Nicolas Dejeans

Nicolas Tajeddine

Raphaël Beck

Julien Verrax

Henryk Taper

Philippe Gailly

Pedro Buc Calderon
email: pedro.buccalderon@uclouvain.be

Buc Pedro

Calderon

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells

Keywords: Oxidative stress, Breast cancer, Calcium homeostasis, Endoplasmic reticulum stress, MCF-7 cells, Ascorbate-driven menadione redox cycling

Increase in cytosolic calcium concentration ([Ca 2+ ] c ), release of endoplasmic reticulum (ER) calcium ([Ca 2+ ] er ) and ER stress have been proposed to be involved in oxidative toxicity.

Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbatedriven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca 2+ ] c , largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90 min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca 2+ ] er emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca 2+ ] er release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca 2+ ] er release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca 2+ ] er decrease in the process of oxidative stress-induced cell death in MCF-7 cells.

A c c e p t e d M a n u s c r i p t

Introduction

It has long been known that disruption of intracellular calcium homeostasis is one of the primary processes in the early development of cell injury [1][2][3] and that elevation of intracellular calcium levels can provoke a switch from normal regulation of cell function to a signal for cell death [3]. Different mechanisms have been implicated in this phenomenon, including activation of calcium-dependent proteases (i.e., calpains), calmodulin-associated enzymes (i.e., the phosphatase and regulator calcineurin), and calcium-dependent endonucleases or apoptotic pathways associated with perturbations in mitochondrial calcium concentrations [3][4][5]. In oxidative stress, intracellular calcium deregulation has been shown to have a central role in the induction of apoptosis or necrosis [5].

Endoplasmic reticulum (ER) stress is an essential adaptive cell response to the accumulation of misfolded proteins and is induced by the quality control system that ensures the transit of correctly folded proteins to the Golgi [6]. The primary role of ER stress is to favor cell survival by increasing the capacity to fold or refold proteins or by facilitating the export of misfolded proteins to the cytosol and their subsequent degradation (ER-associated degradation or ERAD) [6]. This phenomenon requires a complex regulatory process, known as the unfolded protein response (UPR), which regulates the transcription and translation of a great number of genes [7]. This process can finally induce cell death, probably when the initial survival response cannot counteract the protein modifications [6]. The molecular processes of ER stress-induced cell death represent a promising research area as this event may be involved in the physiopathology of atherosclerosis, diabetes and cancer, diseases known to be closely linked to oxidative stress [8]. In tumors, UPR is activated in response to the microenvironment, generally hypoxic and low in energy. Thus, some ER chaperones A c c e p t e d M a n u s c r i p t appear to be potential biomarkers for tumor behavior and resistance to cancer therapies [8][9][10] and some researchers have proposed that ER stress proteins should be targeted to potentiate cancer treatments [9,10].

Cells exposed to oxidative injury have various ER stress inducing factors [11]. Indeed, previous reports showed that oxidants can release ER calcium ([Ca 2+ ] er ) [5,[12][13][14] (presumably by inhibition of sarcoplasmic calcium ATPase [15,16]); this could represent a first mechanism by which oxidative injury can cause ER stress. Another possibility is that reactive oxygen species (ROS) may cause ER stress through generation and accumulation of oxidized proteins [11,17]. The ER appears to be particularly sensitive to such modifications [18][19][20][21], as its molecular environment has a high oxidizing redox potential, specialized in protein folding and disulfide bond formation [18,22,23]. Finally, ROS could directly damage proteins of the ER folding machinery, such as ER chaperones, and induce an accumulation of misfolded proteins and consequent UPR activation. Despite these data, research on the involvement of ER stress in the cellular response to ROS exposure is only emerging [11]. Few authors have reported UPR activation during oxidative stress and even fewer have demonstrated a direct involvement of ER stress in oxidative toxicity [19,[24][25][26][27][28]. In summary, ER homeostasis is a fragile equilibrium, which can be modulated by dysregulation of calcium or oxidative/reductive balance, features previously associated with oxidative stress. However, studies of the links between these factors are scarce.

We and others have shown that the association of ascorbate and menadione is an H 2 O 2generating system that results in necrosis-like cell death in a wide variety of cancer cell types [29][30][31][32][33][34][35] including MCF-7 cells (a human breast derived cell line), and that loss of calcium homeostasis appeared to be a major factor in the cytotoxicity [36]. The aim of the present study was to investigate the role of disruption of calcium homeostasis and a potential involvement of ER stress in the mechanisms leading to cancer cell death from an oxidative A c c e p t e d M a n u s c r i p t stress. Using MCF-7 cells, we showed that 1) oxidative stress caused a slow increase in [Ca 2+ ] c concentration in MCF-7 cells; 2) this increase in [Ca 2+ ] c appeared not to be associated with oxidative stress cytotoxic pathways; 3) oxidative stress led to a partial release of [Ca 2+ ] er ; 4) calcium release was not the main causative factor in ER stress triggered by oxidative conditions; and 5) [Ca 2+ ] er release potentiated oxidative stress induced cell death.

Materials and methods

Chemicals

Menadione sodium bisulfite (Men), sodium ascorbate (Asc), N-acetylcysteine (NAC), BAPTA-AM, dimethylsulfoxide (DMSO), EGTA, ionomycin and W-7 hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). Thapsigargin (TG), aurintricarboxylic acid (ATA) and cyclosporine A (CsA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Calpeptin (Calp) and Ru360 from Calbiochem (San Diego, CA), fluo-3/AM from Tebu-Bio (Boechout, BE), fura-2, AM from Molecular Probes (Eugene, OR), Iodoacetate from Acros Organics (Geel, BE) and propidium iodide from Roche Diagnostics Corp (Indianapolis, IN). All other chemicals were ACS reagent grade.

Cell Culture and Treatments

The MCF-7 cell line was a gift from Dr. F. Brasseur (Ludwig Institute for Cancer Research, LICR-Brussels). The cells were cultured in DMEM (Dulbecco's modified eagle medium, (Gibco BRL, Life Technologies, Merelbeke, BE)) supplemented with 10% fetal calf serum, penicillin (10 000 U/ml), streptomycin (10 mg/ml) and 1.2% glutamine. The cultures were maintained at a density of about 50x10³ cells/cm². The medium was changed at 48-72 h intervals. All cultures were maintained at 37°C in 95% air/5% CO2 with 100% humidity. For each passage, cells were washed twice with PBS (Gibco) and then incubated at 37°C with 0.25% trypsin-EDTA (Sigma-Aldrich, St Louis, MO). Cultures were treated with 1 or 0.5 mM minutes before the addition of ascorbate and menadione. Cells were incubated with TG or iodoacetate at concentrations of 1 and 100 µM, respectively, to induce ER calcium release or glycolysis arrest, respectively.

Cell survival measurement

The effects of Asc/Men and TG on MCF-7 cell survival were monitored by the erythrosine exclusion assay. Cells were seeded onto 6-well plates at a density of 300 000 cells/well for 24 h and then incubated for different durations in the absence or presence of the various compounds. Cells were then gently washed with 1 ml PBS, harvested using trypsin-EDTA solution, collected and mixed with 100 µl of medium. Then, 900 µl of a solution containing 0.9% NaCl and 0.4% erythosin (w/v) was added, the solution was gently mixed and nonerythrosine positive cells were immediately counted microscopically using a Burker chamber.

MTT assay

The effects of Asc/Men, TG or iodoacetate on cell metabolic status were assessed by following the reduction of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to blue formazan [37]. Briefly, cells were seeded into 96-well plates at a density of 10 000 cells/well for 24 h and then incubated for different durations in the absence or in the presence of the various compounds (8 wells were used for each condition). Cells were then washed with PBS and incubated with MTT (0.5 mg/ml) for 2 h at 37°C. Blue formazan crystals were solubilized by adding 100 µl DMSO/well, and the colored solution was subsequently read at 550 nm. Results are expressed as % of MTT reduction compared to untreated control conditions.

ATP measurements

A c c e p t e d M a n u s c r i p t

The ATP content was assessed using the bioluminescence kit ATPLite (Perkin Elmer, Zaventem, BE) according to the procedure provided by the manufacturer. Results are expressed as nmol/10 6 cells.

Immunoblotting assays

At the indicated times, cells were washed twice with ice-cold PBS and then resuspended in an RIPA lysis buffer supplemented with 1% Protease Inhibitor Cocktail (Sigma-Aldrich) and 3%

Phosphatase Inhibitor Cocktail (Calbiochem). The samples were kept on ice for 5 min. They

were then centrifuged at 13,000 × g for 15 min at 4°C or sonicated for 15 seconds for the detection of γ-H2AX. Supernatants or sonicated samples were collected and then stored at -80°C. Equal amounts of proteins were subjected to SDS-PAGE (6-15% separating gel) followed by electroblot to nitrocellulose membranes. The membranes were blocked for 1 hour in TBS buffer (pH 7.4) containing 5% powdered milk protein and then incubated overnight at 4°C with the appropriate antibody. Antibody against PAR was obtained from BD Biosciences Pharmingen (Franklin Lakes, NJ). Rabbit polyclonal antibody against phospho-H2AX (γ-H2AX) was from Upstate (Billerica, MA), against GRP94 from Chemicon International (Temecula, CA), and against Phospho-eIF2a (Ser51) from Cell Signaling Technology (Danvers, MA). After washing, membranes were exposed for 60 min at room temperature to a secondary antibody from Chemicon International (Temecula, CA) linked to HRP or alkaline phosphatase. Finally, the protein bands were detected by chemiluminescence.

[Ca 2+ ] c measurements by microspectrofluorimetry

Measurement of [Ca 2+ ] c was performed as previously described [38] with some modifications.

The cells, plated on glass coverslips, were incubated with 1 µM fura-2 AM in medium at 37°C for 30 min, then washed for 10 min in HBSS at 20°C and maintained at room temperature in the same saline solution until the fluorescence was measured. The coverslip was then mounted in a Pecon microscope chamber (1 ml). When indicated, cells were

[Ca 2+ ] c measurements using a fluorescence microplate reader

This method was adapted from the article by Robinson et al., [40]. For [Ca 2+ ] c measurement, MCF-7 cells were seeded onto 96-well plates at a density of 25 000 cells/well in 100 µl of growth media. At subconfluence, the culture medium was replaced with FBS free media for as described for the quantification protocol.

Oxyblot

The immunoblot detection of carbonyl groups introduced into proteins by oxidative reactions was achieved using OxyBlot™ Protein Oxidation Detection Kit (Chemicon International, Temecula, CA, USA) according to the procedure provided by the manufacturer.

Data analysis

Results are expressed as means ± standard deviation (SD). Differences among the experimental groups were analysed using one-way ANOVA followed, where appropriate, by a Tukey post-hoc test. These tests were performed using GraphPad Prism software (GraphPad Software San Diego, CA, USA). P values less than 0.05 were considered statistically significant.

Results

Oxidative stress induces calcium increase in MCF-7 cells

To investigate the acute effects of the pro-oxidant association of Asc/Men on [Ca 2+ ] c in MCF- ] c was estimated to be close to 50% after 90 min (Fig. 1B).

To verify whether the calcium mobilization was not due to membrane disruption, [Ca 2+ ] c and propidium iodide permeation were simultaneously measured after different durations of exposure to Asc/Men (Fig. 1C). This experiment revealed that calcium increase was triggered during the first hour of Asc/Men treatment and that, in contrast, propidium iodide uptake was observed after more than 4 h. This clearly indicates that the increase in [Ca 2+ ] c largely preceded signs of cell death.

[Ca2+]c increase is unlikely to be associated with activation of calcium-dependent death pathways

Since Asc/Men triggered disruption of calcium homeostasis in MCF-7 cells, we then explored the influence of oxidative stress on different pathways associated with calcium disturbance.

First, calcium itself may directly activate proteases (i.e., calpains) or calmodulin-associated enzymes, which may themselves be involved in the cytotoxic processes of oxidative stress.

Moreover, perturbations of intracellular calcium can activate mitochondrial apoptotic pathways, including the opening of the mitochondrial permeability transition pore (mPTP).

We therefore tested the influence of inhibitors of these pathways on the loss of cell viability observed after Asc/Men treatment. W7 (25 µM), calpeptin (10 µM), Ru360 (5 µM), and cyclosporine A (5 µM) were chosen to inhibit calcium-dependent proteases, calmodulin, entry of calcium into the mitochondria, and mPTP opening, respectively (1 h pre-incubation).

Unlike the antioxidant, NAC, none of these compounds had any protective effect on oxidative stress-mediated decrease in cell viability (Fig. 2A andB). We also hypothesized that the DNA strand breaks induced by Asc/Men (as we have previously shown [31,33]) could be the result of calcium-dependent endonuclease activation. To study this hypothesis, we tested the effects of 1 h pre-incubation with 100 µM aurintricarboxylic acid (a calcium-dependent endonuclease inhibitor) on the loss of viability observed in MCF-7 cells after Asc/Men exposure; this A c c e p t e d M a n u s c r i p t inhibitor had no measurable effect (Fig. 2A). To examine whether extracellular calcium chelation or depletion could affect Asc/Men cytotoxicity, we then measured MCF-7 cell viability after 4 h of exposure to Asc/Men, in media with and without calcium, or with 2 mM of the extracellular calcium chelator, EGTA (Fig. 2C). Results from this experiment demonstrated that extracellular calcium depletion or chelation had no protective effects against Asc/Men. Taken together, our results show that the increase in intracellular calcium after administration of Asc/Men is unlikely to be involved in the mechanisms leading to MCF-7 death.

Effect of BAPTA-AM, an intracellular calcium chelator on Asc/Men cytotoxic pathways.

An intracellular calcium chelator, BAPTA-AM, was also used to explore the importance of an increase in cytosolic calcium on the cytotoxic pathways of Asc/Men. In our model, this chelator appeared to have a broad protective action, as shown by its concentration-dependent protection against the decrease in cell viability induced by Asc/Men exposure (Fig. 3A).

However, since BAPTA-AM has been previously shown to chelate iron, and potentially interferes with Fenton's reaction [41,42], we decided to identify at what level BAPTA-AM acts. Our results reveals that BAPTA-AM could prevent against all features of Asc/Men cytotoxicity, including decreases in ATP, PARP activation, DNA fragmentation, ER stress and even protein oxidation (Fig. 3B,C andD). This suggests that the protective effect of BAPTA-AM observed in our model is most probably mediated by its capacity to interfere with pro-oxidative mechanisms, rather than by its ability to chelate calcium.

ATP decrease and calcium release in MCF-7 cells exposed to oxidative stress

Oxidative stress usually induces a rapid and marked decrease in cellular ATP. Among the consequences of this effect, the decrease in ATP may also be associated with ATP-dependent calcium channel dysfunction, a process potentially involved in the increase in [Ca 2+ ] c observed in MCF-7 cells exposed to oxidative stress. We, therefore, decided to explore the observed after 90 min of Asc/Men treatment (Fig. 4C), confirming that the large increase in calcium observed in oxidative stress exposed MCF-7 cells was not due to ATP depletion.

Oxidative stress partially releases [Ca 2+ ] er and induces ER stress in MCF-7 cells

Since disturbance in cytosolic calcium is unlikely to be involved in the pathways leading to the loss of cell viability induced by Asc/Men, we further studied the effect of Asc/Mengenerated oxidative stress on the ER, the major cellular store of calcium. We first measured whether the response of MCF-7 cells to TG (TG irreversibly inhibits (sarco)endoplasmic reticulum Ca 2+ -ATPase (SERCA) and depletes ER calcium stores) could be altered by Asc/Men. To this end, untreated control cells (Fig. 5A) or cells pre-treated with Asc/Men for 90 min (Fig. 5B) were stimulated with TG (1 µM) after extracellular calcium removal and [Ca 2+ ] c was measured. The results showed that the TG-driven increase in [Ca 2+ ] c was attenuated when cells were pre-treated with Asc/Men. Indeed, Fig. 5C shows that the response to TG was decreased by approximately 45% (124.8 ± 8.0 nM for control and 68.9 ± 4.7 nM for Asc/Men conditions). These findings suggest that Asc/Men slowly depletes calcium from the ER. Second, since [Ca 2+ ] er release can be both a cause and a consequence of ER stress, we explored whether ER stress was associated with Asc/Men treatment. We measured levels of

Involvement of ER calcium emptying in ER stress triggered by oxidative stress and cell death

Our results confirm the observations that Asc/Men triggers ER stress and ER calcium release.

We then decided to address the issue of whether TG-induced complete calcium release had a putative effect on the ER stress induction observed in cells exposed to Asc/Men (Fig. 6A). For this experiment, ascorbate and menadione were used at the lowest concentrations of 0.5 mM and 5 µM, respectively. At this concentration, Asc/Men induced eIF2 phosphorylation after 2 h of treatment, while TG triggered this event after 0.5 h (Fig. 6A). Surprisingly, the combination of Asc/Men and TG dramatically increased the appearance of P-eIF2 after just 30 min of treatment. These results demonstrate that oxidative stress and calcium release are two ER stress-inducing factors that synergize when applied together and that calcium release is not likely to be the only process responsible for UPR activation during oxidative stress.

Regarding GRP94 protein levels, TG caused GRP94 overexpression only after 24 h (Fig. 6A).

Since various authors have proposed that ER stress is one of the oxidative pathways leading to cell death, we then decided to address the question of whether the potentiating effect of TG on the ER stress induced by Asc/Men was also accompanied by an increase in cell death. For this purpose, cell survival was measured in cells exposed for different durations to TG, Asc/Men 0.5/5, or both. Previous studies have revealed that TG exposure is generally associated with DNA fragmentation [43][44][45]. Asc/Men treatment also triggers this event, which results in poly(ADP ribose) polymerase (PARP) activation and NAD + depletion (Fig. 3 and Verrax et al., 2004 and2007 [31, 33]). We, therefore, assessed the effects of TG and Asc/Men treatment on histone H2AX phosphorylation, a marker of DNA damage in the form of double-strand breaks. As presented in Fig. 6C, 4 h of treatment with TG and Asc/Men 0.5/5 induced the appearance of -H2AX (the phosphorylated form) only when both treatments were applied together, demonstrating that increased calcium release and consecutive ER stress also potentiate oxidative stress-induced DNA fragmentation.

Discussion

The present study was designed to elucidate the roles of disturbances in calcium homeostasis and of ER stress in oxidative stress-associated death in MCF-7 cells. Our results reveal that Asc/Men-induced oxidative stress elicits [Ca 2+ ] er release, ER stress and increase in [Ca 2+ ] c after drug exposure. In this model, ER stress and associated [Ca 2+ ] er emptying, but not alterations in [Ca 2+ ] c , appear to have a role in cell death induction.

The first finding of our study is that the pro-oxidant association of Asc/Men induces a slow increase in cytosolic calcium. Use of iodoacetate in order to mimic Asc/Men-induced depletion in ATP did not trigger cytoplasmic calcium overload, suggesting that ATP depletion was not the cause of the large increase in intracellular calcium observed during oxidative exposure. One goal of our study was to clarify the link between the identified oxidative A c c e p t e d M a n u s c r i p t increase in cytosolic calcium and MCF-7 cell death. Various data provide evidence that alterations in [Ca 2+ ] c are not involved in oxidative toxicity: First, using specific inhibitors of pathways that have already been proposed to participate in calcium-mediated oxidative toxicity [5], we could not demonstrate any role of enzymes, such as proteases, calmodulinassociated enzymes or calcium-dependent endonucleases, in these processes. The potential involvement of mitochondria was also tested. Indeed, it is known that exposure of mitochondria to high calcium concentrations during oxidative stress results in their swelling and uncoupling. This phenomenon leads to a loss of maintenance of cellular ATP levels and finally to cell death by necrosis [46]. In our study, use of Ru360, a specific mitochondrial calcium uptake inhibitors (uniport transporter inhibitor) and cyclosporine A (a mPTP inhibitor) was not associated with any effect on Asc/Men toxicity. This suggests that mitochondrial calcium uptake was not involved in the toxicity in our model and contrasts with reports examining the role of mitochondrial permeability transition in the cytotoxicity of the pro-oxidant, tert-butylhydroperoxide [47]. Secondly, depleting or chelating extracellular calcium (by EGTA) was not protective against loss of cell viability during oxidative stress.

Because EGTA, in addition to its effect on extracellular calcium, tends to draw intracellular calcium (as shown in Figs. 5A andB), its lack of effect on cell viability can be considered as another argument against the involvement of altered [Ca 2+ ] c in oxidative toxicity.

Furthermore, we found that media without calcium and with 200 µM EGTA, could not preserve cell viability during Asc/Men exposure (not shown). This observation is in agreement with the report of Persoon-Rothert and colleagues in 1994 [48]. Thirdly, BAPTA-AM, one of the most used intracellular calcium chelator, was shown to protect cells from oxidative modifications during Asc/Men exposure (Fig. 3D). The protective effect of this chelator is generally the principal argument for a connection between [Ca 2+ ] c deregulation and oxidative injury. Our results suggest that BAPTA-AM may interact with pro-oxidative A c c e p t e d M a n u s c r i p t mechanisms in our model. However, it cannot be ruled out that BAPTA-AM could also chelate intracellular calcium, and protects cells in this way from oxidative toxicity. These are still speculations and need further study. It has been proposed that BAPTA-AM interferes with Fenton's reaction [41,42] or may protect against a poorly defined pro-oxidative calciumassociated process, such as calcium-induced iron release [49] or ER stress-generated ROS production [50][51][52].

The typical profile of the intracellular calcium response identified in our study has also been observed in other cell lines exposed to H 2 O 2 , such as HeLa, human erythrocytes and MDCK (a canine epithelial cell line) [53], CRI-G1 (an insulin-secreting cell line) [54] or pancreatic acinar cells [14,55]. However, to our knowledge, this is the first time this effect has been demonstrated in MCF-7 cells. Although high concentrations of H 2 O 2 have been shown to induce a similar calcium response to that seen in our study, there is no consensus regarding the source of calcium, as highlighted and discussed in the study by Castro et al., [53]. These authors reported that the increase in intracellular calcium increase came largely from a cytosolic unrecognized source. Although it was not the main goal of our study, we found that ER calcium release could partly explain the elevation in [Ca 2+ ] c in MCF-7 cells exposed to oxidative stress. This is in agreement with previous reports exploring the effects of H 2 O 2 exposure on modulation of calcium homeostasis [13,14].

Oxidative stress encompasses a number of ER stress inducing factors, including calcium emptying (presumably by inhibition of calcium ATPase [15,16,56]), or direct oxidative damage to newly folded proteins or proteins of the ER machinery. However, the relative contributions of these events in oxidative stress-induced ER stress are not known.

Furthermore, the relationship between oxidative stress and ER stress is a matter of debate, because activation of the UPR can also result in ROS generation [50][51][52]. In our study, we found that pro-oxidant Asc/Men treatment induced the translational control arm of the UPR A c c e p t e d M a n u s c r i p t

(by enhancing eIF2 phosphorylation) and upregulation of ER folding capacity (by the increase in GRP94 protein, one of the major ER chaperones). These events were accompanied by a slow leakage of [Ca 2+ ] er . Given these results, we then focused on the potential link between ER calcium release and ER stress activation. For this purpose, TG was used as an UPR inducer to trigger irreversible calcium release from the ER. We showed that TG and consequent calcium emptying also induced eIF2 phosphorylation shortly after addition of TG.

This effect was weaker than that observed after exposure of cells to ascorbate or menadione at concentrations of 1 mM and 10 µM, respectively (Fig. 5D and6A), suggesting that the In conclusion, in this study we report the major role of disrupted ER calcium homeostasis on Asc/Men oxidative toxicity in MCF-7 cells and highlight the relative contribution of ER stress in this process. Our results confirm the pro-oxidant activity of the association of ascorbate and menadione [29-33, 35, 58, 59] and suggest that it could represent an interesting therapy in tumors in which increased ER folding capacity is associated with cancer resistance.

Introduction

It has long been known that disruption of intracellular calcium homeostasis is one of the primary processes in the early development of cell injury [1][2][3] and that elevation of intracellular calcium levels can provoke a switch from normal regulation of cell function to a signal for cell death [3]. Different mechanisms have been implicated in this phenomenon, including activation of calcium-dependent proteases (i.e., calpains), calmodulin-associated enzymes (i.e., the phosphatase and regulator calcineurin), and calcium-dependent endonucleases or apoptotic pathways associated with perturbations in mitochondrial calcium concentrations [3][4][5]. In oxidative stress, intracellular calcium deregulation has been shown to have a central role in the induction of apoptosis or necrosis [5].

Endoplasmic reticulum (ER) stress is an essential adaptive cell response to the accumulation of misfolded proteins and is induced by the quality control system that ensures the transit of correctly folded proteins to the Golgi [6]. The primary role of ER stress is to favor cell survival by increasing the capacity to fold or refold proteins or by facilitating the export of misfolded proteins to the cytosol and their subsequent degradation (ER-associated degradation or ERAD) [6]. This phenomenon requires a complex regulatory process, known as the unfolded protein response (UPR), which regulates the transcription and translation of a great number of genes [7]. This process can finally induce cell death, probably when the initial survival response cannot counteract the protein modifications [6]. The molecular processes of ER stress-induced cell death represent a promising research area as this event may be involved in the physiopathology of atherosclerosis, diabetes and cancer, diseases known to be closely linked to oxidative stress [8]. In tumors, UPR is activated in response to the microenvironment, generally hypoxic and low in energy. Thus, some ER chaperones A c c e p t e d M a n u s c r i p t appear to be potential biomarkers for tumor behavior and resistance to cancer therapies [8][9][10] and some researchers have proposed that ER stress proteins should be targeted to potentiate cancer treatments [9,10].

Cells exposed to oxidative injury have various ER stress inducing factors [11]. Indeed, previous reports showed that oxidants can release ER calcium ([Ca 2+ ] er ) [5,[12][13][14] (presumably by inhibition of sarcoplasmic calcium ATPase [15,16]); this could represent a first mechanism by which oxidative injury can cause ER stress. Another possibility is that reactive oxygen species (ROS) may cause ER stress through generation and accumulation of oxidized proteins [11,17]. The ER appears to be particularly sensitive to such modifications [18][19][20][21], as its molecular environment has a high oxidizing redox potential, specialized in protein folding and disulfide bond formation [18,22,23]. Finally, ROS could directly damage proteins of the ER folding machinery, such as ER chaperones, and induce an accumulation of misfolded proteins and consequent UPR activation. Despite these data, research on the involvement of ER stress in the cellular response to ROS exposure is only emerging [11]. Few authors have reported UPR activation during oxidative stress and even fewer have demonstrated a direct involvement of ER stress in oxidative toxicity [19,[24][25][26][27][28]. In summary, ER homeostasis is a fragile equilibrium, which can be modulated by dysregulation of calcium or oxidative/reductive balance, features previously associated with oxidative stress. However, studies of the links between these factors are scarce.

We and others have shown that the association of ascorbate and menadione is an H 2 O 2generating system that results in necrosis-like cell death in a wide variety of cancer cell types [29][30][31][32][33][34][35] including MCF-7 cells (a human breast derived cell line), and that loss of calcium homeostasis appeared to be a major factor in the cytotoxicity [36]. The aim of the present study was to investigate the role of disruption of calcium homeostasis and a potential involvement of ER stress in the mechanisms leading to cancer cell death from an oxidative

Materials and methods

Cell Culture and Treatments

The MCF-7 cell line was a gift from Dr. F. Brasseur (Ludwig Institute for Cancer Research, LICR-Brussels). The cells were cultured in DMEM (Dulbecco's modified eagle medium, (Gibco BRL, Life Technologies, Merelbeke, BE)) supplemented with 10% fetal calf serum, penicillin (10 000 U/ml), streptomycin (10 mg/ml) and 1.2% glutamine. The cultures were maintained at a density of about 50x10³ cells/cm². The medium was changed at 48-72 h intervals. All cultures were maintained at 37°C in 95% air/5% CO2 with 100% humidity. For each passage, cells were washed twice with PBS (Gibco) and then incubated at 37°C with 0.25% trypsin-EDTA (Sigma-Aldrich, St Louis, MO). Cultures were treated with 1 or 0.5 mM minutes before the addition of ascorbate and menadione. Cells were incubated with TG or iodoacetate at concentrations of 1 and 100 µM, respectively, to induce ER calcium release or glycolysis arrest, respectively.

Cell survival measurement

The effects of Asc/Men and TG on MCF-7 cell survival were monitored by the erythrosine exclusion assay. Cells were seeded onto 6-well plates at a density of 300 000 cells/well for 24 h and then incubated for different durations in the absence or presence of the various compounds. Cells were then gently washed with 1 ml PBS, harvested using trypsin-EDTA solution, collected and mixed with 100 µl of medium. Then, 900 µl of a solution containing 0.9% NaCl and 0.4% erythosin (w/v) was added, the solution was gently mixed and nonerythrosine positive cells were immediately counted microscopically using a Burker chamber.

MTT assay

The effects of Asc/Men, TG or iodoacetate on cell metabolic status were assessed by following the reduction of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to blue formazan [37]. Briefly, cells were seeded into 96-well plates at a density of 10 000 cells/well for 24 h and then incubated for different durations in the absence or in the presence of the various compounds (8 wells were used for each condition). Cells were then washed with PBS and incubated with MTT (0.5 mg/ml) for 2 h at 37°C. Blue formazan crystals were solubilized by adding 100 µl DMSO/well, and the colored solution was subsequently read at 550 nm. Results are expressed as % of MTT reduction compared to untreated control conditions.

ATP measurements

A c c e p t e d M a n u s c r i p t

The ATP content was assessed using the bioluminescence kit ATPLite (Perkin Elmer, Zaventem, BE) according to the procedure provided by the manufacturer. Results are expressed as nmol/10 6 cells.

Immunoblotting assays

At the indicated times, cells were washed twice with ice-cold PBS and then resuspended in an RIPA lysis buffer supplemented with 1% Protease Inhibitor Cocktail (Sigma-Aldrich) and 3%

Phosphatase Inhibitor Cocktail (Calbiochem). The samples were kept on ice for 5 min. They

were then centrifuged at 13,000 × g for 15 min at 4°C or sonicated for 15 seconds for the detection of γ-H2AX. Supernatants or sonicated samples were collected and then stored at -80°C. Equal amounts of proteins were subjected to SDS-PAGE (6-15% separating gel) followed by electroblot to nitrocellulose membranes. The membranes were blocked for 1 hour in TBS buffer (pH 7.4) containing 5% powdered milk protein and then incubated overnight at 4°C with the appropriate antibody. Antibody against PAR was obtained from BD Biosciences Pharmingen (Franklin Lakes, NJ). Rabbit polyclonal antibody against phospho-H2AX (γ-H2AX) was from Upstate (Billerica, MA), against GRP94 from Chemicon International (Temecula, CA), and against Phospho-eIF2a (Ser51) from Cell Signaling Technology (Danvers, MA). After washing, membranes were exposed for 60 min at room temperature to a secondary antibody from Chemicon International (Temecula, CA) linked to HRP or alkaline phosphatase. Finally, the protein bands were detected by chemiluminescence.

[Ca 2+ ] c measurements by microspectrofluorimetry

Measurement of [Ca 2+ ] c was performed as previously described [38] with some modifications.

The cells, plated on glass coverslips, were incubated with 1 µM fura-2 AM in medium at 37°C for 30 min, then washed for 10 min in HBSS at 20°C and maintained at room temperature in the same saline solution until the fluorescence was measured. The coverslip was then mounted in a Pecon microscope chamber (1 ml). When indicated, cells were

[Ca 2+ ] c measurements using a fluorescence microplate reader

This method was adapted from the article by Robinson et al., [40]. For [Ca 2+ ] c measurement, MCF-7 cells were seeded onto 96-well plates at a density of 25 000 cells/well in 100 µl of growth media. At subconfluence, the culture medium was replaced with FBS free media for For live-cell imaging, cells were seeded on a 4-well Lab-Tek chamber glass slide (Nunc, Naperville, IL), at a density of 50 000 cells/well, cultured for 3 days, and treated as described for the quantification protocol.

Oxyblot

The immunoblot detection of carbonyl groups introduced into proteins by oxidative reactions was achieved using OxyBlot™ Protein Oxidation Detection Kit (Chemicon International, Temecula, CA, USA) according to the procedure provided by the manufacturer.

Data analysis

Results are expressed as means ± standard deviation (SD). Differences among the experimental groups were analysed using one-way ANOVA followed, where appropriate, by a Tukey post-hoc test. These tests were performed using GraphPad Prism software (GraphPad Software San Diego, CA, USA). P values less than 0.05 were considered statistically significant.

Results

Oxidative stress induces calcium increase in MCF-7 cells

To investigate the acute effects of the pro-oxidant association of Asc/Men on [Ca 2+ ] c in MCF- ] c was estimated to be close to 50% after 90 min (Fig. 1B).

To verify whether the calcium mobilization was not due to membrane disruption, [Ca 2+ ] c and propidium iodide permeation were simultaneously measured after different durations of exposure to Asc/Men (Fig. 1C). This experiment revealed that calcium increase was triggered during the first hour of Asc/Men treatment and that, in contrast, propidium iodide uptake was observed after more than 4 h. This clearly indicates that the increase in [Ca 2+ ] c largely preceded signs of cell death.

[Ca2+]c increase is unlikely to be associated with activation of calcium-dependent death pathways

Since Asc/Men triggered disruption of calcium homeostasis in MCF-7 cells, we then explored the influence of oxidative stress on different pathways associated with calcium disturbance.

First, calcium itself may directly activate proteases (i.e., calpains) or calmodulin-associated enzymes, which may themselves be involved in the cytotoxic processes of oxidative stress.

Moreover, perturbations of intracellular calcium can activate mitochondrial apoptotic pathways, including the opening of the mitochondrial permeability transition pore (mPTP).

We therefore tested the influence of inhibitors of these pathways on the loss of cell viability observed after Asc/Men treatment. W7 (25 µM), calpeptin (10 µM), Ru360 (5 µM), and cyclosporine A (5 µM) were chosen to inhibit calcium-dependent proteases, calmodulin, entry of calcium into the mitochondria, and mPTP opening, respectively (1 h pre-incubation).

Unlike the antioxidant, NAC, none of these compounds had any protective effect on oxidative stress-mediated decrease in cell viability (Fig. 2A andB). We also hypothesized that the DNA strand breaks induced by Asc/Men (as we have previously shown [31,33]) could be the result of calcium-dependent endonuclease activation. To study this hypothesis, we tested the effects of 1 h pre-incubation with 100 µM aurintricarboxylic acid (a calcium-dependent endonuclease inhibitor) on the loss of viability observed in MCF-7 cells after Asc/Men exposure; this A c c e p t e d M a n u s c r i p t inhibitor had no measurable effect (Fig. 2A). To examine whether extracellular calcium chelation or depletion could affect Asc/Men cytotoxicity, we then measured MCF-7 cell viability after 4 h of exposure to Asc/Men, in media with and without calcium, or with 2 mM of the extracellular calcium chelator, EGTA (Fig. 2C). Results from this experiment demonstrated that extracellular calcium depletion or chelation had no protective effects against Asc/Men. Taken together, our results show that the increase in intracellular calcium after administration of Asc/Men is unlikely to be involved in the mechanisms leading to MCF-7 death.

Effect of BAPTA-AM, an intracellular calcium chelator on Asc/Men cytotoxic pathways.

An intracellular calcium chelator, BAPTA-AM, was also used to explore the importance of an increase in cytosolic calcium on the cytotoxic pathways of Asc/Men. In our model, this chelator appeared to have a broad protective action, as shown by its concentration-dependent protection against the decrease in cell viability induced by Asc/Men exposure (Fig. 3A).

However, since BAPTA-AM has been previously shown to chelate iron, and potentially interferes with Fenton's reaction [41,42], we decided to identify at what level BAPTA-AM acts. Our results reveals that BAPTA-AM could prevent against all features of Asc/Men cytotoxicity, including decreases in ATP, PARP activation, DNA fragmentation, ER stress and even protein oxidation (Fig. 3B,C andD). This suggests that the protective effect of BAPTA-AM observed in our model is most probably mediated by its capacity to interfere with pro-oxidative mechanisms, rather than by its ability to chelate calcium.

ATP decrease and calcium release in MCF-7 cells exposed to oxidative stress

Oxidative stress usually induces a rapid and marked decrease in cellular ATP. Among the consequences of this effect, the decrease in ATP may also be associated with ATP-dependent calcium channel dysfunction, a process potentially involved in the increase in [Ca 2+ ] c observed in MCF-7 cells exposed to oxidative stress. We, therefore, decided to explore the observed after 90 min of Asc/Men treatment (Fig. 4C), confirming that the large increase in calcium observed in oxidative stress exposed MCF-7 cells was not due to ATP depletion.

Oxidative stress partially releases [Ca 2+ ] er and induces ER stress in MCF-7 cells

Since disturbance in cytosolic calcium is unlikely to be involved in the pathways leading to the loss of cell viability induced by Asc/Men, we further studied the effect of Asc/Mengenerated oxidative stress on the ER, the major cellular store of calcium. We first measured whether the response of MCF-7 cells to TG (TG irreversibly inhibits (sarco)endoplasmic reticulum Ca 2+ -ATPase (SERCA) and depletes ER calcium stores) could be altered by Asc/Men. To this end, untreated control cells (Fig. 5A) or cells pre-treated with Asc/Men for 90 min (Fig. 5B) were stimulated with TG (1 µM) after extracellular calcium removal and [Ca 2+ ] c was measured. The results showed that the TG-driven increase in [Ca 2+ ] c was attenuated when cells were pre-treated with Asc/Men. Indeed, Fig. 5C shows that the response to TG was decreased by approximately 45% (124.8 ± 8.0 nM for control and 68.9 ± 4.7 nM for Asc/Men conditions). These findings suggest that Asc/Men slowly depletes calcium from the ER. Second, since [Ca 2+ ] er release can be both a cause and a consequence of ER stress, we explored whether ER stress was associated with Asc/Men treatment. We measured levels of eIF2 phosphorylation, a central component of the control arm of the UPR, and of GRP94 protein abundance, one of the major ER chaperones that are transcriptionally upregulated during ER stress. Fig. 5D shows that 1 h of Asc/Men treatment induced eIF2 phosphorylation, We then decided to address the issue of whether TG-induced complete calcium release had a putative effect on the ER stress induction observed in cells exposed to Asc/Men (Fig. 6A). For this experiment, ascorbate and menadione were used at the lowest concentrations of 0.5 mM and 5 µM, respectively. At this concentration, Asc/Men induced eIF2 phosphorylation after 2 h of treatment, while TG triggered this event after 0.5 h (Fig. 6A). Surprisingly, the combination of Asc/Men and TG dramatically increased the appearance of P-eIF2 after just 30 min of treatment. These results demonstrate that oxidative stress and calcium release are two ER stress-inducing factors that synergize when applied together and that calcium release is not likely to be the only process responsible for UPR activation during oxidative stress.

Regarding GRP94 protein levels, TG caused GRP94 overexpression only after 24 h (Fig. 6A).

Since various authors have proposed that ER stress is one of the oxidative pathways leading to cell death, we then decided to address the question of whether the potentiating effect of TG on the ER stress induced by Asc/Men was also accompanied by an increase in cell death. For this purpose, cell survival was measured in cells exposed for different durations to TG, Asc/Men 0.5/5, or both. Previous studies have revealed that TG exposure is generally associated with DNA fragmentation [43][44][45]. Asc/Men treatment also triggers this event, which results in poly(ADP ribose) polymerase (PARP) activation and NAD + depletion (Fig. 3 and Verrax et al., 2004 and2007 [31, 33]). We, therefore, assessed the effects of TG and Asc/Men treatment on histone H2AX phosphorylation, a marker of DNA damage in the form of double-strand breaks. As presented in Fig. 6C, 4 h of treatment with TG and Asc/Men 0.5/5 induced the appearance of -H2AX (the phosphorylated form) only when both treatments were applied together, demonstrating that increased calcium release and consecutive ER stress also potentiate oxidative stress-induced DNA fragmentation.

Discussion

The present study was designed to elucidate the roles of disturbances in calcium homeostasis and of ER stress in oxidative stress-associated death in MCF-7 cells. Our results reveal that Asc/Men-induced oxidative stress elicits [Ca 2+ ] er release, ER stress and increase in [Ca 2+ ] c after drug exposure. In this model, ER stress and associated [Ca 2+ ] er emptying, but not alterations in [Ca 2+ ] c , appear to have a role in cell death induction.

The first finding of our study is that the pro-oxidant association of Asc/Men induces a slow increase in cytosolic calcium. Use of iodoacetate in order to mimic Asc/Men-induced depletion in ATP did not trigger cytoplasmic calcium overload, suggesting that ATP depletion was not the cause of the large increase in intracellular calcium observed during oxidative exposure. One goal of our study was to clarify the link between the identified oxidative A c c e p t e d M a n u s c r i p t increase in cytosolic calcium and MCF-7 cell death. Various data provide evidence that alterations in [Ca 2+ ] c are not involved in oxidative toxicity: First, using specific inhibitors of pathways that have already been proposed to participate in calcium-mediated oxidative toxicity [5], we could not demonstrate any role of enzymes, such as proteases, calmodulinassociated enzymes or calcium-dependent endonucleases, in these processes. The potential involvement of mitochondria was also tested. Indeed, it is known that exposure of mitochondria to high calcium concentrations during oxidative stress results in their swelling and uncoupling. This phenomenon leads to a loss of maintenance of cellular ATP levels and finally to cell death by necrosis [46]. In our study, use of Ru360, a specific mitochondrial calcium uptake inhibitors (uniport transporter inhibitor) and cyclosporine A (a mPTP inhibitor) was not associated with any effect on Asc/Men toxicity. This suggests that mitochondrial calcium uptake was not involved in the toxicity in our model and contrasts with reports examining the role of mitochondrial permeability transition in the cytotoxicity of the pro-oxidant, tert-butylhydroperoxide [47]. Secondly, depleting or chelating extracellular calcium (by EGTA) was not protective against loss of cell viability during oxidative stress.

Because EGTA, in addition to its effect on extracellular calcium, tends to draw intracellular calcium (as shown in Figs. 5A andB), its lack of effect on cell viability can be considered as another argument against the involvement of altered [Ca 2+ ] c in oxidative toxicity.

Furthermore, we found that media without calcium and with 200 µM EGTA, could not preserve cell viability during Asc/Men exposure (not shown). This observation is in agreement with the report of Persoon-Rothert and colleagues in 1994 [48]. Thirdly, BAPTA-AM, one of the most used intracellular calcium chelator, was shown to protect cells from oxidative modifications during Asc/Men exposure (Fig. 3D). The protective effect of this chelator is generally the principal argument for a connection between [Ca 2+ ] c deregulation and oxidative injury. Our results suggest that BAPTA-AM may interact with pro-oxidative mechanisms in our model. However, it cannot be ruled out that BAPTA-AM could also chelate intracellular calcium, and protects cells in this way from oxidative toxicity. These are still speculations and need further study. It has been proposed that BAPTA-AM interferes with Fenton's reaction [41,42] or may protect against a poorly defined pro-oxidative calciumassociated process, such as calcium-induced iron release [49] or ER stress-generated ROS production [50][51][52].

The typical profile of the intracellular calcium response identified in our study has also been observed in other cell lines exposed to H 2 O 2 , such as HeLa, human erythrocytes and MDCK (a canine epithelial cell line) [53], CRI-G1 (an insulin-secreting cell line) [54] or pancreatic acinar cells [14,55]. However, to our knowledge, this is the first time this effect has been demonstrated in MCF-7 cells. Although high concentrations of H 2 O 2 have been shown to induce a similar calcium response to that seen in our study, there is no consensus regarding the source of calcium, as highlighted and discussed in the study by Castro et al., [53]. These authors reported that the increase in intracellular calcium increase came largely from a cytosolic unrecognized source. Although it was not the main goal of our study, we found that ER calcium release could partly explain the elevation in [Ca 2+ ] c in MCF-7 cells exposed to oxidative stress. This is in agreement with previous reports exploring the effects of H 2 O 2 exposure on modulation of calcium homeostasis [13,14].

Oxidative stress encompasses a number of ER stress inducing factors, including calcium emptying (presumably by inhibition of calcium ATPase [15,16,56]), or direct oxidative damage to newly folded proteins or proteins of the ER machinery. However, the relative contributions of these events in oxidative stress-induced ER stress are not known.

Furthermore, the relationship between oxidative stress and ER stress is a matter of debate, because activation of the UPR can also result in ROS generation [50][51][52]. In our study, we found that pro-oxidant Asc/Men treatment induced the translational control arm of the UPR (by enhancing eIF2 phosphorylation) and upregulation of ER folding capacity (by the increase in GRP94 protein, one of the major ER chaperones). These events were accompanied by a slow leakage of [Ca 2+ ] er . Given these results, we then focused on the potential link between ER calcium release and ER stress activation. For this purpose, TG was used as an UPR inducer to trigger irreversible calcium release from the ER. We showed that TG and consequent calcium emptying also induced eIF2 phosphorylation shortly after addition of TG.

This effect was weaker than that observed after exposure of cells to ascorbate or menadione at concentrations of 1 mM and 10 µM, respectively (Fig. 5D and6A), suggesting that the

A c c e p t e d M a n u s c r i p t 6 sodium

 6 ascorbate and 10 or 5 µM menadione bisulfite as the H 2 O 2 -generating system. When indicated, the antioxidant NAC was added to cell cultures at a concentration of 3 mM for 15

A c c e p t e d M a n u

  s c r i p t superfused with HBSS without calcium and with 200 µM EGTA (1 ml. minute -1 ) allowing quick changes of extracellular solution. Fura-2 loaded cells were alternately excited at 340 and 380 nm using a Lambda DG-4 Ultra High Speed Wavelength Switcher (Sutter Instrument, Novato, CA) coupled to a Zeiss Axivert 200M inverted microscope (x 20 fluorescence objective) (Zeiss Belgium, Zaventem, BE). Images were acquired every second at different times with a Zeiss Axiocam camera coupled with a 510 nm emission filter and analyzed by the Axiovision software. Fluorescence intensity was recorded over the entire surface of each cell and intracellular calcium concentration was evaluated from the ratio of the fluorescence emission intensities excited at the two wavelengths. Calibration of fura-2 was performed as described by Palmer et al.,[39] except that cells were treated with 10 µM ionomycin and 10 mM EGTA in Ca 2+ -free HBSS to obtain Rmin and with 20 mM Ca 2+ in HBSS to obtain Rmax. The standard equation: K d [(R-Rmin)/(Rmax-R)]•S f /S b was used to convert the fura-2 ratio to [Ca 2+ ] c , where S f and S b are the emission intensities at 380 nm for Ca 2+ -free and Ca 2+ -bound fura-2, respectively. In situ Rmin and Rmax values were multiplied by 0.85 to adjust for the minimum viscosity effect.

24 h to attempt

  to synchronize cells into a nonproliferative stage. MCF-7 cells were then loaded with 25 µM fluo-3/AM in medium at 37°C for 30 min followed by 30 min at room temperature to minimize dye leakage and sequestration into intracellular organelles. After loading, cells were washed twice with 200 µl HBSS in order to remove excess fluorescent dye. Cells were then treated for different durations with various compounds in normal or calcium-free medium. Cells were washed twice with 200 µl HBSS and then 100 µl

A c c e p t e d M a n u

  s c r i p t HBSS/well was added. The fluorescence intensity of fluo-3 was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Relative changes in calcium concentration using fluo-3 were determined by calculations of ΔF/F0, where ΔF/F0=(Ft-F0)/F0. Ft represents the fluorescence reading at each time point and F0 the initial fluorescence. For live-cell imaging, cells were seeded on a 4-well Lab-Tek chamber glass slide (Nunc, Naperville, IL), at a density of 50 000 cells/well, cultured for 3 days, and treated

7 cells, fluo- 3 /A c c e p t e d M a n u s c r i p t 10 fura- 2 ,

 3102 AM loaded cells were exposed to Asc/Men for different time periods. The results presented in Fig.1Ashow that incubation with Asc/Men resulted in a slow increase in [Ca 2+ ] c in MCF-7 cells. Using microspectrofluorimetry with the ratiometric fluorescent probe, AM, the increase in[Ca 2+

A c c e p t e d M a n u s c r i p t

  potential links between ATP depletion and increase in [Ca 2+ ] c during Asc/Men treatment. As shown in Fig. 4A, Asc/Men treatment induced a fast and dramatic decrease in ATP in MCF-7 cells. The glycolysis inhibitor, iodoacetate, was then used to mimic the decrease in cellular ATP level observed during Asc/Men treatment. As shown in Fig. 4B, exposure to 100 µM iodoacetate resulted in a reduction in ATP level from 20.6 ± 0.8 to 8.6 ± 0.2 nmol/10 6 cells after 90 min. This decrease in ATP was not accompanied by the same increase in [Ca 2+ ] c

  eIF2 phosphorylation, a central component of the control arm of the UPR, and of GRP94 protein abundance, one of the major ER chaperones that are transcriptionally upregulated during ER stress. Fig.5Dshows that 1 h of Asc/Men treatment induced eIF2 phosphorylation, A c c e p t e d M a n u s c r i p t 13 which increased dramatically after 2 and 4 h. The appearance of P-eIF2 was associated with the increase in GRP94 protein. These results clearly indicate that oxidative stress induces ER stress in MCF-7 cells, concomitant with ER calcium release.

  Fig. 6B shows that incubation with Asc/Men 0.5/5 or TG resulted in a slow reduction in MCF-7 cell survival, reaching levels of 52.6 ± 7.3 and 66.4 ± 9.5 after 24 h, respectively. The combination of the two treatments resulted in enhancement of cell death, especially in the 6th hour of treatment (p<0.01 for TG + Asc/Men 0.5/5 compared to TG A c c e p t e d M a n u s c r i p t alone and p<0.05 compared to Asc/Men). This experiment supports a role of ER stress in processes of oxidative stress-induced cell death.

  A c c e p t e d M a n u s c r i p t represent one of the pathways leading to this event; this observation also underlines the capacity of TG to trigger DNA fragmentation, as shown by others[43][44][45] 57].

  A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

Fig. 1 -

 1 Fig. 1 -Asc/Men exposure triggers increase in intracellular calcium. A, intracellular

Fig. 2 -Fig. 3 -

 23 Fig. 2 -Influence of different inhibitors of calcium-associated death pathways or the

Fig. 4 -

 4 Fig. 4 -Decrease in ATP was not associated with increase in intracellular calcium. A-B,

Fig. 5 -Fig. 6 -A c c e p t e d M a n u s c r i p t 2 ABSTRACT

 562 Fig. 5 -Asc/Men exposure partially releases ER calcium and induces ER stress. A, B and C, cytosolic calcium was assessed using the ratiometric dye, fura-2. The release of [Ca 2+ ] er by

A c c e p t e d M a n u

  s c r i p t stress. Using MCF-7 cells, we showed that 1) oxidative stress caused a slow increase in [Ca 2+ ] c concentration in MCF-7 cells; 2) this increase in [Ca 2+ ] c appeared not to be associated with oxidative stress cytotoxic pathways; 3) oxidative stress led to a partial release of [Ca 2+ ] er ; 4) calcium release was not the main causative factor in ER stress triggered by oxidative conditions; and 5) [Ca 2+ ] er release potentiated oxidative stress induced cell death.

A c c e p t e d M a n u s c r i p t 6 sodium

 6 ascorbate and 10 or 5 µM menadione bisulfite as the H 2 O 2 -generating system. When indicated, the antioxidant NAC was added to cell cultures at a concentration of 3 mM for 15

A c c e p t e d M a n u s c r i p t 8 superfused

 8 with HBSS without calcium and with 200 µM EGTA (1 ml. minute -1 ) allowing quick changes of extracellular solution. Fura-2 loaded cells were alternately excited at 340 and 380 nm using a Lambda DG-4 Ultra High Speed Wavelength Switcher (Sutter Instrument, Novato, CA) coupled to a Zeiss Axivert 200M inverted microscope (x 20 fluorescence objective) (Zeiss Belgium, Zaventem, BE). Images were acquired every second at different times with a Zeiss Axiocam camera coupled with a 510 nm emission filter and analyzed by the Axiovision software. Fluorescence intensity was recorded over the entire surface of each cell and intracellular calcium concentration was evaluated from the ratio of the fluorescence emission intensities excited at the two wavelengths. Calibration of fura-2 was performed as described by Palmer et al.,[39] except that cells were treated with 10 µM ionomycin and 10 mM EGTA in Ca 2+ -free HBSS to obtain Rmin and with 20 mM Ca 2+ in HBSS to obtain Rmax. The standard equation: K d [(R-Rmin)/(Rmax-R)]•S f /S b was used to convert the fura-2 ratio to [Ca 2+ ] c , where S f and S b are the emission intensities at 380 nm for Ca 2+ -free and Ca 2+ -bound fura-2, respectively. In situ Rmin and Rmax values were multiplied by 0.85 to adjust for the minimum viscosity effect.

24 h to attempt

  to synchronize cells into a nonproliferative stage. MCF-7 cells were then loaded with 25 µM fluo-3/AM in medium at 37°C for 30 min followed by 30 min at room temperature to minimize dye leakage and sequestration into intracellular organelles. After loading, cells were washed twice with 200 µl HBSS in order to remove excess fluorescent dye. Cells were then treated for different durations with various compounds in normal or calcium-free medium. Cells were washed twice with 200 µl HBSS and then 100 µl

A c c e p t e d M a n u

  s c r i p t HBSS/well was added. The fluorescence intensity of fluo-3 was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Relative changes in calcium concentration using fluo-3 were determined by calculations of ΔF/F0, where ΔF/F0=(Ft-F0)/F0. Ft represents the fluorescence reading at each time point and F0 the initial fluorescence.

7 cells, fluo- 3 /A c c e p t e d M a n u s c r i p t 10 fura- 2 ,

 3102 AM loaded cells were exposed to Asc/Men for different time periods. The results presented in Fig.1Ashow that incubation with Asc/Men resulted in a slow increase in [Ca 2+ ] c in MCF-7 cells. Using microspectrofluorimetry with the ratiometric fluorescent probe, AM, the increase in[Ca 2+

A c c e p t e d M a n u

  s c r i p t potential links between ATP depletion and increase in [Ca 2+ ] c during Asc/Men treatment. As shown in Fig. 4A, Asc/Men treatment induced a fast and dramatic decrease in ATP in MCF-7 cells. The glycolysis inhibitor, iodoacetate, was then used to mimic the decrease in cellular ATP level observed during Asc/Men treatment. As shown in Fig. 4B, exposure to 100 µM iodoacetate resulted in a reduction in ATP level from 20.6 ± 0.8 to 8.6 ± 0.2 nmol/10 6 cells after 90 min. This decrease in ATP was not accompanied by the same increase in [Ca 2+ ] c

A c c e p t e d M a n u s c r i p t 13 which

 13 increased dramatically after 2 and 4 h. The appearance of P-eIF2 was associated with the increase in GRP94 protein. These results clearly indicate that oxidative stress induces ER stress in MCF-7 cells, concomitant with ER calcium release.3.6. Involvement of ER calcium emptying in ER stress triggered by oxidative stress and cell deathOur results confirm the observations that Asc/Men triggers ER stress and ER calcium release.

  Fig. 6B shows that incubation with Asc/Men 0.5/5 or TG resulted in a slow reduction in MCF-7 cell survival, reaching levels of 52.6 ± 7.3 and 66.4 ± 9.5 after 24 h, respectively. The combination of the two treatments resulted in enhancement of cell death, especially in the 6th hour of treatment (p<0.01 for TG + Asc/Men 0.5/5 compared to TG A c c e p t e d M a n u s c r i p t alone and p<0.05 compared to Asc/Men). This experiment supports a role of ER stress in processes of oxidative stress-induced cell death.

Fig. 1 -

 1 Fig. 1 -Asc/Men exposure triggers increase in intracellular calcium. A, intracellular

Fig. 2 -

 2 Fig. 2 -Influence of different inhibitors of calcium-associated death pathways or the

Fig. 3 -

 3 Fig. 3 -Effect of BAPTA-AM in the process of Asc/Men induced cell death. A, cells were

Fig. 4 -

 4 Fig. 4 -Decrease in ATP was not associated with increase in intracellular calcium. A-B,

Fig. 5 -Figure1A

 5 Fig. 5 -Asc/Men exposure partially releases ER calcium and induces ER stress. A, B and C, cytosolic calcium was assessed using the ratiometric dye, fura-2. The release of [Ca 2+ ] er by

Figure3A

  Figure3

Figure5 A c c e p t e d M a n u s c r i p

  Figure5

Figure6

  Figure6

[

  Ca 2+ ] er leak was not sufficient to explain the oxidative stress-induced UPR activation in our model. Moreover, the combination of oxidative stress and TG-induced ER [Ca 2+ ] er release resulted in a greater and more rapid eIF2 phosphorylation, in a synergistic manner. This clearly demonstrates that ER calcium depletion is not the main process responsible for ER stress induction by oxidative stress. Ca 2+ ] er emptying also potentiated Asc/Men-induced DNA fragmentation and associated histone H2AX phosphorylation, suggesting that ER stress could represent one of the pathways leading to this event; this observation also underlines the capacity of TG to trigger DNA fragmentation, as shown by others[43][44][45] 57].In conclusion, in this study we report the major role of disrupted ER calcium homeostasis on Asc/Men oxidative toxicity in MCF-7 cells and highlight the relative contribution of ER stress in this process. Our results confirm the pro-oxidant activity of the association of ascorbate and menadione[29-33, 35, 58, 59] and suggest that it could represent an interesting therapy in tumors in which increased ER folding capacity is associated with cancer resistance.

	The involvement of ER stress and consequent UPR activation in the processes of oxidative
	stress-induced cell death has not been elucidated. Here, we showed that [Ca 2+ ] er release
	potentiates Asc/Men-associated ER stress and cytotoxicity. This suggests that ER stress is
	involved in the processes of oxidative stress-induced cell death. Moreover, this is in
	agreement with previous reports which showed that increasing ER folding capacity (by
	transfection with an ER chaperone) or decreasing UPR can markedly attenuate ROS-induced
	apoptosis in other cell lines [19, 24-26]. These findings also confirm results from the study by
	Hung et al., in which protection of ER calcium release by overexpression of the major ER
	Ca 2+ -binding protein, calreticulin, protected against H 2 O 2 -induced cell injury [26]. In
	addition, in our study, [

Acknowledgements

The authors would like to thank Isabelle Blave and Véronique Allaeys for their excellent technical assistance. This work was supported by grants from Televie and the Belgian Fonds National de la Recherche Scientifique (7.4566.07) and and by grant ARC 05/10-328 from the General Direction of Scientific Research of the French Community of Belgium. Raphaël Beck is a FRIA recipient. Julien Verrax and Nicolas Tajeddine are FNRS Post-Doctoral Researchers, and Nicolas Dejeans is a Televie-FNRS Post-Doctoral Researcher. We dedicate this work to the late Dr. Henryk Taper, in honor of his pioneering studies on the association of ascorbate and menadione. Acknowledgements The authors would like to thank Isabelle Blave and Véronique Allaeys for their excellent technical assistance. This work was supported by grants from Televie and the Belgian Fonds National de la Recherche Scientifique (7.4566.07) and and by grant ARC 05/10-328 from the General Direction of Scientific Research of the French Community of Belgium. Raphaël Beck is a FRIA recipient. Julien Verrax and Nicolas Tajeddine are FNRS Post-Doctoral Researchers, and Nicolas Dejeans is a Televie-FNRS Post-Doctoral Researcher. We dedicate this work to the late Dr. Henryk Taper, in honor of his pioneering studies on the association of ascorbate and menadione.


Thomas ; Wenzl
email: thomas.wenzl@ec.europa.eu

Dg-

Szilard ; Szilagyi

Johan ; Rosén

Lubomir Karasek

Collaborative Trial of an Isotope Dilution Liquid Chromatographic Tandem Mass Spectrometric Method to Determine the Content of Acrylamide in Roasted Coffee

Keywords: Chromatography, Inter-laboratory validation, LC/MS Additives/Contaminants: Acrylamide

A method for the determination of acrylamide in roasted coffee was subjected to method validation by collaborative trial. The aim was to extend the scope of a method already standardised for the determination of acrylamide in bakery and potato products to include roasted coffee. Modifications of the already standardised method were therefore kept to a minimum. The method was based on

aqueous extraction of the roasted coffee matrix and solid phase extraction (SPE) clean-up followed by isotope dilution high performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). The test portion of the sample was spiked with stable isotope labelled acrylamide and extracted on a mechanical shaker with n-hexane and water for 1 hour. The sample extract was centrifuged, the organic phase was discarded, and a portion of the aqueous extract was further cleaned-up by SFE on an Isolute Multimode cartridge followed by a second clean-up step on an Isolute ENV+ cartridge. The volume of the acrylamide-containing fraction eluted from the second SPE column was reduced by evaporation and analysed by LC-MS/MS. Three coffee samples and one aqueous acrylamide standard solution were sent to 11 laboratories from 8 EU Member States. All samples were sent as blind duplicates. Based on the reported results the relative standard deviations for reproducibility (RSD R ) were 11.5 % at a level of 160 µg/kg, 10.1 % at a level of 263 µg/kg, and 9.6 % at a level of 585 µg/kg. The values for repeatability (RSD r ) in those materials ranged from 1 % to 3.5 %. The method performance parameters are satisfying with regard to internationally accepted criteria. Hence the method would be suitable for the enforcement of regulatory limits.

Introduction

The accurate determination of acrylamide in food requires robust and reliable analytical methods. Still today workshops are conducted to discuss amongst others the state-of-the-art of analytical procedures for this purpose (e.g. EFSA's Colloquium 11, Tabiano, Italy, 22 to 23 May 2008). The number of papers published so far on analytical aspects of the determination of acrylamide in food is large and led to four review papers [START_REF] Wenzl | Analytical methods for the determination of acrylamide in food products: a review[END_REF][START_REF] Castle | Analytical methods used to measure acrylamide concentrations in food[END_REF][START_REF] Zhang | Occurrence and analytical methods of acrylamide in heattreated foods: Review and recent developments[END_REF]Wenzl et al. 2007). The most frequently applied measurement techniques are gas chromatography -mass spectrometry (GC-MS) after derivatisation of acrylamide, and liquid chromatography -tandem mass spectrometry (LC-MS/MS).

A previous collaborative trial revealed that an analytical method based on LC-MS/MS performs well for bakery and potato products [START_REF] Wenzl | Collaborative trial validation study of two methods, one based on HPLC-MS/MS and on GC-MS for the determination of acrylamide in bakery and potato products[END_REF]). This method was developed at the Swedish National Food Administration and became recently an international standard [START_REF] Rosén | Retention studies of acrylamide for the design of a robust liquid chromatography-tandem mass spectrometry method for food analysis[END_REF]; Nordic Committee on Food Analysis (NMKL) 2007).

At the workshop on acrylamide (Brussels, 16 to 17 March 2006), jointly organised by the European Commission, Directorate General Health and Consumer Protection (EC-DG SANCO) and the European Food and Drink Federation (CIAA), it was felt that standardised methods should be made available also for more challenging food matrices [START_REF] Guenther | Acrylamide in coffee: Review of progress in analysis, formation and level reduction[END_REF]). In particular a standardised method for the determination of acrylamide in roasted coffee was requested, as problems with the analysis of coffee samples were reported in the literature [START_REF] Roach | Rugged LC-MS/MS survey analysis for acrylamide in food[END_REF]. Roach et al. experienced deterioration of the performance of the chromatographic method after injection of coffee extracts at room temperature within a few hours [START_REF] Roach | Rugged LC-MS/MS survey analysis for acrylamide in food[END_REF]. [START_REF] Riediker | Analysis of acrylamide in food by isotope-dilution liquid chromatography coupled with electrospray ionization tandem mass spectrometry[END_REF] reported difficulties in the analysis of coffee samples caused by matrix interferences. The sample clean up procedure, which they applied for many types of food, was based on dual stage solid phase extraction (SPE) applying Isolute Multimode ® (500mg, 3 mL, Biotage AB, Uppsala, Sweden) and AccuBond II SCX ® (100 mg, 1 mL, Agilent, Palo Alto, CA, USA). However, it turned out to be not suitable for difficult food matrices such as roasted coffee or cocoa powder. An additional liquid/liquid extraction step employing ethyl acetate had to be included in the sample clean up procedure for the determination of acrylamide in these food matrices. Severe ion suppression was also noted by [START_REF] Granby | Analysis of acrylamide in coffee and dietary exposure to acrylamide from coffee[END_REF] in the determination of acrylamide in coffee.

Similarly to [START_REF] Riediker | Analysis of acrylamide in food by isotope-dilution liquid chromatography coupled with electrospray ionization tandem mass spectrometry[END_REF], they applied also Isolute Multimode ® SPE cartridges for sample clean up, but in a different format (300 mg, 3 mL). The capacity of these cartridges was limited to a volume of 500 µL of coffee extract, corresponding to 20 mg of roasted However, the proposed clean-up procedure could not completely remove the colour from the extract [START_REF] Granby | Analysis of acrylamide in coffee and dietary exposure to acrylamide from coffee[END_REF].

To reduce interferences, the application of LC-MS with atmospheric pressure chemical ionisation (APCI) was proposed for the determination of acrylamide in coffee by [START_REF] Senyuva | Study of acrylamide in coffee using an improved liquid chromatography mass spectrometry method: Investigation of colour changes and acrylamide formation in coffee during roasting[END_REF]. In a more recent paper, [START_REF] Aguas | Analysis of acrylamide in coffee and cocoa by isotope dilution liquid chromatography-tandem mass spectrometry[END_REF] published an analysis procedure for the determination of acrylamide in coffee that is based on liquid/liquid partitioning and SPE on aminopropyl cartridges (500 mg, 4 mL, Alltech Associates, Deerfield, IL, USA). The authors achieved high levels of precision. However, the analysis protocol included azeotropic drying of the aqueous extract, which might cause losses of the analyte, and measurement of the samples by LC-APCI-MS/MS. Since APCI is primarily used for the determination of nonpolar compounds, it is compared to electrospray ionisation less frequently applied in food control laboratories, which limits the broad applicability of such methods.

However, as many problems were reported in the analysis of acrylamide in coffee and as none of the analytical procedures proposed for that purpose was tested in a collaborative trial, information on the precision of any analysis procedure under reproducibility conditions was not available. Hence it was decided by the European Commission's Joint Research Centre (EC JRC), the Swedish National Food Administration (SNFA), and the Nordic Committee on Food Analysis (NMKL) to organise a second collaborative trial, this time focusing on the determination of acrylamide in roasted coffee. For the sake of user-friendliness, it was agreed to extend the scope of the initially collaboratively validated method to roasted coffee instead of proposing a completely different analysis procedure. It was also decided to apply samples with naturally incurred acrylamide in the study, covering the relevant range of acrylamide levels in roasted coffee. Data available from the EU monitoring database on acrylamide levels in food were used to identify the relevant contamination range [START_REF] Wenzl | European Union database on acrylamide levels in food: Update and critical review of data collection[END_REF]European Commission 2009). About 90 % of the acrylamide content of roasted coffee samples reported to the database lie between 150 µg kg -1 and 550 µg kg -1 . Therefore, the collaborative trial aimed to evaluate the performance of the analytical method for this content range. This paper reports the set up and the outcome of the method validation study by collaborative trial of a LC-MS/MS method for the determination of acrylamide in roasted coffee, and compares the method performance data for the analysis of coffee samples to data gained for bakery and potato products.

Materials and Methods

Design of the study

Twenty laboratories from 12 EU Member States with experience in acrylamide analysis by LC-MS/MS were contacted to participate in the study. Of these, 11 expressed their interest in participation in the study and were therefore supplied with test materials. Finally, nine laboratories submitted results. The study was designed as a blind duplicate study according to international guidelines (International Union for Pure and Applied Chemistry (IUPAC) 1995;

International Organization for Standardization (ISO) 1994). The participants were provided with the method protocol, instruction guidelines, and the test samples. They were requested to follow the method protocol as much as possible. Any deviation from the protocol had to be reported to the organisers of the collaborative trial.

The participants applied for the analyses of the test samples different models of LC-MS/MS instruments from three different suppliers. Hence any contribution of the type of instrument to the precision of the analysis method is included in the precision estimates.

Test samples

Samples were either purchased in local markets or specially prepared and supplied by Nestlé (Orbe, Switzerland). Commercial samples were purchased as ground coffee, whereas grinding was required in the case of the specially prepared materials. A Retsch ZM 200 centrifugal mill (Haan, DE) was applied for this purpose. Since no information on the influence of the grinding process on the acrylamide content of roasted coffee was available, a dual-stage cryo-grinding procedure was applied as a precautionary measure. A 2.0 mm sieve was applied in the first and a 0.5 mm sieve in the second stage. The coffee beans were cooled before and throughout the whole grinding process with liquid nitrogen. This procedure prevented both thermally induced changes of the test material and blockage of the mill.

Cooling with liquid nitrogen entails condensation of humidity from ambient air on the ground sample, which provokes aggregation of particles and which might lead to the growth of mould. Therefore, the ground coffee samples were freeze-dried between grinding and homogenisation. A Turbula mixer (WAB Maschinenfabrik, Muttenz, CH) was employed for the latter. Finally the samples were bottled at below -10°C into 10 mL amber glass vials.

The IUPAC protocol for the design, conduct, and interpretation of method-performance studies specifies the number of test materials to at least five (IUPAC 1995). In contrary, ISO 5725-2:1994 does not fix any particular number of test materials to be included in the collaborative trial (ISO 1994). However, since the targeted acrylamide content range was quite narrow, it was decided to limit the number of test materials included in the collaborative trial to three, representing the lower, and upper level of the tested range, as well as one level in-between, because significant differences in the performance of the method were not expected within such a narrow range of content. The set of test materials shipped to the participants of the study was composed of one commercial ground coffee samples and two samples prepared on request by Nestlé. The test samples were labelled with numbers between 1 and 1000.

Homogeneity of the test samples was tested according to generally accepted procedures for homogeneity testing [START_REF] Thompson | International Harmonized Protocol for the Proficiency Testing of Analytical Chemistry Laboratories[END_REF]. Ten glass vials containing the test samples were randomly selected and analysed in duplicate for the acrylamide content, applying the analysis procedure that was tested in the collaborative trial.

Data were subjected to one-way analysis of variance (ANOVA). The outcome of the statistical evaluation showed that in all cases the bottled test material was sufficiently homogeneous [START_REF] Fearn | A new test for "sufficient homogeneity[END_REF] All samples were sent as blinded duplicates. The participants were requested to analyse each sample once, and report results within eight weeks after reception of the test samples.

However the deadline for reporting of results had to be extended due to the high workload of some participants.

Besides test materials, participants received a training sample and an unknown standard solution. The training sample, was a coffee sample prepared in the same way as the test materials and was supplied for familiarisation with the analytical method. Its amount was sufficient to perform at least four independent analyses. Reporting of results for the test sample was not mandatory. The unknown standard aimed for evaluating correctness of instrument calibrations. Participants were requested to report the acrylamide concentration of this solution to the organisers. For that purpose the internal standard concentration (402 ng mL -1 of d 3 -acrylamide) was given to them. The unknown standard solution was bottled into 2 mL auto-sampler vials and was ready to inject.

Analytical method

The analytical method was to a large extent identical with the analysis procedure developed at the Swedish National Food Administration that previously was validated by collaborative trial for bakery and potato products (Wenzl et al. 2007;NMKL 2007). In particular 2.0 g of roasted coffee test sample are extracted in a 50 mL falcon tube with 40 mL of water. A quantity of 400 ng isotope labelled acrylamide (deuterium content > 98 %) is first vigorously shaking by hand (for 15 sec) followed by shaking on a mechanical shaker (for 60 min at maximum agitation speed). The application of a wrist-arm shaker was proposed, but not mandatory. However, orbital shakers were applied by some participants instead of the proposed type. The three phases (solid, aqueous, and organic) are separated by centrifugation for 20 min at 10 °C and 3250 xg. The organic phase is discarded and 2 ml of the aqueous phase is passed through a preconditioned Isolute Multimode ® solid phase extraction cartridge (6 mL, 500 mg, Biotage AB, Uppsala, Sweden). The eluate is collected and loaded on a Isolute ENV+ ® solid phase extraction cartridge (6 mL, 500 mg, Biotage AB). The cartridge is rinsed with 4 mL of water followed by the elution of acrylamide with 2 mL of 60 % methanol in water. The elution solvent volume is reduced by e.g. a stream of nitrogen to a final volume of about 500 µL. A quantity of 10 µL is injected on a Hypercarb column (e.g. 50 mm x 2.1 mm, Thermo Electron Corporation, Waltham, MA, USA), run isocratically with 0.1 % acetic acid in water at a flow rate of 400 µL min -1 . The participants were only free to choose the length of the Hypercarb column, which is available in 50 mm or 100 mm formats, because other phases do not show the same retention characteristics of both acrylamide and potential interferences. The detection of acrylamide had to be performed by all participants by tandem quadrupole LC-MS/MS and electrospray ionisation in positive ion mode. However, the parameter settings of the mass spectrometer and the interface were not specified, since they are a matter of tuning and change from one instrument to the other significantly. Therefore the participants were only requested to optimise sensitivity of their instrument prior to the analysis of the test samples. For acrylamide the transitions m/z 72>55, 72>54 and 72>44 were recorded, whereas the transition 75>58 was used for the isotope labelled internal standard. Typical chromatograms of the major transitions of acrylamide and isotope labelled acrylamide are depicted in Figure 1. Figure 2 shows the chromatograms for the transition 72>55 of a coffee sample extracted and cleaned up at JRC either with or without partitioning into n-hexane. Each preparation was performed twice, and each preparation was injected into the LC-MS/MS instrument twice. Figure 2a shows chromatograms of samples that were not subjected to the partitioning step. Severe matrix effects suppressed the acrylamide signal. This was not the case when liquid/liquid partitioning into n-hexane was applied (Figure 2b). However, SNFA did not face severe matrix effects when omitting the partitioning step [START_REF] Petersson | Critical factors and pitfalls affecting the extraction of acrylamide from foods: An optimisation study[END_REF].

Hence the lower sensitivity towards matrix effects at SNFA might be reasoned by constructive differences of the LC-MS/MS instrument that was applied for the measurements.

Since the partitioning step was necessary in one laboratory it was decided to include it in the study. However, since the application of n-hexane is not permitted in some laboratories, the partitioning step might be omitted after having demonstrated that the method performance is comparable to the outcome of the present study.

The second modification concerned the volume of extract that was loaded on the Isolute Multimode ® cartridge. To avoid overloading of the SPE cartridge with matrix constituents, the applied extract volume was reduced from 10 mL to 2 mL. This of course affects sample enrichment. However, most commercial roasted coffee samples contain acrylamide at a level at which high enrichment factors are no longer crucial for analyte detection. Hence lower sample enrichment should not affect the performance of the method dramatically.

The third modification consisted of the adjustment of the concentrations of calibration standards to the tested acrylamide content range. The concentration of acrylamide standards was limited to maximum 100 ng/mL due to, compared to the analysis procedure for bakery and potato products, lower sample enrichment, and the quite narrow acrylamide content range of roasted coffee samples.

Results and Discussion

The first part of the data evaluation aimed at identifying non-compliant or outlying results from the reported dataset. Appropriateness of instrument calibration was checked before performing any statistics by scrutinising the results reported for the unknown standard solution for unacceptable large deviations from the gravimetric preparation value (211 ng/mL). The tolerated deviation was set to 10 % of the assigned concentration. Exceeding this limit would result in exclusion of the data for the coffee samples from further evaluations. This procedure aimed to minimise the influence of biased standard preparation on the The data reported by one participant was excluded from the statistical analysis because of non-compliance. The reason was that the respective laboratory reported for all test samples acrylamide contents that were about twice the contents reported by the other participants. The laboratory considered a mistake in the preparation of the isotope labelled standard as the source for the deviations, which led to the data exclusion. Results were also excluded from the data evaluation when only one of the blinded duplicate samples was analysed.

The collaborative trial results, which are listed for the individual participant in Table 1, were examined using Cochran's and Grubbs' tests (p<0.025) for evidence of outliers. Data identified by these tests as outliers were also excluded from the data evaluation.

Precision estimates were obtained in the second part of the data evaluation by using a oneway ANOVA approach according to the IUPAC Harmonised Protocol (IUPAC 1995). Details of the average analyte content, the standard deviations for repeatability (s r ) and for reproducibility (s R ), as well as the respective relative standard deviations (RSD r and RSD R ), the number of outliers, and the HORRAT value (HoR) for reproducibility are presented in Table 2. The HoR value is frequently used as a fitness-for-purpose criterion for the selection of appropriate methods of analysis for the official control of food and feed (Commission of the European Communities 2007).

The reproducibility relative standard deviation (RSD R ) was, depending on the acrylamide content level, between 9.6 % and 11.5 %. This translates into HoR values for reproducibility of 0.5 to 0.6, which is significantly below the broadly accepted maximum value of 2.0 [START_REF] Horwitz | Performance characteristics of methods of analysis used for regulatory purposes. Part II. Pesticide formulations[END_REF]. However, for all coffee samples large differences between the repeatability and reproducibility relative standard deviations were found, which could be caused by two factors. On the one hand, the participants had already much experience with the determination of acrylamide in food, which is reflected in small repeatability standard deviations. On the other hand deviations between results reported by the participants for coffee test samples and the assigned acrylamide contents of the coffee test samples correlated well with the deviations of results for the unknown standard solution from its gravimetrically established concentration. This become clear for participants 5 and 9, whose results deviated from the assigned values by -11.1 % and -18.8 %, and by 7.8 % and 12.8 % for the coffee samples, and by -10.0 % and 10.0 % for the unknown standard solution. Hence, the underrespectively overestimation of the analyte contents of the coffee samples is at least partially caused by biased instrument calibration. This shortcoming affects only the precision parameter that accounts for between-laboratory effects, which consequently explains the large difference between intra-laboratory and inter-laboratory precision estimates. Therefore special attention has to be given to correct preparation of calibration standards.

The performance criteria of the method for roasted coffee agree well with those for bakery and potato products [START_REF] Wenzl | Collaborative trial validation study of two methods, one based on HPLC-MS/MS and on GC-MS for the determination of acrylamide in bakery and potato products[END_REF]). The method performance was investigated for the latter matrices for acrylamide contents between about 40 µg kg -1 and 9100 µg kg -1 . Details are given for better illustration in Table 3. The HoR values for reproducibility were for all samples below 1.0, which means that the precision of the method is better than the precision value predicted by the Horwitz equation. Hence the method would be suitable for the enforcement of regulatory limits.

Conclusions

A method for the determination of acrylamide in roasted coffee samples by LC/MS-MS was validated by collaborative trial. The trial was organised and evaluated according to internationally accepted standards. Nine laboratories from eight EU Member States reported analytical results for roasted coffee test samples covering the acrylamide content range of 160 µg kg -1 to 585 µg kg -1 . The corresponding reproducibility relative standard deviations were between 11.5 % and 9.6 %. The calculated method performance parameters satisfy internationally accepted criteria with Horrat values for reproducibility between 0.5 and 0.6, which is much below the generally accepted maximum value of 2.0. However, the interlaboratory precision estimate was significantly higher than the intra-laboratory precision, which was most likely the consequence of biased instrument calibration. Hence special regard shall be given to this part of the analytical procedure.

  of higher volumes of coffee extract caused severe matrix interferences.

  standard to the extractant. Additionally 5 mL of n-hexane are added to the falcon tube for simultaneous clean up of the aqueous extract. The extraction is performed by

  The main differences between the previously collaborative trial tested procedure and the current procedure consisted first of partitioning of fat and other matrix components between n-hexane and the aqueous extract, which is performed simultaneously with the aqueous extraction of the roasted coffee sample. Second a smaller aliquot of the aqueous extract was cleaned up by solid phase extraction and third the concentrations of calibration standards were adjusted to the lower concentration of the final extract.The clean up of the aqueous extract by partitioning of co-extractives into n-hexane proved beneficial from the point of encountered matrix effects in the consecutive LC-MS/MS

  All reported results were within the acceptable range, although the results of two participants were at the limits.

Figure 1 :

 1 Figure captions

Table 1 :

 1 Individual results of the acrylamide content of the coffee test samples and the unknown standard solution

			Food Additives and Contaminants
									Unknown
	Laboratory	Sample 1	Sample 2	Sample 3
									standard
		µg kg -1	µg kg -1	µg kg -1	µg kg -1	µg kg -1	µg kg -1	µg kg -1
	1	159	176	280	278	572	584		202
	2	185	181	292	294	645	648		210
	3	281 a	322 a	519 a	499 a	923 a	1250	a	219
	4	246 b	271 b	415 b	484 b	545	548		200
	5	130	130	230	220	520 a	-		190
	6	164	159	261	266	582	569		210
	7	159	151	265	269	587	594		213
	8	148	144	227	232	493	500		210
	9	177	173	277	290	662	658		232

a non-compliant result b outlying result Page 17 of 19 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 2 :

 2 Performance parameters for the determination of the acrylamide content of roasted coffee at three different content levels Legend: Mean -mean mass fraction [µg kg -1 ]; N -number of labs; nc -non-compliant laboratories; outl. -outlying laboratories; n -number of laboratories used for statistics; sr -repeatability standard deviation [µg kg -1 ], RSDr -relative standard deviation under repeatability conditions [%]; sR, RSDR-relative standard deviation reproducibility conditions, HoR -the Horrat value for reproducibility

		Mean	N nc outl.	n	s r	RSD r	s R	RSD R	HoR
		µg kg -1					µg kg -1	%	µg kg -1	%
	Sample 1	160	9	1	1	7	5.5	3.5	18.4	11.5	0.5
	Sample 2	263	9	1	1	7	4.9	1.9	26.6	10.1	0.5
	Sample 3	585	8	1	0	7	5.6	1.0	56.3	9.6	0.6

Table 3 :

 3 Performance parameters for the determination of the acrylamide content of bakery and potato products[START_REF] Wenzl | Collaborative trial validation study of two methods, one based on HPLC-MS/MS and on GC-MS for the determination of acrylamide in bakery and potato products[END_REF]

		Mean	RSD r	RSD R	HoR
		µg kg -1	%	%
	Butter biscuits (A)	16	-	-	-
	Toasted bread	38	5.5	8.5	0.3
	Butter biscuits (B)	96	7.8	11.8	0.5
	Spiced biscuits	249	3.7	10.4	0.5
	Potato crisps (A)	324	6.0	12.7	0.7
	Spiked mashed potato powder	500	5.4	8.8	0.5
	Commercial potato crisps (A)	628	8.9	13.2	0.8
	Crisp bread (ERM ® -BD272)	980	3.1	5.4	0.3
	Potato crisps (B)	2512	5.9	11.7	0.8
	Commercial potato crisps (B)	4051	4.3	8.9	0.7
	Potato crisps (C)	9082	5.0	9.1	0.8

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Acknowledgements

The organisers are very grateful for the support given by Nestlé. We would like to thank in particular Dr. Richard Stadler for the preparation and supply of suitable test materials. The authors would like to thank NMKL for technical support in the organisation of the study.

The authors would also like to thank following institutes for participating in the study: Eurofins Analytik GmbH Hamburg Germany Finnish Food Safety Authority EVIRA Helsinki Finland ICT Prague Prague Czech Republic Laboratoire Départemental de la Sarthe Le Mans France