more dispersed. 19 Also, the chemical composition of the surface coating of the NPs is another relevant factor. The coating surface of silver NPs determined cytotoxicity. 20 For example, the oleate coating of nickel ferrite NPs decreased cell viability compared with uncoated NPs. 21 Oleate-coated ZnO NPs induced micronuclei, whereas uncoated ZnO NPs were not genotoxic. 22 Here, we showed that the state of USPIO NPs agglomeration was important for their uptake by the human colon cells but not for their cytotoxicity for the cells. However, the size of the NPs is not the only reason for the low uptake of OA-USPIO NPs; the negative charges at their surface can be hypothesized  Notes: (A) human hT29 or caco 2 colon cancer cells were exposed to Oa-UsPIO NPs (50 µg iron/ml). TeM images were acquired after 0 h, 2 h, 6 h, or 24 h exposure. White arrows: lipid vacuoles; scale bar =2 µm. The white arrows indicate vacuoles/droplets. (B) human hT29 or caco 2 colon cancer cells were unexposed (control) or exposed to Oa-UsPIO NPs (50 µg iron/ml) or free Oa (23 µM) for 6 h or 24 h, then TeM images of the cells (at least 20 cells per treatment) were acquired and the statistical evaluation of the number and diameter of the vacuoles per cell was performed. Statistical significance was assessed using the Wilcoxon-Mann-Whitney test: **P0.01; ***P0.001. Black bar: median, : outliers. control: cells exposed only to culture medium.  influenced their metabolic response. We did not observe a modification of cytoskeletal actin in cells exposed to either of the USPIO NPs. PARP is involved in DNA repair and is inactivated by caspase cleavage in apoptosis, and the histone H2AX (γH2AX) is phosphorylated in response to DNA damage. 23,24 The GST-π is regulated by the HIF-1α, by a hypoxic stress and the level of cell iron. 16,25 Neither of the USPIO NPs modified the PARP, γH2AX, GST-π, or HIF-1α. The transferrin receptor protein-1/CD71, an iron transporter, is downregulated by high concentrations of intracellular iron. 26 The transferrin receptor protein-1/ CD71 expression slightly decreased in HT29 cells exposed to NS-USPIO NPs, which are internalized at high levels by the cells, but not in cells exposed to OA-USPIO NPs, which are internalized at much lower levels. We have previously shown a comparable effect in human melanoma and endothelial cells using cationic USPIO NPs, which are highly taken up by human cells. 27,28 A cell autophagic response to NP exposure will result in the activation of lysosomal proteases and an increase in the expression of the autophagic marker LC3I/II. 29 A weak increase of the expression of the lysosomal protease procathepsin D and cathepsin D was observed in CaCo 2 cells exposed to NS-USPIO NPs, but not to OA-USPIO NPs, suggesting that the level of intracellular iron is responsible for the cathepsin D as well as CD71 responses. We have previously shown in human endothelial cells an autophagic response to the highly internalized cationic amino-PVA-USPIO NPs. 28 In human colon cells, we observed an autophagic response to both NS-USPIO NPs and OA-USPIO NPs, not related to the level of cell uptake of the NPs, and interestingly also to free oleic acid in HT29 cells, suggesting a protective response of the cells dependent on the exposure of cells to NPs, but not to the physicochemical characteristics of the NPs. Chaperones, such as the HSP, assist in the non-covalent folding or unfolding of others proteins, and are overexpressed in response to different environmental stress. 30 The expression of the mitochondrial chaperone HSP60, the toxic stress-induced chaperone HSP70, and the general protective chaperone HSP90 31,32 were evaluated. Neither of the USPIO NPs modified the HSP60 and HSP70 expression or activation state. Only the expression of HSP90 significantly increased in CaCo 2 cells, but not in HT29 cells, exposed to NS-USPIO NPs and even increased at a higher level in those cells exposed to OA-USPIO NPs. Thus, the cellular response to stress, including changes in cell protein activation or expression, did not seem to depend upon the biophysical characteristics of NS-USPIO NPs and OA-USPIO NPs since these responses were mainly cell-specific. Accordingly, it has been previously demonstrated that cell reaction to NPs may depend on the biological characteristics of the cells used as models, even with cells of the same tissue origin. 20 HT29 cells were generally less responsive than CaCo 2 cells, possibly as a consequence of the different functional characteristics of these cells. The HT29 cells originate from a human colon adenocarcinoma and can differentiate into goblet cells, whereas CaCo 2 cells, although also derived from a colon carcinoma, differentiate into cells resembling the absorptive enterocytes. Thus, we could demonstrate not only a cell-selective and NP-selective effect of USPIO NPs in the lysosomal, iron transport, and autophagic responses of colon cells, but also a protective stress response mediated by the chaperone HSP90.
Finally, the formation of large lipid vacuoles/droplets (LD) in high number was induced only by the OA-USPIO NPs. They were clearly dependent on the oleic acid as a stabilizer of these NPs, and not on the iron oxide core of the NPs or on free oleic acid. This intrinsic property of the oleic acid as a NP stabilizer may result from a high local concentration of oleic acid and/or the possibility for the NPs to be inserted into cell and cell organelle membranes, modifying the cell lipid metabolism. Thus, even if OA-USPIO NPs were poorly taken up by the cells, the oleic acid coating of USPIO NPs changed the cell response to the otherwise nontoxic NS-USPIO NPs. LDs are cytoplasmic dynamic organelles and are found in most cells, 33 either under normal or pathological conditions. Their number and size varies under cell stress conditions. It has been shown that small metallic NPs, in particular cadmium, tellurium, or mercury-based NPs, or long-chain unsaturated fatty acids such as oleic acid can induce the formation of LDs under cellular oxidative stress. 34 LDs are motile organelles undergoing microtubule-dependent movements in the cytoplasm and can associate with other cell organelles, including the endoplasmic reticulum. Exposure of murine glial cells to cadmium-telluride (CdTe) or InGaP/ZnS NPs, but also to oleic acid, induced the formation of LDs, some of them located close to lysosomes, possibly playing a protective function in stressed cells and a rescue role for cells exposed to CdTe NPs or controlling the cellular distribution of the InGaP/ZnS NPs. 33,35 Oleic-acid-induced lipid accumulation was shown in smooth muscle cells, which became foam cells, a mechanism not limited to macrophages. 36 It was shown in human hepatoma cells that both oleic-acid-stabilized and unstabilized USPIO NPs induced cell arrest in the G1 phase, but OA-USPIO NPs induced less cell damage than unstabilized USPIO NPs. 37 Unfortunately, in this study, the authors did not quantify the amount of NPs associated with the cells, an important factor to consider according to our previous and present results.

Conclusion
In conclusion, the results presented here show that the biophysicochemical characteristics of USPIO NPs in their media of dispersion can predict some of the cell response to their exposure. Agglomerated NPs will be taken at high levels, inducing an iron-specific response, such as the repression of the iron transporter. Cell protective responses such as autophagy and the induction of the HSP90 chaperone are specific responses of colon cells to the stress of being exposed to NPs, independent of the biophysical characteristics of the USPIO NPs. The cell responses to the exposure to such NPs also depend on the surface properties of the NPs, in particular LDs will form in cells if the coating used to stabilize the USPIO NPs is lipophilic.
International Journal of Nanomedicine 2014:9 submit your manuscript | www.dovepress.com Dovepress Dovepress 3497 stress reaction of human colon cells to iron oxide nanoparticles


extfieldsofstudy: Chemistry Medicine

text:
The Nitric Oxide Donor SNAP-Induced Amino Acid Neurotransmitter Release in Cortical Neurons. Effects of Blockers of Voltage-Dependent Sodium and Calcium Channels

Background The discovery that nitric oxide (NO) functions as a signalling molecule in the nervous system has radically changed the concept of neuronal communication. NO induces the release of amino acid neurotransmitters but the underlying mechanisms remain to be elucidated. Findings The aim of this work was to study the effect of NO on amino acid neurotransmitter release (Asp, Glu, Gly and GABA) in cortical neurons as well as the mechanism underlying the release of these neurotransmitters. Cortical neurons were stimulated with SNAP, a NO donor, and the release of different amino acid neurotransmitters was measured by HPLC. The involvement of voltage dependent Na+ and Ca2+ channels as well as cGMP in its mechanism of action was evaluated. Conclusions Our results indicate that NO induces release of aspartate, glutamate, glycine and GABA in cortical neurons and that this release is inhibited by ODQ, an inhibitor of soluble guanylate cyclase. Thus, the NO effect on amino acid neurotransmission could be mediated by cGMP formation in cortical neurons. Our data also demonstrate that the Na+ and Ca2+ voltage- dependent calcium channels are involved in the NO effects on cortical neurons.

Introduction
Nitric oxide (NO) is a short-lived gas produced by the family of NO synthases from the amino acid L-arginine [1]. Its identification as a signalling molecule in the nervous system changed the concept of neuronal communication [2]. NO is synthesized on demand and diffuses from nerve terminals in the vicinity of the NO producing cells. The mechanism occurs at neuronal and nonneuronal levels and NO release has pleiotrophic effects [3][4][5], suggesting that it acts as a neuromodulator and/or neurotransmitter [6,7]. NO has also been implicated in behaviour, learning and feeding [8][9][10]. The soluble guanylate cyclase (sGC) enzyme has long been considered the major physiological target for neuronal NO [11][12][13]. Thus, nitrergic nerve stimulation or administration of NO donors increases intracellular cGMP concentrations [14][15][16][17][18] and could enhance nitrergic effects.
NO has been shown to modify the release of several neurotransmitters such as acetylcholine [19,20], noradrenaline [21,22], dopamine [23], glutamate [24][25][26][27], GABA [28][29][30], serotonin [31], adenosine [30], carbon monoxide [32] and opioids [33]. Conversely, Jin et al. [34] report that the NO precursor arginine and the NO donor SNAP reduce glutamate release from primary afferent terminals through S-nitrosylation of voltageactivated Ca 2+ channels. On the other hand, Sistiaga et al [16] reported that NO inhibits glutamate release in cortical neurons stimulated with 4-aminopyridine. The mechanisms underlying in these effects are still not fully understood. Nonetheless, the direct S-nitrosylation of receptors, the activation of cGMP-dependent protein phosphorylation, the regulation of neuronal energy and the modulation of transporters are potential mechanisms affecting neurotransmitter release [35][36][37]. In this study, we look into the role of NO as a regulator of excitatory (Asp and Glu) and inhibitory (Gly and GABA) amino acid release in cortical neurons and the possible involvement of calcium and sodium channels on neurotransmitter release (Glu, Asp, Gly, GABA). For this purpose, we used SNAP, a NO donor to increase NO levels in cortical neurons. We evaluated the levels of different neurotransmitters in these cells (Asp, Glu, Gly, GABA) by HPLC.

Ethics statement
Pregnant rats were obtained from the ''Laboratory Animal from the Universidad Complutense de Madrid (U.C.M)''; Licence number #ES280790000086. The work was also approved by the University Animal Care Committee (C.E.A = Commite of Experimental Research and Ethics) from the Universidad Complutense de Madrid (U.C.M; form number RD # 53/2013 for research) and it was carried out in strict accordance with Guidelines for the Care and Use of Laboratory Animals from the European Communities Council Directive (86/609/EEC). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering of animals.

Methods
Cell isolation and culture of cortical neurons. Foetal rat brains from the Wistar rats at 19 days of gestation (E19) were used in the present study. Cortical neurons were obtained following a procedure from Segal [38] with minor modifications. Isolated neurons were suspended in EMEM containing 0.3 g/l glutamine, 3 g/l glucose, 10% foetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were placed at a density of 10 6 cells/ml on plastic multiwell Petri dishes. These plates were previously treated with 10 mg/ml of poly-D-lysine, to allow the attachment of the neurons to the plates. Cortical neurons were grown in a humidified chamber with 95% air/5% CO 2 at 37uC. After 72 hours, the culture medium was replaced by fresh medium containing 10 mM of cytosine arabinoside to prevent glial cells growth. Cell viability was tested by the trypan blue exclusion method.
Glial contamination was measured following a protocol from Figueroa et al [39] using the specific anti-GFAP antibody. Briefly, cells were incubated for 1 h with anti-GFAP antibody diluted 1:500 in PBS at room temperature. After a further wash with PBS, anti-rabbit IgG FITC conjugated was applied and incubated for 30 min as before. The secondary antibody was diluted 1:100 in PBS prior to addition. After a final wash with PBS, the glial cells were identified by flow cytometry analysis.
Under these conditions, the glial cells represented 9%63% of the total cell population of the culture.
Measurement of amino acid secretion. The secreted amino acids (Asp, Glu, Gly and GABA) were analysed by HPLC performed following the procedure described by Márquez and coworkers [40]. Cells, after 10 culture days, were washed twice with 1 ml of Locke medium. The medium was removed and cells were stimulated for 15 min at 37uC with 0.5 ml of fresh Locke medium containing the different SNAP concentrations indicated in each case. After stimulation, we followed these steps: (i) the solution containing the released amino acids were collected (ii) the cells were lysed by adding 0.5 ml of distilled water and this suspension, containing the unreleased neurotransmitters, was centrifuged at 130006g for 5 min. Supernatants were collected out.
The amino acid concentrations were determined by reversephase high-performance liquid chromatography using a precolumn derivation with dansyl chloride and UV detection at 254 nm. Peaks were integrated using a Spectraphysic integrator and then quantified and compared with standards for these neurotransmitters. The separation of dansyl derivatives was carried out using a Waters Spherisorb ODS 2 column (5 mM particle size; 1560.46 cm).
Results were expressed as % of amino acid release as compared to the total amino acid content (amino acid in the medium plus amino acid inside the cells).
Assessment of cell viability (XTT tests). This assay is based on the ability of live metabolically active cells to reduce yellow tetrazolium salt (XTT) to form an orange formazan dye. Thus, this conversion can only occur in living cells. The newlyformed formazan dye is directly quantified using a scanning multiwell spectrophotometer at a wavelength of 492 nm (reference wavelength 690). The amount of orange formazan formed, as monitored by the absorbance, directly correlates with the number of living cells. Control and treated neurons were washed with PBS and incubated with the XTT solution (final concentration 0.3 mg/ ml) according to Kit manufacture's instructions. After this incubation period, orange dye solution was spectrophotometrically quantified. Results were expressed as percentages with respect to the control cells, which were considered as 100%.
Measurement of the membrane potential. Changes in the membrane potential were monitored with the fluorescent dye bisoxonol (bis-[1, 3-diethyl-thio-barbiturate]-trimethineoxonol), a lipophylic anion that enter cell membrane in the presence of a depolarization. The increase of bisoxonol fluorescence indicates membrane depolarization, thus allows more of the negatively charged dye to enter the cells [41]. The control and treated neurons were washed and incubated for 15 min with 0.2 mM bisoxonol in the culture medium. We used 60 mM KCl as a positive control for cell depolarization. After this exposure time, the bisoxonol was removed and the neurons washed with PBS. Fluorescence was measured at 530/25 nm excitation and 590/ 20 nm emission and were monitored with a FL600-BioTek spectrofluorimeter. Fluorescence intensity was expressed as arbitrary fluorescence units (AFU).

Statistical analysis
Data are shown as mean 6 SEM from either two or four independent experiments using different cultures, each experiment performed in triplicate with different cell batches (total 6-12 measurements/condition). Statistical analyses were made with a One Way ANOVA on Ranks followed by the Dunn's test. One Way ANOVA on Ranks analyses the ''normality'' and ''variance'' of the data. If both data are passed the program uses the tests for parametric data but if ''normality or ''variance'' are failed, the program performs the Kruskal-Wallis test. A value of p,0.05 was considered statistically significant. Statistical analysis were performed using SigmaPlot 11.0 software.

SNAP action on amino acid release
Our aim was to study whether NO (SNAP) could induce amino acid neurotransmitter release (Asp, Glu, Gly, GABA). Cortical neuron cultures were exposed to different SNAP concentrations (from 1 mM to 1 mM) for 15 minutes and the amino acid content measured by HPLC in the extracellular medium as well as inside the cells. Amino acid release was expressed as the percentage of amino acid in the external medium in comparison to the total amino acid release (outside amino acid plus that inside the cells, which was assumed to make 100%).
The results shown in Figure 1A and 1B indicate that SNAP, at concentration between 1 mM to 1 mM, induces release of aspartate (Asp), glutamate (Glu), glycine (Gly) and GABA. The Asp release increased significantly at all concentrations tested between 10 mM to 1 mM ( Figure 1A). Glutamate release remained constant at all SNAP dosages from 10 mM up to 1 mM. The maximal release of glycine was achieved at 10 mM SNAP and was always much higher than the basal values at all concentrations tested up to 1 mM. (Figure 1B). On the other hand, GABA was only released at SNAP concentrations of 1 mM ( Figure 1B). The release of these amino acids neurotransmitter was Ca 2+ -dependent since SNAP had no effect in a medium without calcium (data not show).

Action of SNAP (NO) on cell viability
As it has been demonstrated that SNAP (NO) may be toxic in cortical neurons [42], we studied the action of 1 mM SNAP on cell viability at different exposure times. This experiment was performed in order to demonstrate that the amino acid neurotransmitter release mediated by SNAP was not caused by cellular death. Exposure times between 15 m to 24 hr were evaluated. Results from Fig. 2 show that cortical neurons subjected to 1 mM SNAP for 15 min to 4 hr do not present signs of lost cell viability. However, longer SNAP exposure times induced a progressive loss in cell viability that reached 40% at 24 hr.

Effect of carboxy-PTIO on amino acid release mediated by SNAP (NO)
As Carboxy-PTIO is a NO scavenger molecule its action on amino acid release mediated by SNAP (NO) was evaluated, in order to learn the specificity of the NO effect on SNAP-mediated amino acid release. Cortical neurones were stimulated with 1 mM SNAP in the absence and presence of 100 mM of carboxy-PTIO and the resulting amino acid neurotransmitter release was measured. Results from Figure 3A show that Asp and Gly release were inhibited in the presence of carboxy-PTIO. Similar results were found for glutamate and GABA (Fig. 3B).

Action of guanylate cyclase on SNAP-mediated amino acid release
Many NO actions have been attributed to guanylate cyclase. In order to study this mechanism, ODQ, an irreversible inhibitor of soluble guanylate cyclase (sGC), was used in the present study. We chose 1 mM SNAP concentrations to study the possible involvement of cGMP because this was the only concentration that induced GABA release in our study. Our data showed that 10 mM ODQ largely reversed the effect of 1 mM SNAP over the release of all the amino acid neurotransmitters analyzed. (Figure 4). ODQ did not significantly affect basal aspartate ( Figure 4A), glutamate ( Figure 4B), glycine ( Figure 4C) or GABA ( Figure 4D) release.

Action of SNAP (NO) on membrane potential
Cortical neurons exposure to 1 mM SNAP for 15 minutes did not alter the cell membrane potential. However, treatment with 60 mM KCl increased fluorescence ( Figure 5). This fluorescence confirmed the existence of KCl-mediated membrane depolariza-  The possible role of voltage-dependent sodium and calcium channels on SNAP-mediated amino acid release Since SNAP (NO)-mediated release of amino acid neurotransmitters is Ca 2+ dependent, the involvement of the voltage-dependent calcium channels (VDCC) on the amino acid release mediated by 1 mM SNAP in cortical neurons was analyzed. When the action of 1 mM SNAP was assayed in the presence of w-AGA IVA, a blocker of P/Q VDCC, amino acid release was strongly inhibited ( Figure 6A, 6B, 6C, 6D). Similar results were obtained with w-CTX GVIA, a blocker of N type VDCC. Nevertheless, verapamil, a blocker of L type VDCC only inhibited glutamate   Figure 6B) and glycine ( Figure 6C) release without affecting aspartate ( Figure 6A) or GABA release ( Figure 6D).
In addition, we ascertained whether voltage-dependent Na+ channels are involved in SNAP (NO) mediated amino acid release. We used tetrodotoxin, (TTX), a toxin which blocks the voltage dependent Na+ channels (VDSC). TTX inhibited the aspartate ( Figure 6A), glutamate ( Figure 6B) and glycine ( Figure 6C) release induced by 1 mM SNAP. However, this toxin did not affect the GABA release mediated by 1 mM SNAP in the cortical neurons ( Figure 6D).

NO formed from SNAP induces amino acid neurotransmitter release
We analyzed whether NO would trigger neurotransmitter release in cortical neurons. Our results showed that SNAP (NO) induces significant Asp, Glu and Gly release at SNAP concentrations ranging from 10 mM to 1 mM while GABA release was only detected at concentration of 1 mM SNAP.
This suggests that NO could regulate the balance between excitatory and inhibitory neurotransmitters in cortical neurons by through the levels of the NO donor SNAP. NO effects on excitatory and inhibitory synaptic neurotransmitter release have also been reported by several other researchers. In fact, Segieth and co-workers [43], using NO donors, found that NO regulates excitatory amino acid release in a biphasic manner in the hippocampus of freely-moving rats; Horn et al. [44], demonstrated that NO may control the activity of hypothalamic neurohypophyisis neurons because these neurons also release Glu, GABA and taurine ''in vivo'' after NO stimulation. On the other hand, in vivo, Engelmann et al. [45], found that NO may differentially regulate excitatory and inhibitory neurotransmitter release into the hypothalamic supraoptic nuclei. Taken together, these observations, accompanied by our findings in cortical neurons, support the hypothesis that SNAP (NO) could exert a differential effect on GABA (inhibitory) as well as glutamate (excitatory) release. The amino acid release induced by SNAP was mediated by NO because the NO scavenger carboxy-PTIO strongly inhibited the effects of the NO donor. SNAP-mediated amino acid release was not the result of cell death because exposing cortical neurons to 1 mM SNAP did not produce any loss of cellular viability (death) for at least 4 hr and our studies were performed at 15 minutes after SNAP exposure.

Involvement of VDCCs and VDSCs in amino acid neurotransmitter release mediated by SNAP (NO)
Our results indicate that SNAP (NO) induced neurotransmitter release through calcium dependent mechanisms since there was no effect in a calcium-free medium. This means that NO probably stimulates Ca 2+ entry necessary to provoke neurotransmitter release in cortical neurons. Calcium entry could be mediated by the action of different types of Ca 2+ channels, such as voltagedependent calcium channels (VDCC). In fact, when VDCC were blocked by different specific toxins, the release of all SNAP (NO) mediated neurotransmitters was inhibited in our study; since both AGA-toxin and conotoxin were effective, P/Q-and N-type VDCC probably cooperate in stimulating neurotransmitter release. The blockade of L-type VDCC by verapamil strongly reduced glutamate and glycine release without affecting aspartate or GABA release. This observation suggests that the L-type voltage dependent calcium channels do not seem to be involved in Asp and GABA release mediated by NO (SNAP), maybe because verapamil may work in a specific cell subpopulation. Differences in the involvement of different calcium channels in neurotransmitter release have been reported by others. Waterman et al. [46] indicates that parasympathetic neurons in the mouse bladder employ different Ca 2+ channels to release neurotransmitters in these neurons. Additionally, Turner et al. [47] reported that glutamate release from whole rat brain synaptosomes was inhibited by v-Aga toxin but unaffected by v-CTX GIVA.
Our results on the inhibition of amino acid neurotransmitter release by the calcium channel-blocker suggests that NO induces calcium entry though VDCC since their blockade inhibits neurotransmitter release in the presence of SNAP (NO).
In addition, our results also showed that when cortical neurons were stimulated with 1 mM SNAP in the presence of TTX -a blocker of voltage dependent Na+ channels-, there was a strong inhibition of aspartate, glutamate and glycine release but GABA release was unafected. TTX data may suggest that NO activates voltage-gated Na + channels and ensures VDCC and Ca 2+dependent neurotransmitter release. Thus, the differential sensitivity of GABA release may be related to different mechanism in GABAergic neurons were supported it by literature data [48,49]. GABA release, resistant to Na + channel blockade (TTX), has also been reported by others. Bieda and Copenhage [50], using retinal amacrine cells, found that voltage dependent Na + blockade by TTX partially suppressed the Glycinergic and GABAergic input in these cells. Therefore, they concluded that amacrine cells are a cell type that uses both dependent and independent action potentials for light-evoked release of neurotransmitters. In addition, Martinez Martos et al. [51] have demonstrated that the basal GABA release in the frontal cortex of awake rats was TTX resistant.

The activation of VDCCs and VDSC may be mediated by sGC activation
The involvement of VDCC and VDSC in SNAP (NO) release can suggest that 1) SNAP (NO) may induce depolarization and/or 2) SNAP (NO) directly modulates these channels through activation of sGC. In our study, we saw no depolarization in SNAP-treated cells. However, Mongin et al. [52] found membrane depolarization in synaptosome preparations treated with NO donors (sodium nitroprusside, S-nitroso-L-cysteine and hydroxylamine). They attributed this plasma membrane depolarization to decreased potassium permeability and inhibition of the sodium pump. Although we cannot exclude the possibility that SNAP/ NO-mediated amino acid neurotransmitter release is a consequence of depolarization in cortical neurons, our results, showing the inhibitor effect of both the channel blockers and ODQ on amino acid neurotransmitter release, suggest that formation of cGMP is an intermediate step in the modulation of neurotransmitter release via GMPc on VDCC and VDNC. Although we did not directly measure VDCC and VDNC activity, the data available in the literature report that several VDCC are involved in these mechanisms [53][54][55][56][57].
Taken together, these findings suggest that SNAP(NO) mediates amino acid release in cortical neurons (Figure 7): the NO formed from the donor (SNAP) (see 1 on Fig. 7) binds to its intracellular receptor, the soluble guanylate cyclase (sGC) activating it. The activation of sGC forms cGMP, which may directly activate Na + (see 2 in Fig.7) and Ca 2+ voltage dependent channels (see 3 on Fig. 7) by a mechanism which needs to be further elucidate. This effect would promote activation of these channels and the entry of Ca 2+ (see 4 on Fig.7) and Na + (see 5 on Fig. 7) to induce neurotransmitter release. Finally, the increase of Ca 2+ through VDCC may be involved in this neurotransmitter release (see 4 on Fig. 7) while the increase of Na+ via VDSC would have induced action potentials in cortical neurons¨in vitrö (see 5 on Fig. 7).
The working model presented in Figure 7 is supported by several observations from our study: 1) SNAP (NO) induces amino acid release that is inhibited by the presence of ODQ (an inhibitor of sGC) and 2) voltage dependent calcium channels of the types N,  (1) Increased in NO induces activation of sGC and, as a consequence, cGMP formation. The increases in cGMP levels may activate VDSC (2) and VDCC (3) channels by a mechanism which yet to be elucidated. An increase in intracellular calcium levels (4) would be responsible for the amino acid release. Activation of VDSC may trigger local depolarization and promote or amplify opening of VDCC (5). doi:10.1371/journal.pone.0090703.g007


extfieldsofstudy: Biology Medicine

text:
Stromal Cell-Derived Factor 1 Gene Polymorphism Is Associated with Susceptibility to Adverse Long-Term Allograft Outcomes in Non-Diabetic Kidney Transplant Recipients

Although the genetic polymorphism of Stromal Cell-Derived Factor 1 (SDF-1) is associated with higher mortality of liver allograft recipients, the role of SDF-1 in the modulation of renal allograft outcomes is unclear. Between March 2000 and January 2008, we recruited 252 non-diabetic renal transplant recipients (RTRs). Baseline characteristics and blood chemistry were recorded. Genomic DNA extraction with polymerase chain reaction-restriction fragment length polymorphism was utilized to analyze the genetic polymorphisms of SDF-1 (rs1801157). The influence of SDF-1 on an adverse renal allograft outcome, defined as either a doubling of serum creatinine, graft failure, or patient death was evaluated. Sixteen patients with the SDF-1 AA/AG genotype and nine with the SDF-1 GG genotype reached an adverse outcome. According to Kaplan-Meier analysis, patients carrying the SDF-1 AA/AG genotype or A allele showed a significantly higher risk of reaching an adverse outcome than those carrying the SDF-1 GG genotype or G allele (p = 0.041; p = 0.0051, respectively; log rank test). Stepwise multivariate Cox proportional regression analysis revealed that patients carrying the SDF-1 AA/AG genotype and A allele had a 2.742-fold (95% CI. 1.106–6.799, p = 0.03) and 2.306-fold (95% CI. 1.254–4.24, p = 0.008) risk of experiencing an adverse outcome. The SDF-1 AA/AG genotype and A allele have a detrimental impact on the long-term outcome of RTRs.

Introduction
The outcome of renal allograft is determined by a combination of risk factors including immunologic risk factors, such as delayed graft function (DGF), acute rejection, hepatitis B (HBV), and hepatitis C (HCV) infection as well as non-immunologic risk factors, such as hypertension (HTN), hyperlipidemia, and hyperglycemia [1][2][3]. Recently, it has been reported that single nucleotide polymorphisms (SNPs) contribute to short and long term outcomes [4,5], including DGF, chronic allograft dysfunction, and graft rejection [6,7].
Chemokines, which are involved in leukocyte migration during tissue inflammation [8], are known to be required for development and tissue homeostasis [8]. Anders et al. reported that SDF-1-CXCR4 function is also necessary to maintain homeostatic tissue renewal and regeneration upon renal injury [8]. SDF-1 (rs1801157) is located on chromosome 10q 11.1. A guanine to adenine (G to A) SNP at position 801 of the 3' untranslated region has been found to result in a SDF-1 chemokine gene polymorphism [9]. This SNP was first reported in a study by Winkler et al., which demonstrated that the onset of acquired immunodeficiency syndrome (AIDS) was delayed in 2587 patients with SDF-1 3' A/3' A (homozygous) [10]. A similar finding was observed in a study conducted by Gianesin et al., who showed that children born to a human immunodeficiency virus type 1 (HIV-1) seropositive mother carrying the SDF-1 GA genotype had a higher risk of late AIDS than those carrying a GG genotype [11].
This SDF-1 A/A or AG gene polymorphism has been suggested to alter the production of SDF-1 [12]. Soriano et al. found that in: exposed but not infected HIV-1 patients, in HIV-1 positive patients, and all patients, there were no significant differences between wild-type homozygotes and SDF-1 3' A heterozygotes, but each one of these two groups showed very significantly increased plasma SDF-1 concentrations compared with those in SDF-1 3' A homozygotes [12]. In addition, a study from Italy conducted by Xiao et al. showed that SDF-1 gene variation has an influence on SDF-1 level [13]. These studies indicate that variant SDF-1 genotypes play a role in the functional expression of this gene.
In addition to these studies showing the relationship of the SDF-1 SNP with infection, this SNP appears to be associated with a higher mortality among liver allograft recipients [14]. Additionally, along with its principal receptor CXCR4, SDF-1 regulates trafficking of stem cells during development, homeostasis, inflammation, and regeneration [15]. Togel et al. conducted an ischemic/reperfusion induced acute kidney injury and reported that SDF-1 is a major factor involved in kidney repair through the recruitment of cells involved in tissue regeneration [16]. Moreover, an anti-SDF-1 antibody was found to retard the process of chronic allograft nephropathy [17]. However, there remains limited knowledge of the role of SDF-1 and its genetic polymorphisms in the modulation of renal allograft.
Hyperglycemia has an adverse effect on graft function and patient survival [18,19]. Indeed, we recently found that pre-diabetic patients and post-transplant diabetic mellitus (PTDM) had more adverse long-term renal allograft outcomes than those without DM [20]. In order to investigate the influences of genetic polymorphisms of SDF-1 and to eliminate the adverse effects of DM, we recruited renal transplant recipients (RTRs) who were free of DM throughout the study. We hypothesized that the variant genetic polymorphism of SDF-1 could contribute to the long-term renal allograft outcome; this information could be useful in identifying individuals at higher risk of developing adverse outcomes.
A total of 25 RTRs reached the primary outcome, 16 (13.2%) of whom had the SDF-1 AA/AG genotype while the remaining 9 (6.9%) had the SDF-1 GG genotype. Compared to the RTRs with SDF-1 GG genotype, RTRs who carried the SDF-1 AA/AG genotypes showed a non-significant trend for being more likely to reach the primary outcome (AA/AG:GG = 13.2%:6.9%; p = 0.092). According to Kaplan-Meier analysis, RTRs who carried the SDF-1 GG genotype or G allele showed a significantly lower risk of reaching the primary composite outcome compared to those with the SDF-1 genotype AA/AG or allele A (p = 0.041; p = 0.0051, respectively; log rank test, Figure 2A After adjustment for age, gender, HLA, CV events, HCV infection, and usage of immunosuppressive regimens, stepwise multivariate Cox proportional regression analysis revealed that RTRs carrying the SDF-1 AA/AG genotype and an A allele had a 2.742-fold (95% CI. 1.106-6.799, p = 0.03) and 2.306-fold (95% CI. 1.254-4.24, p = 0.008) risk at the development of primary outcome, respectively ( Table 2).

Discussion
In the current study, we report that non-DM RTRs who carried the SDF-1 genotype AA/AG or an allele A had a higher risk of an adverse long-term allograft outcome in comparison to those who had the SDF-1 GG genotype or a G allele.
SDF-1 is a highly potent chemo-attractant for CXCR4 positive monocytes and naïve T cells that is constitutively expressed by many cell types, including endothelial and dendritic cells. In addition to its role in T-lymphocyte migration, SDF-1 is also a co-stimulator of CD4 + T cells and plays a role in HIV progression [10,11], microvascular complications among systemic sclerosis patients [21], and progression of carotid artery stenosis [22].
Chemokines, which are known to be involved in leukocyte migration during tissue inflammation, are required for development and tissue homeostasis [8]. Stromal derived factor 1 (SDF-1), which is a 93 amino acid chemokine, was first described as a growth factor for pre-B cells but turned out to be an essential regulator for important functions, such as recruiting progenitor cells to hypoxic tissues, epithelial progenitor cell migration, angiogenesis, and natural killer cell/B cell developments. When SDF-1-CXCR4-related maladaptive control occurred, podocyte progenitors of the Bowman's capsule would proliferate in crescentic glomerulonephritis [23], hypoxia-induced upregulation of CXCR4 in nephrosclerosis was observed [24], and there was insufficient podocyte regeneration in diabetic nephropathy [25]. Together, these results indicate that SDF-1-CXCR4 function is necessary to maintain homeostatic tissue renewal and regeneration upon renal injury [8].
Moreover, in an ischaemia/reperfusion injury mice model, Stokman et al. found that renal SDF-1 protein levels increased significantly in the early phase of kidney injury and a reduction of corticomedullary SDF-1 expression occurred, which was accompanied by severely increased tubular injury and decreased renal function after antisense treatment. They postulated a possible role of SDF-1 in mediating tubular epithelial cells against ischemic injury [26]. For chronic kidney disease, Chen et al. found that: SDF-1 mRNA doubled in human focal segmental glomerulosclerosis, which might be due to the use of angiotentin converting enzyme inhibitor; and local SDF-1 delivery induces glomerular eNOS activation in a CXCR4-dependent manner. They postulated that local SDF-1/CXCR4 could function to prevent renal fibrosis [27].
In addition to its association with infections, the inflammatory process, or acute and chronic kidney diseases, the SDF-1 3' A genotype of liver transplant recipients was found to contribute to a 36-month reduction in survival time. It is postulated that SDF-1 might promote integrin-mediated retention and adhesion of alloreactive lymphocytes to the biliary epithelium [14]. In fact, the portal tract appears to be the main target of infiltrating cells during acute rejection, meanwhile portal inflammation is associated with a progressive loss of bile ducts in the course of chronic rejection [14].
In studying human renal transplantation, Hoffmann et al. reported that recipients diagnosed with a chronic allograft rejection demonstrated an upregulation of SDF-1 in interstitial infiltrates and infiltrating neo-intimal cells of arteries [28]. Moreover, Gao et al. found that the anti-SDF-1 antibody could down-regulate the expression of SDF-1 and CXCR-4, and also delayed the progression of chronic allograft nephropathy in rats [17]. In this study, we found that SDF-1 AA/AG genotypes and the A allele of RTRs predisposed patients to impaired long-term allograft outcomes compared with those patients with GG genotype and G allele.
On the contrary, the CD26/SDF-1 axis is reported to be involved in the endothelial repair process following kidney transplantation [29]. Thus, future trials should evaluate whether SDF-1 has pro-inflammatory or protective properties in renal transplantation, as well as the possible underlying pathophysiology.
Since this was a retrospective single-center cohort study, one limitation of the current investigation was the number of patients. Additionally, this study lacked a functional assay evaluating SDF-1. Thus, further studies that include more patients and evaluate serum activities of SDF-1 are warranted.

Subjects
Between March 2000 and January 2008, RTRs who were diagnosed as pre-DM, PTDM and those who did not offer informed consents were excluded, resulting in the recruitment of a total of 252 RTRs. Pre-DM and PTDM RTRs were excluded since both conditions are known to significantly impair allograft outcome [20]. Clinical data including age, gender, human leukocyte antigen, HBV, HCV, parental DM, follow-up duration after transplantation, HTN, cardiovascular (CV) events (including stroke, coronary artery disease and peripheral artery occlusive disease) and usage of tacrolimus-based or cyclosporine-based immunosuppressive regimens were recorded. After transplantation, serum creatinine (SCr) was monitored at 1, 3, 6, and 12 months during regular outpatient follow up. The difference in SCr between 6 and 12 months after transplantation was defined as delta SCr.
The follow-up period began on the date of kidney transplantation and continued until a primary outcome or the end of the study (December 2009). The blood samples for determination of the SDF-1 genotype were obtained once informed consent was acquired from each RTR during regular follow up. This study was approved by the institutional review board of Chung Shan Medical University Hospital.

Genomic DNA Extraction
Genomic DNA was extracted from whole blood samples, which were collected from study subjects using QIAamp DNA blood mini kits (Qiagen, Valencia, CA, USA) in accordance with the manufacturer's instructions. DNA was dissolved in TE buffer (10 mM Tris, pH 7.8; 1 mM EDTA) and then quantitated via measurement of optical density (OD260). The final preparation was stored at −20 °C and was used to create templates in the polymerase chain reaction.

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP)
The SDF-1 (rs1801157) gene polymorphism was determined by a PCR-RFLP assay. The sequences of primers used to amplify the SDF-1 genotype were 5'-CAGTCAACCTGGGCAAAGCC-3' and 5'-CCTGAGAGTCCTTTTGCGGG-3'. PCR was performed in a 10 μL reaction mixture containing 100 ng DNA template, 1.0 μL of 10× PCR buffer (Invitrogen, Carlsbad, CA, USA), 0.25 U of Taq DNA polymerase (Invitrogen), 0.2 mM deoxyribonucleotide triphosphates (dNTPs; Promega, Madison, WI, USA), and 200 nM of each primer (MDBio Inc., Taipei, Taiwan). The PCR cycling started at 94 °C for 5 min followed by 35 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min, with a final step at 72 °C for 20 min to allow for a complete extension of all PCR fragments. The PCR products of SDF-1 gene polymorphisms were subjected to enzymatic digestion by incubation with HpaII for 4 h at 37 °C and subsequent electrophoresis in 3% agarose gels.

Adverse Composite Outcome
The primary outcome in this study was the time to the first event including any of the following: doubling of SCr, graft failure, or death. Graft survival was calculated from the date of transplantation until the date of graft loss. Living or deceased patients with a functioning graft at date of last follow-up were right censored. Doubling of SCr, graft failure, and patient survival were chosen as the adverse composite outcome because they encompassed well-defined, clinically relevant outcomes to both patients and physicians [6,20].

Statistical Analysis
Hardy-Weinberg equilibrium (HWE) was assessed using a goodness-of-fit chi-square test for SDF-1. Distributions of alleles were calculated by direct counting. Results were presented as mean ± standard deviation or percentages, and were analyzed by Mann-Whitney U test or chi-square test, as appropriate. The cumulative incidence and difference between time-to-event probabilities of the adverse outcome was analyzed by Kaplan-Meier curves and log-rank test. Stepwise multivariate Cox regression analysis was used to calculate the hazard ratio of experiencing an adverse composite outcome according to the SDF-1 genetic polymorphism. Age, gender, HCV infection, CV events, and usage of immunosuppressive regimens were included as covariates in this analysis. A p value of less than 0.05 was considered to be statistically significant. Statistical analysis was performed using MedCalc Statistical Software version 12.7.2 (MedCalc Software bvba, Ostend, Belgium).

Conclusions
To our knowledge, our results revealed a unique immune-modulating role of SDF-1 genetic polymorphisms on the outcome of renal allograft, suggesting that SDF-1 AA/AG genotype and an A allele possessed an unfavorable impact on the long-term allograft outcome.

Author Contributions
Chung-Jieh, Jen-Pi Tsai, and Horng-Rong Chang designed this study, analyzed the data and wrote this manuscript. Shun-Fa Yang andn Chung-Jieh performed the experiments of this study. Jong-Da Lian and Horng-Rong Chang recruited patients.