Ascorbic acid-pretreated quartz enhances cyclo-oxygenase-2 expression in RAW 264.7 murine macrophages ` Sonia Scarfı1,2, Umberto Benatti2, Marina Pozzolini1,2, Emanuela Clavarino2, Chiara Ferraris1,2, Mirko Magnone2, Laura Valisano3 and Marco Giovine1,4 Advanced Biotechnology Center, Genoa, Italy Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Italy Department for the Study of the Territory and its Resources, University of Genoa, Italy Department of Biology, University of Genoa, Italy Keywords ascorbic acid; inflammation; macrophages; reactive oxygen species; silica Correspondence ` S Scarfı, Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Viale Benedetto XV n°1, 16132 Genoa, Italy Fax: +39 010 354415 Tel: +39 010 3538151 E-mail: soniascarfi@unige.it (Received 22 May 2006, revised 26 October 2006, accepted 30 October 2006) doi:10.1111/j.1742-4658.2006.05564.x Exposure to quartz particles induces a pathological process named silicosis Alveolar macrophages initiate the disease through their activation, which is the origin of the later dysfunctions Ascorbic acid is known to selectively dissolve the quartz surface During the reaction, ascorbic acid progressively disappears and hydroxyl radicals are generated from the quartz surface These observations may be relevant to mammalian quartz toxicity, as substantial amounts of ascorbic acid are present in the lung epithelium We studied the inflammatory response of the murine macrophage cell line RAW 264.7 incubated with ascorbic acid-treated quartz, through the expression and activity of the enzyme cyclo-oxygenase-2 (COX-2) COX-2 expression and prostaglandin secretion were enhanced in cells incubated with ascorbic acid-treated quartz In contrast, no changes were observed in cells incubated with Aerosil OX50, an amorphous form of silica Quantification of COX-2 mRNA showed a threefold increase in cells incubated with ascorbic acid-treated quartz compared with controls The transcription factors, NF-jB, pCREB and AP-1, were all implicated in the increased inflammatory response Reactive oxygen species (H2O2 and OH•) were involved in COX-2 expression in this experimental model Parallel experiments performed on rat alveolar macrophages from bronchoalveolar lavage confirmed the enhanced COX-2 expression and activity in the cells incubated with ascorbic acid-treated quartz compared with untreated quartz In conclusion, the selective interaction with, and modification of, quartz particles by ascorbic acid may be a crucial event determining the inflammatory response of macrophages, which may subsequently develop into acute inflammation, eventually leading to the chronic pulmonary disease silicosis Long-term exposure to quartz particles induces a pathological process characterized by the development of fibrotic nodules in the lung, due to the accumulation of inflammatory cells, deposition of extracellular matrix and cellular proliferation The ensuing disease, silicosis, is the result of a complex interaction of molecular pathways, the molecular mechanisms of which have been only partially elucidated [1] The main difficulties arising from these studies are due to the solid nature of quartz particles Particulates are intrinsically heterogeneous in dimension, shape and composition Furthermore, they never act as a constant Abbreviations AA, ascorbic acid; BAL, bronchoalveolar lavage; COX-2, cyclo-oxygenase-2; EMSA, electrophoretic mobility-shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN-c, interferon-c; PGE2, prostaglandin E2; ROS, reactive oxygen species 60 FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS ` S Scarfı et al entity, and their cytotoxic potential in a biological environment depends on their mechanical, thermal and chemical history as well as on the micromorphology at the atomic level [2,3] It is now generally accepted that silicosis originates from inhalation of quartz particles, which are subsequently incorporated by alveolar macrophages The cell’s inability to dissolve the crystalline particulate leads to the chronic inflammation responsible for the development of the disease [4] Ten years ago, Bavestrello et al [5] reported that ascorbic acid (AA) is able to partially dissolve the surface of quartz, greatly increasing the concentration of soluble silica in the surrounding medium More recently, Fenoglio et al [6] demonstrated that during this peculiar chemical reaction, while AA progressively disappears, important modifications of the quartz surface occur, leading to an increased production of free hydroxyl radicals and H2O2 These findings are relevant to mammalian quartz toxicity: by reacting with AA, quartz could deprive the alveolar epithelium of one of its most effective antioxidant defences, and the surface modifications induced by AA increase the concentration of particle-derived reactive oxygen species (ROS) in the alveolar space, which is one of the mechanisms proposed for quartz fibrogenicity and carcinogenicity [1] Furthermore, AA-derived quartz dissolution is specific for crystalline silica, as the amorphous silica particulate is not modified at its surface by ascorbate treatment and does not produce hydroxyl radicals [6] These findings prompted us to further investigate the cytotoxicity of AA-treated quartz particles in the murine macrophage cell line RAW 264.7, a cell model widely used for molecular studies on cell–particle interaction [7,8] In this study, we showed that AA-pretreated quartz establishes a significantly higher cytotoxicity compared with untreated quartz [9] These results suggested an active role for AA as a cofactor involved in the early stages of quartz-induced pathology, and they represent the basis of the present study on the effect of AA on the quartz-induced inflammatory response in the same cell model The inducible enzyme cyclo-oxygenase-2 (COX-2) is one of the molecules principally involved in the immediate cellular inflammatory response It is encoded by an immediate-early gene induced by various proinflammatory agents, including endotoxin, cytokines, mitogens and particulates This enzyme has emerged as primarily responsible for the synthesis of the prostanoids involved in acute and chronic inflammatory states, and recently it has been documented that its expression is also increased in cellular and animal models after quartz exposure [7,10–12] Ascorbate-treated quartz enhances COX-2 expression The aim of the present work was to evaluate the production of COX-2 and prostaglandin E2 (PGE2) in RAW 264.7 cells incubated with AA-treated quartz and to compare it with that induced by untreated quartz particles and by a different polymorph of silica, Aerosil OX50 This form of silica does not possess a crystalline structure and has been demonstrated to be unable to chemically interact with AA [6] An assessment of the major transcription factors (NF-jB, CREB, AP-1) involved in COX-2 biosynthesis was performed, and the relative amount of COX-2 mRNA was quantified The role of quartz-induced ROS in the modulation of COX-2 expression was investigated Catalase, mannitol and desferrioxamine were used to assess the importance of different ROS in triggering the inflammatory response towards crystalline silica pretreated or not with AA Furthermore, experiments performed on rat alveolar macrophages obtained from bronchoalveolar lavage (BAL) confirmed the enhanced COX-2 expression and activity in the cells incubated with AA-pretreated quartz compared with cells challenged with untreated quartz These results suggest that the selective interaction of AA with quartz could be a crucial event determining the inflammatory response of macrophages, which is recognized as the first necessary event eventually leading to the quartz-induced chronic pulmonary disease silicosis Results Quartz-induced COX-2 expression and PGE2 production in RAW 264.7 cells The inducible enzyme COX-2 is expressed in the early stages of the inflammatory response and catalyses the first step of the synthesis of PGE2, an important inflammatory mediator COX-2 and PGE2 were quantified in RAW 264.7 macrophages challenged with quartz particles pretreated or not with AA RAW 264.7 cells were incubated with different concentrations of AA-treated Min-U-Sil quartz or with AA-treated Aerosil OX50 (a commercial amorphous silica) COX-2 expression was evaluated, by western blot analysis, after h of treatment Results were compared with data obtained on cells incubated with untreated particles and on untreated cells Untreated quartz suspensions, at concentrations of 15, 50 and 100 lgỈmL)1, induced COX-2 synthesis in RAW 264.7 cells (Fig 1A, black bars) as well as AA-treated quartz (Fig 1A, striped bars) The statistical analysis of variance showed a significant difference in COX-2 synthesis both between untreated and AA-treated quartz particles (P < 0.001) and among the different quartz FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS 61 ` S Scarfı et al Ascorbate-treated quartz enhances COX-2 expression Fig COX-2 expression in quartz-treated and aerosil-treated cells (A) COX-2 expression in RAW 264.7 cells stimulated with AA-treated (striped bars) or untreated (black bars) Min-U-Sil quartz was evaluated after h of incubation by western blot analysis of total cell lysates Cells were challenged with untreated (q) or AA-treated (qa) quartz particles at 15, 50 and 100 lgỈmL)1 Results are expressed as density ratio between each COX-2 band and the corresponding b-actin band, relative to control Values are the mean ± SD from eight experiments The asterisk indicates a statistically significant difference between q and qa values (Tukey test, P < 0.05) (B) COX-2 expression in RAW 264.7 cells stimulated with AA-treated (striped bars) or untreated (black bars) Aerosil OX50 silica particles was evaluated after h of incubation by western blot analysis of total cell lysates Cells were challenged with untreated (a) or AA-treated (aa) aerosil particles at 15, 50 and 100 lgỈmL)1 Results are expressed as density ratio between each COX-2 band and the corresponding b-actin band, relative to control Values are the mean ± SD from eight experiments The symbol # indicates a significant increase in COX-2 at increasing aerosil concentrations (15 versus 50 lgỈmL)1, P < 0,05; 50 versus 100 lgỈmL)1, P < 0,05, Tukey test) concentrations (P < 0.001), and, in particular, cells stimulated with AA-treated quartz showed higher COX-2 expression than cells challenged with untreated particles (Tukey test, P < 0.05) Similar experiments were performed with Aerosil OX50, which is not known to cause silicosis and does not react with AA [6] Exposure of RAW 264.7 cells to 62 the same concentrations of AA-treated (Fig 1B, striped bars) or untreated Aerosil OX50 particles (Fig 1B, black bars) stimulated the production of high amounts of COX-2 with no significant difference between AA-treated and untreated aerosil samples However, COX-2 expression in cells challenged with AA-treated or untreated aerosil showed a significant, dose-dependent COX-2 synthesis (analysis of variance, P < 0.001), with values at 15 lgỈmL)1 lower than those at 50 lgỈmL)1 (Tukey test, P < 0.05) and values at 50 lgỈmL)1 lower than those at 100 lgỈmL)1 (Tukey test, P < 0.05) Aerosil particles have a surface area 10 times higher than quartz particles Thus, 100 lgỈmL)1 quartz and 15 lgỈmL)1 aerosil had a similar surface area (5.2 and 6.5 cm2, respectively; Fig 1A,B) and induced a similar increase in COX-2 expression At 100 lgỈmL)1 and a surface area of 43 cm2ỈmL)1, aerosil particles induced the highest increase in COX-2 expression in RAW cells (Fig 1B) To quantify the enzymatic activity of COX-2 in our experimental conditions, we also determined PGE2 production in quartz-treated RAW 264.7 cells after and 18 h of treatment PGE2 concentration in the medium of cells incubated for h (Fig 2A, black bars) or for 18 h (Fig 2A, white bars) was significantly increased by untreated quartz (analysis of variance, P ¼ 0.000); furthermore, at h, values measured at 100 lgỈmL)1 were higher than values at 15 and 50 lgỈmL)1 (Student–Newman–Keuls test, P < 0.05) The relevant values were the following: at h Q15 was 44.3 pgỈmL)1, Q50 was 54.1 pgỈmL)1 and Q100 was 73.9 pgỈmL)1; at 18 h Q15 was 53.1 pgỈmL)1, Q50 was 56.2 pgỈmL)1 and Q100 was 75.6 pgỈmL)1 Prostaglandin production in cell cultures challenged with AA-treated particles (Fig 2A, dotted bars) was significantly different (1.3-fold to 1.65-fold higher) from that measured in cultures stimulated with untreated quartz at both h (black, dotted bars) and 18 h (white, dotted bars) incubation (analysis of variance, P < 0.01) In this case, the PGE2 concentration in the medium of cells incubated with AA-treated quartz increased in a significant, dose-dependent fashion both at and 18 h, with a significant difference between increasing quartz concentrations (analysis of variance, P ¼ 0.000, Student–Newman–Keuls test, P < 0.05) The relevant values were: at h, QA15 was 57.6 pgỈmL)1, QA50 was 79.7 pgỈmL)1 and QA100 was 117.9 pgỈmL)1; at 18 h, QA15 was 72.9 pgỈmL)1, QA50 was 92 pgỈmL)1 and QA100 was 116.8 pgỈmL)1 Murine macrophages were also challenged with AApretreated or untreated quartz costimulated with 100 pgỈmL)1 interferon-c (IFN-c) a cytokine released by activated lymphocytes, mimicking in our model a FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS ` S Scarfı et al Fig PGE2 production in quartz-incubated cells (A) PGE2 concentration detected in RAW 264.7 cell supernatants after h (black bars) and 18 h (white bars) stimulation with quartz was determined using a PGE2 monoclonal EIA kit Cells were challenged with untreated (q) or AA-treated (qa, dotted bars) quartz particles at 15, 50 and 100 lgỈmL)1 Values are the mean ± SD from eight experiments The symbol § indicates a significant difference between the Q100 and both the Q15 and the Q50 values at h (Student–Newman–Keuls test, P < 0.05) The asterisk indicates a significant difference between Q and QA values, at all particle concentrations and at both time points (analysis of variance, P < 0.01) The symbol # indicates a significant difference between PGE2 values at increasing QA concentrations, at both time points (QA15 versus QA50 versus QA100, Student– Newman–Keuls Test, P < 0.05) (B) PGE2 concentration detected in RAW 264.7 cell supernatants after h (black bars) and 18 h (white bars) stimulation with AA-treated (dotted bars) or untreated quartz, in the presence of murine IFN-c (100 pgỈmL)1), was determined using a PGE2 monoclonal EIA kit Values are the mean ± SD from eight experiments The symbol § indicates a significant difference between Q15 versus Q50 versus Q100 values at 18 h (Student–Newman–Keuls test, P < 0.05) The asterisks (**) indicate a significant difference between Q values at 18 h versus Q values at h (analysis of variance, P < 0.001) The single asterisk indicates a significant difference between QA and Q values at all particle concentrations and at both time points (analysis of variance, P < 0.001) The symbol # indicates a significant difference between the QA values at increasing concentrations, at both time points (Tukey test, P < 0.05) Ascorbate-treated quartz enhances COX-2 expression later stage of inflammation, when these cells infiltrate the lung tissue in large numbers and contribute to the development of a chronic inflammatory state In the presence of IFN-c, PGE2 release into the medium was significantly increased by treatment of the cells with quartz, AA-treated or untreated, by the particle concentration and by the incubation time (Fig 2B; analysis of variance, P < 0.001) Specifically, costimulation of RAW 264.7 cells with untreated quartz particles together with IFN-c for h (black bars) or 18 h (white bars) induced a greater increase in PGE2 production in all samples ranging between a 2.8fold and a 19.6-fold increase compared with the corresponding values in the absence of IFN-c (Fig 2A) The relevant values of PGE2 concentrations were the following: at h, Q15 was 122.2 pgỈmL)1, Q50 was 149.6 pgỈmL)1 and Q100 was 174.8 pgỈmL)1; at 18 h, Q15 was 225.7 pgỈmL)1, Q50 was 598 pgỈmL)1 and Q100 was 1485 pgỈmL)1 PGE2 production by cells challenged with untreated quartz was significantly increased at 18 h compared with h (Fig 2B, analysis of variance, P < 0.001): the increase in PGE2 production at increasing concentrations (15–100 lgỈmL)1) was only significant (analysis of variance, P ¼ 0.000) after 18 h of incubation (Student–Newman–Keuls test, P < 0.05) Furthermore, in the presence of IFN-c, PGE2 release from cells stimulated with AA-treated Min-USil quartz (Fig 2B, dotted bars) was further significantly increased, 1.4-fold to 4.8-fold, over that from cells challenged with untreated quartz (analysis of variance, P < 0.001) The relevant values were: at h, QA15 was 256.5 pgỈmL)1, QA50 was 502.2 pgỈmL)1 and QA100 was 836.3 pgỈmL)1; at 18 h, QA15 was 529.7 pgỈmL)1, QA50 was 1337 pgỈmL)1 and QA100 was 2065 pgỈmL)1 PGE2 release from cells stimulated with AA-treated quartz in the presence of IFN-c was affected by both incubation time and particle concentration (Fig 2B, dotted bars, analysis of variance, P < 0.001); in particular, values recorded at 18 h were significantly higher than those at h (Tukey test, P < 0.05), and at both time points a significant concentration-dependence of the effect of AA-treated quartz was observed (Tukey test, P < 0.05) Summarizing, PGE2 production and release by RAW cells stimulated with both AA-treated and untreated quartz occurred mainly during the first h and did not significantly increase during the subsequent 12 h (Fig 2A) Conversely, in the presence of IFN-c, both AA-treated and untreated quartz particles induced a sustained PGE2 release, leading to a significantly higher PGE2 concentration in the FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS 63 ` S Scarfı et al Ascorbate-treated quartz enhances COX-2 expression medium at 18 h than at h incubation (Fig 2B) Under the same conditions (particle concentration, incubation time, presence or absence of IFN-c), PGE2 release from cells stimulated with AA-treated quartz was always higher than that from cells challenged with untreated quartz COX-2 expression and activity were also evaluated in primary cultures of rat alveolar macrophages isolated from BAL of healthy animals and challenged for h with or without 100 lgỈmL)1 AA-treated or untreated quartz COX-2 expression (Fig 3A) was significantly increased by incubation of the cells with untreated or AA-treated quartz (analysis of variance, P ¼ 0.000), with increasing protein expression being observed in control versus untreated versus AA-treated quartz samples (Student–Newman–Keuls test, NT versus Q100 versus QA100, P < 0.05) In line with this result, PGE2 release (Fig 3B) in the culture medium of BAL macrophages was significantly increased in AA-treated or untreated quartz samples compared with control, untreated cells (Kruskal–Wallis analysis of variance, 0.02 < P < 0.05) with progressively increasing PGE2 concentrations being observed in the media from control cells, cells stimulated with untreated quartz, and cells challenged with AA-treated quartz suspensions (multiple comparison test, NT versus Q100 versus QA100, P < 0.05) In particular, PGE2 release into the culture medium from cells incubated with AA-treated quartz was 2.8-fold higher than that measured in the supernatant from cells challenged with untreated 272.9 pgỈmL)1) quartz (754.2 pgỈmL)1 versus Time-course of COX-2 mRNA synthesis in RAW 264.7 macrophages COX-2 mRNA synthesis was measured by quantitative RT-PCR analysis in RAW 264.7 macrophages stimulated with 100 lgỈmL)1 AA-treated or untreated quartz COX-2 mRNA expression at the various incubation times, normalized to the respective glyceraldehyde-3phosphate dehydrogenase (GAPDH) internal standard, was compared with COX-2 expression at time zero (Fig 4) COX-2 transcription was significantly induced 30 and 60 after cell exposure to both AA-treated (striped bars) and untreated quartz (black bars), with no significant difference observed for each stimulus between the two time points For both stimuli, COX-2 mRNA decreased below time-zero values h after cell exposure to the particles (Fig 4) Thus, COX-2 mRNA concentrations in cells challenged with untreated and AA-treated quartz were statistically compared at 30 and 60 of incubation A significant difference between samples challenged with untreated and AA-treated quartz was observed (Scheirer–Ray–Hare test, 0.001 < P < 0.01), with AA-treated quartz inducing a COX-2 transcription 2.5-fold higher than that at time zero, and untreated quartz increased COX-2 transcription 1.5-fold over time zero (mean of values recorded at 30 and 60 min) Fig COX-2 expression and PGE2 production in quartz-incubated rat alveolar macrophages (A) COX-2 expression in rat alveolar macrophages stimulated with AA-treated (striped bars) or untreated (black bars) Min-U-Sil quartz was evaluated by western blot analysis of total lysates after h of incubation The final concentration of quartz particles was 100 lgỈmL)1 (Q100, QA100 untreated and treated) Results are expressed as the density ratio between each COX-2 band and the corresponding b-actin band relative to control Values are the mean ± SD from three experiments The asterisk indicates a significant increase in COX-2 expression in QA100 versus Q100 versus NT samples (Student–Newman–Keuls test, P < 0.05) (B) PGE2 concentration was detected in rat alveolar macrophage supernatants after h of stimulation with 100 lgỈmL)1 untreated (black bars) or AA-treated quartz (striped bars) using a PGE2 monoclonal EIA kit Values are the mean ± SD from six experiments The symbol § indicates a significant increase in PGE2 production in QA100 versus Q100 versus NT samples (multiple comparison test, P < 0.05) 64 FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS ` S Scarfı et al Ascorbate-treated quartz enhances COX-2 expression Fig RT-PCR of COX-2 mRNA in quartz-treated RAW 264.7 cells COX-2 mRNA transcription was monitored in RAW 264.7 macrophages by RT-PCR analysis from 30 to 18 h after cell stimulation with 100 lgỈmL)1 AA-treated (striped bars) or untreated quartz (black bars) Results are the mean of three independent experiments performed in triplicate and expressed as COX-2 mRNA synthesis normalized to the GAPDH transcription, relative to control cells at time zero At 30 and 60 of incubation, a significant difference (*) between samples challenged with untreated and AA-treated quartz was observed (Scheirer–Ray–Hare test, 0.001 < P < 0.01) Nuclear translocation of NF-jB, AP-1 and pCREB in RAW 264.7 macrophages The nuclear translocation of the transcription factors known to be responsible for COX-2 synthesis triggered by inflammatory stimuli, i.e NF-jB, AP-1 and pCREB [13], was assessed by electrophoretic mobilityshift assay (EMSA) on RAW 264.7 macrophages stimulated with different concentrations of AA-treated or untreated quartz for 30 The results of these experiments are shown in Fig Nuclear translocation of NF-jB (Fig 5A) in RAW 264.7 was significantly higher in cells incubated with both AA-treated (striped bars) and untreated (black bars) quartz at increasing particle concentration (analysis of variance, P < 0.05) AA-treated quartz suspensions at 15, 50 and 100 lgỈmL)1 induced a concentration-dependent increase in NF-jB translocation (analysis of variance, P < 0.001; Tukey test, 15 versus 50 versus 100 lgỈmL)1, P < 0.05), with 100 lgỈmL)1 inducing a higher NF-jB translocation than untreated quartz (t-test, P < 0.01) In untreated quartz suspensions, the dose-dependence of the effect was significant only between 15 and 50 lgỈmL)1 (analysis of variance, P < 0.05; Tukey test, P < 0.05) pCREB nuclear translocation (Fig 5B) was also significantly increased by incubation of the cells with AA-treated and untreated quartz, at all particle Fig NF-jB, pCREB and AP-1 nuclear translocation in quartz-treated RAW 264.7 cells EMSA analyses of NF-jB (A), pCREB (B) and AP-1 (C) were performed on nuclear extracts of RAW 264.7 cells stimulated for 30 with 15, 50 and 100 lgỈmL)1 of AA-treated (QA, striped bars) or untreated quartz (Q, black bars) Bars indicate transcription factor band density relative to control, untreated cells and are the mean ± SD from four experiments (A) The asterisk indicates a significant difference between Q and QA samples versus control (analysis of variance, P < 0.05) The symbol § indicates a significant difference between QA100 and Q100 values (t-test, P < 0.01) (B) The asterisk indicates a significant difference between Q and QA samples versus control (analysis of variance, P < 0.001) The symbol § indicates a significant difference between QA100 and Q100 values (t-test, P < 0.01) (C) The asterisk indicates a significant difference between Q and QA samples versus control (analysis of variance, P < 0.05) The symbol § indicates a significant difference between QA15 and Q15 values (t-test, P < 0.01) concentrations (analysis of variance, P < 0.001) In both AA-treated and untreated quartz samples, pCREB density values at 100 lgặmL)1 particle concentration FEBS Journal 274 (2007) 6073 ê 2006 The Authors Journal compilation ª 2006 FEBS 65 ` S Scarfı et al Ascorbate-treated quartz enhances COX-2 expression were significantly higher than at 15 and 50 lgỈmL)1, but, in the case of untreated quartz, the difference between the effect of 50 and 100 lgỈmL)1 was lower (analysis of variance, P < 0.05; Tukey test, P < 0.05) than the one measured for the same concentrations of AA-treated quartz (analysis of variance, P < 0.001; Tukey test, P < 0.05) Besides, at 100 lgỈmL)1, AA-treated quartz induced a higher pCREB translocation than untreated quartz (t-test, P < 0.01) Finally, nuclear translocation of AP-1 (Fig 5C) was also significantly increased in cells incubated with AAtreated or untreated quartz, at all particle concentrations (analysis of variance, P < 0.05) For both the AA-treated and untreated quartz suspensions, the highest effect was observed at 15 lgỈmL)1 (analysis of variance, P < 0.05; Tukey test, P < 0.05, for both series of data), with AA-treated quartz inducing a higher AP-1 translocation than untreated quartz (t-test, P < 0.01) Summarizing, at 100 lgỈmL)1 particle concentration, translocation of NF-jB (Fig 5A) and pCREB (Fig 5B) was significantly higher in cells stimulated with AA-treated quartz (striped bars) than with untreated quartz (black bars), whereas no significant difference was observed at lower particle concentrations; conversely, AP-1 (Fig 5C) was more abundant in the nuclei of cells stimulated with AA-treated quartz compared with untreated quartz only at the lowest quartz concentration (15 lgỈmL)1) Role of oxygen radicals in quartz-induced COX-2 synthesis In preliminary experiments, ROS production by quartz-stimulated RAW cells was evaluated with a ROS-specific fluorescent probe in the presence or absence of the radical scavengers catalase, mannitol and desferrioxamine, which remove hydrogen peroxide, hydroxyl radicals and iron, respectively (the latter being required in the Fenton reaction, which converts H2O2 into OHã) AA-treated quartz at 100 lgặmL)1 induced a threefold increase in ROS generation compared with the same concentrations of untreated quartz, after h incubation (not shown) Desferrioxamine did not affect ROS generation by either untreated or AA-treated quartz Conversely, mannitol completely quenched ROS production triggered by untreated quartz, whereas it reduced the probe’s fluorescence in the presence of AA-treated quartz only by  24% (not shown) Unfortunately, the effect of catalase could not be tested, because of severe interference of the enzyme with the probe’s fluorescence 66 The increased ROS generation induced by AA-treated versus untreated quartz on RAW cells prompted us to explore the effect of ROS scavengers on the increased COX-2 expression triggered by AA-treated quartz in RAW 264.7 macrophages Cells were incubated for h with AA-treated and untreated quartz in the presence of excess of the radical scavengers, and COX-2 expression was analyzed by western blot Preliminary experiments had demonstrated no effect of the radical scavengers themselves on COX-2 synthesis in RAW cells (not shown) At all quartz concentrations tested, COX-2 expression in RAW 264.7 cells was increased by AA-treated or untreated quartz and was affected by the presence of the three scavengers (Fig 6A, analysis of variance P < 0.005; Fig 6B, analysis of variance P < 0.001; Fig 6C, analysis of variance P < 0.01) At 15 lgỈmL)1 particle concentration (Fig 6A), no significant difference in the COX-2 synthesis triggered by untreated quartz was observed in the absence (Q) or presence of the various scavengers (Qc, Qm, Qd) Conversely, the COX-2 expression stimulated by AAtreated quartz was significantly inhibited by the presence of scavengers (analysis of variance, P < 0.001) Indeed, the COX-2 density values in the presence of catalase (QAc) and mannitol (QAm) were significantly lower than the values in the absence of scavengers (QA) or in the presence of desferrioxamine (QAd) (Dunnett test, P < 0.05) At 50 lgỈmL)1 particle concentration (Fig 6B), no significant difference in the COX-2 synthesis stimulated by AA-treated quartz was observed in the absence (QA) or presence of the various scavengers (QAc, QAm, QAd), whereas COX-2 expression triggered by untreated quartz (Q) was significantly inhibited by the presence of scavengers (analysis of variance, P < 0.001) In fact, COX-2 density values in the presence of catalase (Qc) and mannitol (Qm) were significantly lower than in the absence of scavengers (Q) or in the presence of desferrioxamine (Qd) (Dunnett test, P < 0.05) Also at 100 lgỈmL)1 particle concentration (Fig 6C), no significant difference in the COX-2 synthesis stimulated by AA-treated quartz was observed in the absence (QA) or presence of the various scavengers (QAc, QAm, QAd) Conversely, in untreated quartz samples, a significant difference was observed in the absence or presence of scavengers (Q, Qc, Qm, Qd, analysis of variance, P < 0.005), although the only significant reduction in COX-2 expression relative to the quartz-treated samples (Q) was observed in the presence of catalase (Qc) (Dunnett test, P < 0.05) FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS ` S Scarfı et al Ascorbate-treated quartz enhances COX-2 expression Fig COX-2 expression in quartz-stimulated RAW 264.7 cells in the presence of ROS scavengers COX-2 was analyzed by western blot in RAW 264.7 cells after h incubation with different concentrations of AA-treated (QA, striped bars) or untreated quartz (Q, black bars) in the presence of 4000 mL)1 catalase (c), 50 mM mannitol (m) and mM desferrioxamine (d) Bars indicate the COX-2 band density, normalized on the corresponding b-actin bands, relative to the density of the COX-2 band in cells stimulated with untreated quartz (Q) Values are the mean ± SD from four experiments (A) The symbol § indicates a significant difference between Qc, Qm, Qd, QA, QAm, QAc, QAd values versus the Q value (analysis of variance, P < 0.005) COX-2 expression in QA samples was significantly inhibited (*) by the presence of catalase and mannitol (QAc and QAm versus QA, analysis of variance, P < 0.001, Dunnett test, P < 0.05) (B) The symbol § indicates a significant difference between Qc, Qm, Qd, QA, QAm, QAc, QAd values versus the Q value (analysis of variance, P < 0.001) COX-2 expression in Q samples was significantly inhibited (*) by the presence of catalase and mannitol (Qc and Qm versus Q, analysis of variance, P < 0.001, Dunnett test, P < 0.05) (C) The symbol § indicates a significant difference between Qc, Qm, Qd, QA, QAm, QAc, QAd values versus the Q value (analysis of variance, P < 0.01) COX-2 expression in Q samples was significantly inhibited (*) by the presence of catalase (Qc versus Q, analysis of variance, P < 0.005, Dunnett test, P < 0.05) Summarizing, catalase reduced COX-2 expression in cells stimulated with AA-treated quartz (QA, striped bars) at the lowest particle concentration (15 lgỈmL)1, Fig 6A), while at higher particle concentrations (50– 100 lgỈmL)1) it reduced COX-2 expression only in cells stimulated with untreated quartz (Fig 6B,C) Mannitol reduced COX-2 expression in cells incubated with untreated quartz only at 50 lgỈmL)1 particle concentration (Fig 6B), while in cells stimulated with AA-treated quartz it was effective only at the lowest particle concentration (15 lgỈmL)1, Fig 6A) Desferrioxamine was always without effect The failure of desferrioxamine to inhibit quartz-induced COX-2 expression apparently rules out the involvement of a Fenton reaction in mediating quartz effects on COX-2 expression These data confirm results obtained by others indicating that oxygen radicals play an important role in mediating quartz-induced COX-2 expression in macrophages, and indicate that their production is strictly related to particle concentration [14] Furthermore, the above results indicate that ROS generation is higher in cells challenged with AA-treated compared with untreated quartz and that, at the lowest particle concentration, ROS scavengers are able to prevent COX-2 synthesis, whereas at higher particle concentrations FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS 67 ` S Scarfı et al Ascorbate-treated quartz enhances COX-2 expression protein production is apparently triggered by other signals Discussion Quartz toxicity towards mammalian cells is well known in the field of occupational health research, although the molecular mechanisms of quartz-induced cell tissue damage are not yet completely understood Many experimental models have been investigated to address the complex interactions between the heterogeneous lung tissue cell population and quartz particles One of the most consolidated pieces of evidence from these studies is the strict dependence of cell reactivity on the properties of particle surface The presence of AA in the lung fluids has been demonstrated, and its possible involvement in the development of quartz-induced lung injury had already been hypothesized some years ago [15–17] Ghio et al [16] observed a remarkable increase in the accumulation of inflammatory cells in the lung fluids of guinea pigs fed an AA-rich diet and exposed to Min-U-Sil Conversely, control animals fed low doses of AA and exposed to the same amount of quartz showed a reduced proliferation of inflammatory cells In connection with this, we have recently demonstrated that specific chemical modifications occurring on the quartz surface after exposure to AA cause increased quartz cytotoxicity in the murine macrophage cell line RAW 264.7 [9] As cytotoxicity is only one of the many aspects of macrophage reactivity to quartz, we here investigated the inflammatory response of RAW 264.7 macrophages, a cell model widely used for in vitro studies of the biological response to quartz particles and of primary rat alveolar macrophages to AA-treated quartz [7,8,18–20] Transcription of COX2, which catalyzes the first step of PGE biosynthesis, occurs in the early stages of macrophage activation and is known to be triggered by quartz [11] Thus, we investigated COX-2 transcription, expression and enzymatic activity in terms of PGE2 production in the RAW cell line and BAL macrophages The transcription factors involved in COX-2 induction and the possible role of specific ROS in triggering the COX-2 synthetic pathway were also investigated In RAW 264.7 cells, AA-treated quartz particles induced higher biosynthesis of COX-2 and greater production of PGE2 than untreated particles (Figs and 2) Similar results were obtained with rat alveolar macrophages freshly collected from healthy animals by BAL (Fig 3) These results are particularly relevant because COX-2 over-expression seems to be strictly related not only to inflammation development but also 68 cancer progression [21,22] Interestingly, the enhancing effect of AA-treated over untreated quartz on COX-2 synthesis was even more evident in the presence of IFN-c (Fig 2), a cytokine produced by activated lymphocytes, which are believed to be recruited by macrophages at a later stage of the lung inflammatory process The result of this experiment suggests that the quartz surface modifications caused by AA are relevant not only in the first steps of the lung inflammatory response, but also subsequently The fact that macrophages are unable to dissolve the internalized quartz particles indeed prolongs macrophage activation through multiple ingestion–re-ingestion cycles and exposes the same cells to cytokines produced by activated lymphocytes [4] The long-lasting presence of quartz in the lung may expose the particles to AA present in the bronchoalveolar fluid, inducing the chemical modifications demonstrated by previous in vitro experiments [6,9]; the resulting in vivo AA-modified quartz could eventually favour an escalation of the inflammatory response by macrophages, also stimulated by cytokines released by other cell types during the ongoing inflammation Results obtained here also demonstrate the specificity of action of AA on crystalline (Min-U-Sil quartz), as opposed to amorphous (Aerosil OX50), silica Indeed, no difference in COX-2 synthesis was observed between cell cultures incubated with AAtreated or untreated Aerosil OX50, although, rather surprisingly, we observed significant COX-2 expression triggered by the amorphous silica In contrast with what was observed with quartz, however, cell activation was transient, decreasing rapidly 18 h after exposure to the particles (data not shown), suggesting an acute cell response followed by a rapid recovery This time-course of COX-2 synthesis led us to rule out a possible endotoxin contamination of the amorphous silica particles, as it is well known that lipopolysaccharide induces high production of prostaglandins, which lasts well after 18 h of stimulation of the cells [23] A possible explanation for the inflammatory response elicited by amorphous silica in RAW 264.7 cells comes from the dimensions of the aerosil particles, which are smaller than the crystalline ones [24] Particle dimension is critical during macrophage phagocytosis, with smaller particles being internalized better than larger ones and consequently inducing greater cellular activation [25] In fact, the highest increase in COX-2 expression in RAW cells was observed with an aerosil particle concentration (100 lgỈmL)1) resulting in a surface area 10 times higher than that of the same concentration of quartz particles (Fig 1) FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS ` S Scarfı et al In line with our in vitro results, both Johnston et al [26] and Warheit et al [27] reported in in vivo experiments a large, but transient, pulmonary inflammatory response to amorphous silica, followed by a rapid post-exposure decrease in the principal inflammatory and cytotoxic biomarkers In RAW 264.7, activation of COX-2 by quartz is controlled by NF-jb translocation to the nucleus and also by AP-1 [11,14,28] Indeed, nuclear translocation of NF-jB and AP-1 in RAW 264.7 cells was stimulated to a higher degree when macrophages were challenged with AA-treated, as compared with untreated, quartz particles (Fig 5) Moreover, we describe for the first time, to our knowledge, involvement of pCREB in the inflammatory response triggered by quartz As observed for NF-jB and AP-1, AA-treated quartz was more effective than untreated quartz in stimulating pCREB translocation Interestingly, whereas NF-jB and pCREB were activated by the highest quartz concentration (100 lgỈmL)1), the AP-1 complex was activated by the lowest (15 lgỈmL)1) These data indicate that quartz can trigger different signal-transduction pathways in macrophages depending on the number of particles coming into contact with the phagocytic cells In line with its stimulation of transcription factor translocation, AA-treated quartz induced a higher COX-2 mRNA synthesis than untreated crystalline silica (Fig 4) It is generally accepted that free-radical production by quartz is responsible for both quartz-induced toxicity and NF-jB translocation leading to macrophage activation [29–32] COX-2 production by RAW 264.7 cells stimulated with 15 lgỈmL)1 AA-treated quartz was indeed significantly reduced in the presence of the ROS scavengers catalase and mannitol (Fig 6A), indicating that AA treatment of quartz enhances COX-2 synthesis by means of radicals derived from H2O2 A crucial role of iron in the generation of hydroxyl free radicals triggered by quartz has been reported [32] However, the absence of inhibition of COX-2 synthesis stimulated by AA-treated quartz by desferrioxamine (Fig 6), together with our previous results demonstrating hydroxyl radical production by AA-treated quartz in the presence of H2O2, suggests that the AA-modified quartz surface has itself a specific reactivity towards H2O2, generating OH• without the need for iron [9] If this hypothesis is correct, the iron content of quartz particles may play a minor role in its radical-induced toxicity Summarizing the results obtained, our work demonstrates that, indeed, COX-2 transcription, synthesis and enzymatic activity, as assessed by PGE2 production, are significantly increased in murine and rat macrophages challenged with AA-pretreated quartz compared with untreated quartz particles Further- Ascorbate-treated quartz enhances COX-2 expression more, we confirm the recruitment of NF-jB and AP-1 transcription factors into the quartz-triggered macrophage activation pathway and provide evidence indicating involvement of another transcription factor, pCREB, which has already been implicated in COX-2 induction in macrophages but never associated with quartz stimulation in these cells Finally, a causal role for H2O2-derived ROS in the mechanism by which AA-modified quartz stimulates COX-2 transcription is demonstrated In conclusion, the AA concentration in the lung epithelium may play a pivotal role in enhancing the cytotoxic and pro-inflammatory properties of the quartz particles, instead of preventing them by means of its antioxidant properties, as currently believed Experimental procedures Materials All reagents were acquired from Sigma-Aldrich (Milan, Italy), unless otherwise stated Cell cultures The mouse macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (Rockville, MD, USA) Rat alveolar macrophages were obtained by BAL from healthy animals (see below) Cells were cultured at 37 °C in a humidified, 5% CO2 atmosphere in Dulbecco’s modified essential medium containing mm glutamine, supplemented with 10% defined fetal bovine serum (HyClone, Logan, UT, USA) (complete medium) Cell stimulation using different concentrations of both sterilized quartz (Min-U-Sil 5; US Silica, Berkeley Spring Plant, specific surface area calculated by Brunaner, Emmett and Teller, SSABET ẳ 5.2 m2ặg)1) and Aerosil OX50 (SSABET ẳ 43.3 m2ặg)1, Degussa AG, Bitterfeld, Germany) was obtained by adding 15, 50 or 100 lgỈmL)1 particles treated with distilled water or AA (prepared as described in [9]) In detail, in terms of surface area ⁄ incubation volume, 15, 50 and 100 lgỈmL)1 Min-U-Sil quartz particles corresponded to 0.75, 2.6 and 5.2 cm2ỈmL)1, and for Aerosil OX50 they corresponded to 6.5, 21.65 and 43.3 cm2ỈmL)1, respectively After or 18 h culture, media were collected to detect PGE2 release, and cells were processed to obtain cell lysates for western blot analyses, or nuclei were separated and extracted for EMSA Collection of alveolar macrophages from BAL samples Male Sprague-Dawley rats (8–10 weeks) were purchased from Harlan Italy (S Pietro al Natisone, Italy) and housed FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS 69 ` S Scarfı et al Ascorbate-treated quartz enhances COX-2 expression at the animal facility of the Biochemistry Section in the Department of Experimental Medicine of the University of Genoa The program of animal use was approved by the CBA ethics committee, and all procedures involving animals were performed under protocols approved by the European Community directives Three groups of four males were killed with sodium pentobarbital (100 mgỈkg)1, intraperitoneally) Then, a tracheal cannula was inserted, and BAL was performed using ice-cold Ca2+ ⁄ Mg+-free Hanks’ medium Lavages of 6– mL were performed until a total of 50 mL lavage fluid was collected from each rat The samples were centrifuged at 300 g for 10 at °C (Allegra X-22R, swinging bucket rotor, Beckman Coulter SpA, Milan, Italy) The supernatants were removed, and the cells from the four rats were combined, resuspended in Hanks’ medium (with Ca2+ and Mg+) and centrifuged Cells were then resuspended in complete medium, then (1–1.5) · 106 cells ⁄ well (depending on the number of cells collected from each group) were seeded on to 60 · 15 mm tissue culture dishes (Falcon Becton Dickinson, Franklin Lakes, NJ, USA), and cultured for h at 37 °C Medium containing nonadherent cells was discarded Then 100 lgỈmL)1 AA-treated or untreated quartz in complete medium was added to the cultures, which were further incubated for h at 37 °C COX-2 expression and PGE2 production were quantified as described below Measurement of murine COX-2 expression and PGE2 production Expression of murine COX-2 in RAW 264.7 macrophages after h incubation with 15, 50 or 100 lgỈmL)1 AA-treated or untreated quartz or aerosil was measured by western blot analysis of total cell lysates PGE2 production was quantified in the culture medium Untreated cultures were used as controls Briefly, · 106 cells were seeded on to 60 · 15 mm tissue culture dishes (Falcon BD) and cultured as described above; after 18 h, the stimuli were added to the culture medium, and cells were further incubated for h at 37 °C Thereafter, adherent cells were washed three times with ice-cold NaCl ⁄ Pi and lysed with 400 lL lysis buffer (100 mm dithiothreitol, 2% SDS, 10% glycerol and 50 mm Tris ⁄ HCl, adjusted to pH 6.8) The lysates were heated at 100 °C for 10 min, sonicated, and the protein concentration was determined [33] Identical amounts of lysate proteins (40 lg per sample) were loaded on to SDS ⁄ 10% polyacrylamide gels, electrophoretically separated, and transferred to Immun-Blot poly(vinylidene difluoride) membranes (BioRad, Milan, Italy) Membranes were blocked and cut at the level of the 50-kDa precoloured marker The upper part was incubated with an anti-COX-2 mouse monoclonal IgG, and the lower part was stained with an anti-(b-actin) goat polyclonal IgG, both at lgỈmL)1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Western blots were developed with 70 the ECL-PLUS kit (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK), according to the manufacturer’s instructions Band detection and densitometry were performed using the Chemi-Doc System and the quantity one software package (Bio-Rad) The PGE2 concentration in the culture medium from cells incubated for or 18 h with quartz particles, in the presence or absence of 100 pgỈmL)1 murine IFN-c, was quantified using the PGE2 Monoclonal EIA Kit (Cayman Chemical Company, Ann Arbor, MI, USA), according to the manufacturer’s instructions Nuclear extracts RAW 264.7 cells (3 · 106 per assay) were seeded on to 60 · 15 mm tissue culture dishes (Falcon BD) and cultured as described above After 18 h, the medium was discarded and cells were incubated at 37 °C with 15, 50 and 100 lgỈmL)1 AA-treated or untreated quartz for 30 At the end of the incubation, cells were washed times with icecold NaCl ⁄ Pi, recovered from the dishes with a cell scraper in mL NaCl ⁄ Pi, and pelleted in a microfuge at 14 000 g for at °C (MICROcentrifugette 4212, fixed angle rotor, ALC, Milan, Italy) The cell pellets were then resuspended in 400 lL ice-cold buffer A (20 mm Tris ⁄ HCl, pH 7.8; 50 mm KCl; 10 lgỈmL)1 leupeptin; 0.1 m dithiothreitol; mm phenylmethanesulfonyl fluoride), and 400 lL buffer B (buffer A plus 1.2% Nonindet P40; Sigma) was then added The suspension was vortex-mixed for 10 s, centrifuged in a Microfuge at 14 000 g for 30 s at °C (MICROcentrifugette 4212, fixed angle rotor, ALC), and the supernatant was discarded The pelleted nuclei were then washed with 400 lL buffer A and centrifuged again at 14 000 g for 30 s at °C (MICROcentrifugette 4212, fixed angle rotor, ALC) After removal of the supernatant, the nuclear pellet was resuspended in 100 lL buffer B, mixed thoroughly in ice for 15 to disrupt nuclear membranes, sonicated for 10 s, and finally centrifuged at 14 000 g for 20 at °C (MICROcentrifugette 4212, fixed angle rotor, ALC) The supernatant containing the nuclear extracts was collected and the total protein content was measured [33] EMSA EMSAs were performed by the method of Singh et al [34], slightly modified Briefly, 67 ng of the specific oligonucleotide (Santa Cruz) for NF-kB, AP-1 or pCREB was mixed with lL Forward Reaction Buffer (Invitrogen, Carlsbad, CA, USA) and 0.5 lCi [c-32P]ATP (Amersham) in a final volume of 15 lL and preincubated at 37 °C for Then 10 U T4 polynucleotide kinase (Invitrogen srl, Milan, Italy) was added, and the mixture was incubated at 37 °C for 20 The radiolabelled oligonucleotide was then loaded and purified on a Sephadex G25 mini-column (Amersham), and FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS ` S Scarfı et al the elution fraction from the column centrifuged in a microfuge at 3800 g at room temperature (MICROcentrifugette 4212, fixed angle rotor, ALC) The radioactivity of the eluted fraction was quantified with a b-Counter (Beckman Coulter Inc., Fullerton, CA, USA) The binding reaction was carried out at 25 °C for 20 in a final volume of 20 lL containing 10 lg nuclear extract, 0.25 lCi purified radiolabelled oligonucleotide and 0.5 lg poly(dI)poly(dC) (Amersham) in binding buffer (25 mm Hepes, pH 7.9; 0.5 mm EDTA; 0.5 mm dithiothreitol; 5% glycerol; 50 mm NaCl; 0.5 mm phenylmethanesulfonyl fluoride) Finally, the radiolabelled protein–DNA complexes were electrophoretically resolved on a 4.5% nondenaturing polyacrylamide gel The gel was dried on a Gel Dryer 543 (Bio-Rad), and the radioactive complexes were visualized and quantified with the Packard Cyclone Storage Phosphor System and optiquant acquisition and analysis software (PerkinElmer Inc., Boston, MA, USA) Quantitative RT-PCR Total RNA from RAW 264.7 macrophages was extracted using Rnaeasy Mini Kit (Qiagen spa, Milan, Italy) and RNase-Free DNase Set (Qiagen) according to the manufacturer’s instructions, from a starting material of · 106 RAW 264.7 cells grown on 60 · 15 mm tissue culture dishes (Falcon BD) in the presence of 100 lgỈmL)1 quartz pretreated with distilled water or AA for 30 min, 60 min, h and 18 h Total cell cDNA was synthesized from lg RNA in the appropriate buffer containing mm MgCl2, 40 U ribonuclease inhibitor (RNASEOUT; Invitrogen), 10 mm dithiothreitol, and 200 U SuperscriptTM III (Invitrogen), at 50 °C for 50 Then complementary RNA was removed using lL Escherichia coli RNase H (Invitrogen) at 37 °C for 20 The amount of COX-2 mRNA, normalized to the relative GAPDH control, was determined by real-time quantitative PCR using a Chromo instrument (MJ Research, Bio-Rad) PCR was performed in a 20-lL volume in nuclease-free water containing 10 lL · master mix iQ SYBR GreenÒ (Bio-Rad), 0.2 lm each primer, and 0.5 lL cDNA or negative control All samples were analysed in triplicate The following PCR conditions were used: 10 initial denaturation, followed by 40 cycles with denaturation at 95 °C for 15 s, annealing and elongation at 60 °C for 60 s The fluorescence was measured at the end of each elongation step The next step was slow heating (1 °C per s) of the amplified product from 55 °C to 92 °C, in order to generate a melting temperature curve This curve served as a specificity control The entire cycling process, including data analysis, was monitored using the dna engine opticonÒ real-time detection system Software program (2.03 version) The sequences of the GAPDH (M32599) primers were: 5¢-TCTCCCTCACAATTTCCATCCCAG-3¢ (forward pri- Ascorbate-treated quartz enhances COX-2 expression mer) and 5¢-GGGTGCAGCGAACTTTATTGATGG-3¢ (reverse primer) The sequences of the COX-2 (M64291) primers were: 5¢CCAGCAAAGCCTAGAGCAAC-3¢ (forward primer) and (5¢-AGCACAAAACCAGGATCAGG-3¢) reverse primer To detect the PCR efficiency for each couple of primers, an amplification curve was performed, using four different dilutions of cDNA Data analysis to detect the relative gene expression of COX-2, using the cDNA from untreated cells as calibrator sample, was performed with the comparative threshold Ct method [35] via gene expression analysis software for the iCycler iQ Real Time Detection System (Bio-Rad) [36] Scavenger treatment COX-2 expression in RAW 264.7 macrophages after stimulation with 15, 50 and 100 lgỈmL)1 AA-treated or untreated quartz was also assessed in the presence of 4000 mL)1 catalase (Sigma), 50 mm mannitol (Sigma) and mm desferrioxamine (Sigma) Cells were cultured as described above, and the scavengers were added together with the quartz particles for a total incubation time of h Subsequently, cells were processed for western blot analysis, as described above Statistical analysis COX-2 expression in RAW 264.7 cells (Fig 1) In both Fig 1A and 1B, data were square-root-transformed and analysed with analysis of variance, choosing P < 0.01 as basal level of significance as described by Underwood [37] Where significant F-ratios were obtained with analysis of variance, multicomparison analyses were performed using the Tukey test PGE2 production in RAW 264.7 cells (Fig 2) In Fig 2A, data were checked for normality and homoscedasticity of variance with the Cochran test and analysed with analysis of variance Where suitable, multicomparison analyses were performed using the Tukey test or Student– Newman–Keuls test; in Fig 1B, data were log-transformed and analysed with analysis of variance Again, where suitable, multicomparison analyses were performed using the Tukey test or Student–Newman–Keuls test COX-2 expression and PGE2 production in rat alveolar macrophages (Fig 3) In Fig 3A, data were checked with the F-test and analysed with analysis of variance Multicomparison analyses were performed using Student–Newman–Keuls test; in Fig 3B, FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS 71 ` S Scarfı et al Ascorbate-treated quartz enhances COX-2 expression data were analysed by Kruskal–Wallis analysis of variance and multicomparison analysis [38] Time-course of COX-2 mRNA synthesis in RAW 264.7 macrophages (Fig 4) Data were analysed with the Scheirer–Ray–Hare test and multicomparison analysis NF-jB, pCREB and AP-1 nuclear translocation in RAW 264.7 cells (Fig 5) In Fig 5A,B, data were checked with the F-test and analysed with analysis of variance and the Tukey test; the t-test was used to check differences between untreated and AA-treated quartz samples at the same concentration In Fig 5C, data were square-root-transformed and analysed with analysis of variance and the Tukey test; the t-test was used to check differences between untreated and AA-treated quartz samples at the same concentration COX-2 expression in the presence of ROS scavengers (Fig 6) In Fig 6A–C, data were square-root-transformed and analysed with analysis of variance, choosing P < 0.01 as the basal level of significance as described by Underwood [37] Where significant F-ratios were obtained with analysis of variance, multicomparison analyses were performed using the Dunnett test Acknowledgements This work was partially supported by 2004 CIPE Regione Liguria and 2003 MURST-PRIN funds We are deeply indebted to Professor Antonio De Flora and Professor Elena Zocchi for scientific discussion and critical reading of the manuscript We acknowledge the Egenmann & Veronelli s.r.c Company (Rho, Milan, Italy) for kindly providing Aerosil OX50, and the US Silica Company (Berkeley Springs, WV, USA) for providing Min-U-Sil References Ding M, Chen F, Shi X, Yucesoy B, Mossman B & Vallyathan V (2002) Diseases caused by silica: mechanisms of injury and disease development Int Immunopharmacol 2, 173–182 Fubini B (1998) Surface chemistry and quartz hazard Ann Occup Hyg 42, 521–530 Donaldson K & Borm PJ 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Oceanogr Mar Biology 19, 513–605 38 Siegel S & Castellan NJ (1988) Nonparametric Statistics for the Behavioural Sciences, 2nd edn McGraw-Hill, New York, NY FEBS Journal 274 (2007) 60–73 ª 2006 The Authors Journal compilation ª 2006 FEBS 73 ... eventually leading to the quartz- induced chronic pulmonary disease silicosis Results Quartz- induced COX-2 expression and PGE2 production in RAW 264.7 cells The inducible enzyme COX-2 is expressed in the... et al Ascorbate-treated quartz enhances COX-2 expression Fig RT-PCR of COX-2 mRNA in quartz- treated RAW 264.7 cells COX-2 mRNA transcription was monitored in RAW 264.7 macrophages by RT-PCR analysis... enhances COX-2 expression Fig COX-2 expression in quartz- stimulated RAW 264.7 cells in the presence of ROS scavengers COX-2 was analyzed by western blot in RAW 264.7 cells after h incubation with