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Akt pathway
Food and Chemical Toxicology 50 (2012) 4003–4009
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Proinflammatory activation of macrophages by bisphenol A-glycidyl-methacrylate involved NFjB activation via PI3K/Akt pathway Yu-Hsiang Kuan a, Fu-Mei Huang b, Yi-Ching Li a, Yu-Chao Chang b,c,⇑ a
Department of Pharmacology, Chung Shan Medical University, Taichung, Taiwan Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan c School of Dentistry, Chung Shan Medical University, Taichung, Taiwan b
a r t i c l e
i n f o
Article history: Received 10 March 2012 Accepted 9 August 2012 Available online 24 August 2012 Keywords: BisGMA Macrophage NO and ROS Cytokines NFjB PI3K/Akt
a b s t r a c t Aim: Bisphenol A-glycidyl-methacrylate (BisGMA), a dental composite resin and dentin bonding agent, might prompt inflammatory effects to adjacent tissues. Macrophages are a major cellular component of the inflammatory sites. Little is known about the mechanisms of BisGMA on macrophages activation. The aim of this study was to evaluate BisGMA on proinflammatory mediators generation of murine macrophage RAW264.7 cells. Methods: IL-1b and IL-6 were analyzed by enzyme-linked immunosorbent assay. Nitric oxide, extracellular superoxide anion, and intracellular reaction oxygen species were measured by Griess assay, ferricytochrome c, and 20 ,70 -dichlorofluorescein assay, respectively. Expression of iNOS, p-p65, IjB, and p-Akt was analyzed by Western blotting. Results: BisGMA augmented the generation of IL-1b, IL-6, nitric oxide and the expression of iNOS in a time- and dose-dependent manner (p < 0.05). BisGMA enhanced the generation of intracellular and extracellular ROS in a dose-dependent manner (p < 0.05). The levels of p65 phosphorylation, IjB degradation, and Akt phosphorylation were found to be increased in a time- and dose-dependent manner (p < 0.05). Conclusions: These results indicate that BisGMA could induce nitric oxide, ROS, and inflammatory cytokines in macrophages. In addition, BisGMA may active macrophage via NF-jB activation, IjB degradation, and p-Akt activation. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction Various composite materials are frequently used for the restoration of dental cavity due to excellent adhesion to tooth surface, rapid polymerization, and esthetic appearance. Bisphenol Aglycidyl-methacrylate (BisGMA) is one of the resin-based dental composite resins and dentin bonding agents. However, BisGMA was found to leach from light-cured dental composite resins after 24 h polymerization (Yap et al., 2004). BisGMA could be rapidly incorporated from stomach and intestine and then are widely distributed anywhere in the human body (Reichl et al., 2008). The released BisGMA may induce inflammatory or immunologic responses. Recent studies have shown that BisGMA could induce cytotoxicity, genotoxicity, and inflammatory reactions in human dental pulp cells (Chang et al., 2009, 2010, 2012), lymphocytes (Drozdz et al., 2011), and macrophages (Li et al., 2012a; Kuan et al., 2012) in vitro. Despite the above evidences, ⇑ Corresponding author at: School of Dentistry, Chung Shan Medical University, 110, Sec. 1, Chien-Kuo N. Rd., Taichung, Taiwan. Tel.: +886 4 24718668x55011; fax: +886 4 24759065. E-mail address: [email protected] (Y.-C. Chang).
the mechanisms of BisGMA-induced inflammation still remain to be elucidated. In the early stages of inflammation, the functions of macrophages are to destroy pathogens, remove apoptotic neutrophils, and present antigens to T lymphocytes (Wells et al., 2005). Macrophages can initiate an immune response by the secretion of proinflammatory cytokines, such as interleukin (IL)-1b and IL-6 (Kopf et al., 2010). The production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) in macrophages is important for the killing of intracellular infectious agents. In addition, reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, and hydroxyl radical also play a crucial role against microenvironment changes (Billack, 2006). However, excess NO and ROS generation can damage cells and tissues then resulting in the development of inflammation (Billack, 2006). Nuclear factor kappa B (NF-jB) activation is an important transcription factor for the expression of iNOS and cytokines in macrophages (Kim et al., 2007). NF-jB activation is regulated by inhibitory kappa B (IjB) degradation and its upstream phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) pathway (Karin and Ben-Neriah, 2000; Park et al., 2009). Recently, we have demonstrated that BisGMA could upregulate of TNF-a and surface antigens (Kuan et al., 2012). In the present
0278-6915/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.08.019
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study, the effects of BisGMA on murine macrophage cell line RAW264.7 were determined through measuring the production of proinflammatory mediators, such as IL-1b, IL-6, NO, and ROS. Furthermore, it is interesting to know whether BisGMA may induce proinflammatory mediators therefore resulting in the upregulation of PI3K/Akt, the degradation of IjB, and the activation of NFjB.
to reduce nonspecific binding, washed with PBS containing 0.1% Tween-20 (PBST), then probed with antibodies including iNOS, p65, IjB, NFjB, Akt, b-actin, p-p65 (Ser536), and p-Akt (Ser473) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After the membranes were washed again with PBST, a 1:10,000 (v/v) dilution of horseradish peroxidase-labeled IgG (Jackson Immunoresearch, West. Grove, PA, USA) was added at room temperature for 1 h, and the blots were developed using ECL Western blotting reagents (Kuo et al., 2011).
2. Materials and methods
2.7. Statistical analysis
2.1. Cell culture of RAW264.7
At least three independent experiments were performed as indicated in the figure legends. All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by using ANOVA followed by the Bonferroni’s t-test for multigroup comparisons; p < 0.05 was considered significantly for each test.
The murine macrophage cell line RAW264.7 (Bioresource Collection and Research Center, Hsinchu, Taiwan) was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories, Grand Island, NY, USA) at 37 °C in 5% CO2-enriched air. The culture medium contained 10% heat-inactivated fetal bovine serum (FBS; Gibco Laboratories, Grand Island, NY, USA) supplemented with 100 lg/ml streptomycin and 100 U/ml penicillin (Gibco Laboratories, Grand Island, NY, USA).
3. Results 3.1. Effects of BisGMA on inflammatory cytokines secretion
2.2. Measurement of inflammatory cytokines The protein concentrations of IL-1b and IL-6 were measured by using enzymelinked immunosorbent assay (ELISA) (Li et al., 2012b). Briefly, 5 104 cells were incubated with or without 3 lM BisGMA for the indicated periods. The cells were also incubated with or without different concentrations of BisGMA for 2 h. The cytokine levels in culture medium were measured by ELISA kit (eBiosciences, Diego, CA, USA) according to the manufacturer’s instructions.
2.3. Measurement of extracellular O2 generation
The generation of extracellular O2 level was accorded by the superoxide dismutase-inhibitable reduction of ferricytochrome c (Lee et al., 2010). Briefly, 5 104 cells were incubated with 40 lM of ferricytochrome c (Sigma–Aldrich, St. Louis, MO, USA) in 24-well plates for 5 min. The reference well contained 17.5 U/ ml superoxide dismutase (Sigma–Aldrich, St. Louis, MO, USA). After the addition of various concentrations BisGMA or vehicle, the absorbance changes in the reduction of ferricytochrome c were continuously monitored at 550 nm every 1 min and were determined with a microplate reader. The amount of O2 in the reaction mix ture was calculated from the formula: O2 (nmol) = 71.55 absorbance.
The effect of BisGMA on the expression of IL-1b and IL-6 in RAW264.7 macrophages by ELISA assay was shown in Fig. 1. The secretion of IL-6 and IL-1b was demonstrated in a time-dependent manner (p < 0.05). The levels of IL-6 were 581.49, 817.39, and 1476.50 pg/ml incubated with 3 lM BisGMA for 0, 60, and 120 min, respectively. The levels of IL-1b were 185.06, 323.06, and 654.13 pg/ml incubated with 3 lM BisGMA for 0, 60, and 120 min, respectively (Fig. 1A). The secretion of IL-6 and IL-1b was also demonstrated in a concentration-dependent manner (p < 0.05). The levels of IL-6 were 611.50, 1449.38, and 1430.15 pg/ml at concentrations of 0, 1, and 3 lM BisGMA for
2.4. Measurement of intracellular ROS generation The semi-quantitative 20 ,70 -dichlorfluorescein-diacetate (DCFH-DA) fluorescence technique was used to detect the intracellular level of ROS (Cannito et al., 2008). Briefly, 2 104 cells were grown in 96-well plates for 24 h, and then incubated with 20 lM DCFH-DA for 30 min in the dark. DCFH-DA is a non-fluorescent dye easily permeating into cells and then being hydrolyzed by intracellular esterase to DCFH for storage in the cells. At the end of the DCFH-DA incubation, cells were washed with PBS. Cells were then treated with different concentrations of BisGMA for 2 h. After washing, the formation of fluorescence dichlorofluoroscein (DCF), which is the oxidized product of DCFH-DA in the presence of several ROS primarily hydroperoxide, was measured by using the fluorescence microplate reader (Molecular Devices, CA, USA) at excitation/emission wavelengths of 400/505 nm. Results were expressed as the fluorescence intensity. 2.5. Nitrite assay The nitrite concentration in the supernatant was measured as an indicator of NO production detected by Griess reaction (Chen et al., 2011). Briefly, 5 104 cells were seeded in 24-well plates for 24 h, treated with different concentrations of BisGMA or vehicle for an additional 2 h. In addition, the cells were incubated with or without 3 lM BisGMA for the indicated periods. 50 lL of culture media was mixed with an equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl-ethylenediamine dihydrochloride in distilled water; Sigma–Aldrich, St. Louis, MO, USA) and then shaken for 10 min at room temperature. The absorbance at 540 nm was determined with a microplate reader. The nitrite concentration was compared with a standard curve generated with known concentrations of sodium nitrite. 2.6. Western blotting Briefly, cells were mixed with RIPA buffer (50 mmol/L Tris–HCl pH 7.4, 150 mmol/L NaCl, 0.5% deoxycholate, 0.1% SDS, 1% Protease inhibitor cocktail, and 1% NP-40) by incubating on ice for 30 min. After centrifugation at 12,000g (4 °C, 30 min), the protein concentration in the supernatant was determined by Bradford assay. Each well was loaded with 50 lg of protein, separated by SDS–PAGE, and electrophoretically transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% (w/v) nonfat dried milk for 1 h at room temperature
Fig. 1. Effects of BisGMA on the expression of IL-1b and IL-6. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. (B) RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. Values are expressed as mean ± SD (n = 5). Represents significant difference from control values with p < 0.05.
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To investigate whether BisGMA may induce NO production via the induction of the corresponding gene expression, we determined iNOS expression by Western blot. As shown in Fig. 3C, RAW 264.7 macrophages expressed extremely low levels of iNOS protein in an un-stimulated condition. However, iNOS protein expression was demonstrated in a time-dependent manner (p < 0.05). The levels of iNOS was significantly increased about 1.36 and 1.72-fold after exposure to 3 lM BisGMA, incubated for 60 and 120 min, respectively. As shown in Fig. 3D, iNOS protein expression was also demonstrated in a concentration-dependent manner (p < 0.05). The levels of iNOS was significantly increased about 1.41 and 1.53-fold after exposure to 1 and 3 lM BisGMA for 2 h, respectively. 3.4. Effect of BisGMA on NF-jB activation To investigate BisGMA on the stimulation of NF-jB activity in RAW264.7 macrophages, we determined the phosphorylation of NF-jB p65 at Ser536 by Western blot. As shown in Fig. 4A and B, the treatment of RAW264.7 macrophages with or without BisGMA was demonstrated to augment p65 phosphorylation at Ser536 in a time- and dose-dependent manner (p < 0.05). The phosphorylation of p65 was significantly increased about 1.38, 1.94, and 1.91-fold after exposure to 3 lM BisGMA for 30, 60, and 120 min, respectively (Fig. 4B). The phosphorylation of p65 was significantly increased about 1.6 and 1.4-fold after exposure to 1 and 3 lM BisGMA for 2 h, respectively. 3.5. Effect of BisGMA on IjB degradation Fig. 2. Effects of BisGMA on (A) extracellular and (B) intracellular ROS generation. RAW264.7 cells were stimulated with the indicated concentrations of BisGMA. Values are expressed as mean ± SD (n = 5). Represents significant difference from control values with p < 0.05.
2 h, respectively. The levels of IL-1b were 177.62, 634.19, and 724.67 pg/ml at concentrations of 0, 1, and 3 lM BisGMA for 2 h, respectively (Fig. 1B). 3.2. Effects of BisGMA on intracellular and extracellular ROS generation As shown in Fig. 2, the exposure of RAW264.7 macrophages to BisGMA evoked both extracellular O2 and intracellular ROS generation in a concentration-dependent manner (p < 0.05) by ferricytochrome c and DCFH-DA, respectively. The concentration of BisGMA higher than 1 lM significantly upregulated the intracellular and extracellular ROS generation (p < 0.05). The levels of extracellular O2 were 0.21, 1.02, and 2.18 nmol at the concentrations of 0, 1, and 3 lM BisGMA, respectively. The levels of intracellular ROS were 233.62, 450.46, and 660.34 RFU at the concentrations of 0, 1, and 3 lM BisGMA, respectively. 3.3. Effects of BisGMA on NO production and iNOS expression To evaluate the effects of BisGMA on NO production in RAW 264.7 macrophages, nitrite accumulation was examined by the Griess assay. As shown in Fig. 3A, BisGMA was found to induce nitrite generation in a time-dependent manner (p < 0.05). The levels of NO were 5.57, 6.29, and 7.16 lM incubated with 3 lM BisGMA for 0, 60, 120 min, respectively. After treatment with BisGMA for 2 h, BisGMA was found to induce nitrite generation in a concentrationdependent manner (p < 0.05) (Fig. 3B). The levels of NO were 5.81, 6.27, and 7.07 lM at the concentrations of 0, 1, and 3 lM BisGMA, respectively.
To clarify the promoting mechanism of BisGMA for NF-jB, phosphorylation of IjB degradation was also examined. As shown in Fig. 5A and B, the degradation of IjB by BisGMA was increased in a time- and dose-dependent manner (p < 0.05). The degradation of IjB was significantly increased about 0.79, 0.34, and 0.38-fold after exposure to 3 lM BisGMA for 30, 60, and 120 min, respectively (Fig. 5A). The degradation of IjB was significantly increased about 0.51, 0.36-fold after exposure to 1 and 3 lM BisGMA for 2 h, respectively (Fig. 5B). 3.6. Effect of BisGMA on Akt phosphorylation Akt is an upstream factor for NFjB activation. Therefore, the effect of BisGMA on Akt phosphorylation at Ser473 was assessed by Western blot. As shown in Fig. 6A and B, the addition of BisGMA was demonstrated to upregulate Akt phosphorylation in a timeand dose-dependent manner (p < 0.05). The amount of p-Akt was elevated about 2.97 and 2.55-fold after exposure to 3 lM BisGMA for 5 and 15 min, respectively (Fig. 6A). The amount of p-Akt was elevated about 1.99 and 2.94-fold after exposure to 1 and 3 lM BisGMA for 2 h, respectively (Fig. 6B). 3.7. Effects of LY294002 on BisGMA-induced NO, IL-1b, and IL-6 generation To address the relevance of the PI3K signal pathway activated by BisGMA, PI3K inhibitor LY294002 was added to evaluate the effect of NO, IL-1b, and IL-6 generation by BisGMA. As shown in Fig. 7 and 20 lM LY294002 was found to significantly downregulate the BisGMA-induced NO, IL-1b, and IL-6 generation (p < 0.05). 4. Discussion Dental resin materials release residual monomers and other components into the oral environment even after polymerization.
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Fig. 3. Effects of BisGMA on NO generation and iNOS expression. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. The supernatant was harvested and detected by Griess reaction. Values are expressed as mean ± SD (n = 5). Represents significant difference from control values with p < 0.05. (C) Cells were harvested and protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against iNOS and b-actin. RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. (B) The supernatant was harvested and detected by Griess reaction. Values are expressed as mean ± SD (n = 5). Represents significant difference from control values with p < 0.05. (D) Cells were harvested and protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against iNOS and b-actin.
Fig. 4. Effect of BisGMA on p65 phosphorylation. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. (B) RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. Cells were harvested and protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against phosphorylated and total p65. The ratio of immunointensity between the phosphorylation and total protein was calculated. Values are expressed as means ± SD (n = 3 in each group). ⁄p < 0.05 versus control group.
Previously, Kim and Chung (2005) have reported that BisGMA could be eluted form composite based dental resins at the concentration of 175 lM. Moreover, 5.8 lM of unbound monomers was detected in water. Tabatabaee et al. (2009) have showed that BisGMA could be eluted from light cured resin at the concentrations from 0.98 to 9.75 nM. However, it is difficult, if not impossible, to determine how much BisGMA acts on immune cells clinically. Lower concentrations of BisGMA might be more suitable to elucidate BisGMA-induced inflammatory reactions. Recently, our studies Li et al. (2012a) and Kuan et al. (2012) demonstrated that the
concentration level of 3 lM BisGMA could easily reach the effective cytotoxic and genotoxic levels to cause adverse effects. Therefore, the concentrations of BisGMA lower than 3 lM were chosen for the activation of macrophages in this study. Inflammation is a complex process mediated by proinflammatory cytokines and free radicals produced by macrophages. The release of IL-1b and IL-6 can active macrophages during infection, injury, and inflammation (Kim et al., 2007; Li et al., 2012a). In this study, BisGMA was found to induce proinflammatory cytokines IL-1b and IL-6 in RAW264.7 macrophages. Similar results were
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Fig. 5. Effect of BisGMA on IjB degradation. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. Cells were harvested and 100 lg protein extracts were subjected to SDS–PAGE. (B) RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. Cells were harvested and 50 lg protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against IjB and b-actin. The ratio of immunointensity between the IjB and b-actin was calculated. Values are expressed as means ± S.D. (n = 3 in each group). ⁄p < 0.05 versus control group.
Fig. 6. Effect of BisGMA on Akt phosphorylation. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. (B) RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. Cells were harvested and protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against phosphorylated and total Akt. The ratio of immunointensity between the phosphorylation and total protein was calculated. Values are expressed as means ± S.D. (n = 3 in each group). ⁄p < 0.05 versus control group.
reported by Bailey et al. (2006) who found the upregulation of IL1b by BisGMA in macrophages. Consistently, one of our recent reports demonstrated that TNF-a was augmented by BisGMA in RAW264.7 macrophages (Kuan et al., 2012). Taken together, BisGMA could activate various proinflammatory cytokines in macrophages. Dysregulated production of these proinflammatory cytokines may lead to an excessive local amplification of immune response which may be partly responsible for tissue destruction. One important function of activated macrophages is to kill foreign invasive bacteria through the secretion of ROS and NO (Billack, 2006). However, overexpression of ROS and NO can cause various illnesses by injuring surrounding cells resulting in acute or chronic inflammatory diseases (Johar et al., 2004). The ROS include O2, hydroxyl radical, and hydrogen peroxide. O2, the main species of ROS and the precursor of other ROS, is generated by oxygen consumption through the activity of nicotinamide adenine dinucleotide phosphate oxidase (Roberts et al., 2010). In this study, the exposure of RAW264.7 macrophages to BisGMA could evoke both extracellular O2 and intracellular ROS generation by ferricytochrome c and DCFH-DA, respectively. Recently, BisGMA was demonstrated to generate cellular ROS by labeling with DCFH-DA in human pulp
cells (Chang et al., 2009, 2010). Consistently, BisGMA could decrease intracellular glutathione in human gingival fibroblasts (Engelmann et al., 2004). NO is generated by iNOS conversion of L-arginine to L-citrulline (Wink and Mitchell, 1998). To the best of our knowledge, we first found that BisGMA could stimulate NO secretion and iNOS expression in RAW264.7 macrophages. Consistently, dental adhesive systems were found to produce NO generation in primary murine macrophages (Porto et al., 2011). Taken together, macrophage activated by BisGMA may lead to generate ROS/NO and result in inflammatory reaction. Expression of iNOS, IL-1b, and IL-6 in macrophages is regulated by NF-jB activation (Kim et al., 2007; Baeuerle and Henkel, 1994). NF-jB is composed of homo- and hetero-dimers of a family of proteins which include p65, p50, p52, c-Rel, p65 (RelA), and RelB (Karin and Ben-Neriah, 2000). The p65 subunit is a major component associated with transcription activation. In un-stimulated state, p65 is presented in the cytoplasm and complexed with IjB. After stimulation, phosphorylation and translocation to the nucleus of p65 is caused by IjB degradation (Karin and Ben-Neriah, 2000). In addition, the phosphorylation of p65 at Ser536 following LPS stimulation could raise the p65 transcriptional activity (Hsieh
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kinase and a major downstream factor of PI3K, can promote IjB kinase phosphorylation (Ozes et al., 1999) and then result in IjB degradation (Park et al., 2009). The treatment of RAW264.7 macrophages with BisGMA was found to induce Akt phosphorylation. Taken together, it is imply that NF-jB activation by BisGMA may be via PI3K/Akt pathway. In the present study, we demonstrated that the activation of RAW264.7 macrophages by BisGMA may involve NFjB via PI3K/ Akt pathway (Fig. 8). BisGMA was demonstrated to significantly upregulate the secretion of IL-1b and IL-6, the expression of iNOS and NO, the activation of NF-jB, the degradation of IjB, and the phosphorylation of Akt. Further studies on the other signaling pathways responsible for the induction of ROS, cytokines, and NO generation by BisGMA should be done to further punctuate our comprehension of the macrophages response to inflammation of dental composites. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This study was supported by NSC 100-2314-B-040-006. Microplate reader was performed in the Instrument Center of Chung Shan Medical University, which is supported by National Science Council, Ministry of Education and Chung Shan Medical University. References
Fig. 7. Effects of LY294002 on BisGMA-induced NO, IL-1b, and IL-6 generation. RAW264.7 cells were pretreated with or without 20 lM LY294002 for 30 min before stimulated the 3 lM BisGMA for 2 h. The supernatant was harvested and detected by Griess reaction and ELISA assay. Values are expressed as mean ± SD (n = 5). represents significant difference from control values with p < 0.05. #represents significant difference from BisGMA values with p < 0.05.
et al., 2011). After exposure to BisGMA, RAW264.7 macrophages trigger p65 phosphorylation at Ser536 and IjB degradation. These results indicated that BisGMA could induce iNOS, IL-1b, and IL-6 generation via NFjB activation. On the basis of our results, BisGMA could induce NF-jB activation via IjB degradation. The investigation of upstream signaling pathway is worth further assessment. Akt, a serine/threonine
Fig. 8. Schematic diagram illustrating the signaling pathways involved in BisGMAinduced inflammation associated proteins in RAW264.7 macrophages.
Baeuerle, P.A., Henkel, T., 1994. Function and activation of NF-jB in the immune system. Annu. Rev. Immunol. 12, 141–179. Bailey, L.Q., Weir, M.D., Washburn, N.R., 2006. Quantification of macrophage viability and inflammatory response to dental bonding resins. J. Bioact. Compat. Pol. 21, 185–206. Billack, B., 2006. Macrophage activation: role of toll-like receptors, nitric oxide, and nuclear factor kappa B. Am. J. Pharm. Educ. 70, 102. Cannito, S., Novo, E., Compagnone, A., Valfrè di Bonzo, L., Busletta, C., Zamara, E., et al., 2008. Redox mechanisms switch on hypoxia-dependent epithelialmesenchymal transition in cancer cells. Carcinogenesis 29, 2267–2278. Chang, M.C., Chen, L.I., Chan, C.P., Lee, J.J., Wang, T.M., Yang, T.T., et al., 2010. The role of reactive oxygen species and hemeoxygenase-1 expression in the cytotoxicity, cell cycle alteration and apoptosis of dental pulp cells induced by BisGMA. Biomaterials 31, 8164–8171. Chang, M.C., Lin, L.D., Chan, C.P., Chang, H.H., Chen, L.I., Lin, H.J., et al., 2009. The effect of BisGMA on cyclooxygenase-2 expression, PGE2 production and cytotoxicity via reactive oxygen species- and MEK/ERK-dependent and independent pathways. Biomaterials 30, 4070–4077. Chang, M.C., Lin, L.D., Chuang, F.H., Chan, C.P., Wang, T.M., Lee, J.J., et al., 2012. Carboxylesterase expression in human dental pulp cells: role in regulation of BisGMA-induced prostanoid production and cytotoxicity. Acta Biomater. 8, 1380–1387. Chen, C.J., Ou, Y.C., Chang, C.Y., Pan, H.C., Liao, S.L., Raung, S.L., et al., 2011. TNF-a and IL-1b mediate Japanese encephalitis virus-induced RANTES gene expression in astrocytes. Neurochem. Int. 58, 234–242. Drozdz, K., Wysokinski, D., Krupa, R., Wozniak, K., 2011. Bisphenol A-glycidyl methacrylate induces a broad spectrum of DNA damage in human lymphocytes. Arch. Toxicol. 85, 1453–1461. Engelmann, J., Janke, V., Volk, J., Leyhausen, G., von Neuhoff, N., Schlegelberger, B., et al., 2004. Effects of BisGMA on glutathione metabolism and apoptosis in human gingival fibroblasts in vitro. Biomaterials 25, 4573–4580. Hsieh, C.Y., Hsu, M.J., Hsiao, G., Wang, Y.H., Huang, C.W., Chen, S.W., et al., 2011. Andrographolide enhances nuclear factor-kappaB subunit p65 Ser536 dephosphorylation through activation of protein phosphatase 2A in vascular smooth muscle cells. J. Biol. Chem. 286, 5942–5955. Johar, D., Roth, J.C., Bay, G.H., Walker, J.N., Kroczak, T.J., Los, M., 2004. Inflammatory response, reactive oxygen species, programmed (necrotic-like and apoptotic) cell death and cancer. Rocz. Akad. Med. Bialymst. 49, 31–39. Karin, M., Ben-Neriah, Y., 2000. Phosphorylation meets ubiquitination: the control of NF-jB activity. Annu. Rev. Immunol. 18, 621–663. Kim, J.B., Han, A.R., Park, E.Y., Kim, J.Y., Cho, W., Lee, J., et al., 2007. Inhibition of LPSinduced iNOS, COX-2 and cytokines expression by poncirin through the NFkappa B inactivation in RAW264.7 macrophage cells. Biol. Pharm. Bull. 30, 2345–2351. Kim, J.G., Chung, C.M., 2005. Elution from light-cured dental composites: comparison of trimethacrylate and dimethacrylate as base monomers. J. Biomed. Mater. Res. B Appl. Biomater. 72, 328–333.
Y.-H. Kuan et al. / Food and Chemical Toxicology 50 (2012) 4003–4009 Kopf, M., Bachmann, M.F., Marsland, B.J., 2010. Averting inflammation by targeting the cytokine environment. Nat. Rev. Drug Discov. 9, 703–718. Kuan, Y.H., Li, Y.C., Huang, F.M., Chang, Y.C., 2012. The upregulation of tumour necrosis factor-a and surface antigens expression on macrophages by bisphenol A- glycidyl-methacrylate. Int. Endod. J. 45, 619–626. Kuo, M.Y., Liao, M.F., Chen, F.L., Li, Y.C., Yang, M.L., Lin, R.H., et al., 2011. Luteolin attenuates the pulmonary inflammatory response involves abilities of antioxidation and inhibition of MAPK and NFjB pathways in mice with endotoxin-induced acute lung injury. Food Chem. Toxicol. 49, 2660–2666. Lee, J.P., Li, Y.C., Chen, H.Y., Lin, R.H., Huang, S.S., Chen, H.L., et al., 2010. Protective effects of luteolin against lipopolysaccharide-induced acute lung injury involves inhibition of MEK/ERK and PI3K/Akt pathways in neutrophils. Acta Pharmacol. Sin. 31, 831–838. Li, Y.C., Kuan, Y.H., Huang, F.M., Chang, Y.C., 2012a. The role of DNA damage and caspase activation in cytotoxicity and genotoxicity of macrophages induced by bisphenol-A-glycidyldimethacrylate. Int. Endodon. J. 45, 499–507. Li, Y.C., Yeh, C.H., Yang, M.L., Kuan, Y.H., 2012b. Luteolin suppresses inflammatory mediator expression by blocking the Akt/NFjB pathway in acute lung injury induced by lipopolysaccharide in mice. Evid. Based Complement Alternat. Med. 2012, 383608. Ozes, O.N., Mayo, L.D., Gustin, J.A., Pfeffer, S.R., Pfeffer, L.M., Donner, D.B., 1999. NFjB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401, 82–85.
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Park, S.Y., Park, G.Y., Ko, W.S., Kim, Y., 2009. Dichroa febrifuga Lour. inhibits the production of IL-1beta and IL-6 through blocking NF-kappaB, MAPK and Akt activation in macrophages. J. Ethnopharmacol. 125, 246–251. Porto, I.C., Oliveira, D.C., Raele, R.A., Ribas, K.H., Montes, M.A., De Castro, C.M., 2011. Cytotoxicity of current adhesive systems: in vitro testing on cell cultures of primary murine macrophages. Dent. Mater. 27, 221–228. Reichl, F.X., Seiss, M., Kleinsasser, N., Kehe, K., Kunzelmann, K.H., Thomas, P., Spahl, W., Hickel, R., 2008. Distribution and excretion of BisGMA in guinea pigs. J. Dent. Res. 87, 378–380. Roberts, R.A., Smith, R.A., Safe, S., Szabo, C., Tjalkens, R.B., Robertson, F.M., 2010. Toxicological and pathophysiological roles of reactive oxygen and nitrogen species. Toxicology 276, 85–94. Tabatabaee, M.H., Mahdavi, H., Zandi, S., Kharrazi, M.J., 2009. HPLC analysis of eluted monomers from two composite resins cured with LED and Halogen curing lights. J. Biomed. Mater. Res. B Appl. Biomater. 88B, 191–196. Wells, C.A., Ravasi, T., Hume, D.A., 2005. Inflammation suppressor genes: please switch out all the lights. J. Leukoc. Biol. 78, 9–13. Wink, D.A., Mitchell, J.B., 1998. Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med. 25, 434–456. Yap, A.U., Han, V.T., Soh, M.S., Siow, K.S., 2004. Elution of leachable components from composites after LED and halogen light irradiation. Oper. Dent. 29, 448– 453.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Proinflammatory activation of macrophages by bisphenol A-glycidyl-methacrylate involved NFjB activation via PI3K/Akt pathway Yu-Hsiang Kuan a, Fu-Mei Huang b, Yi-Ching Li a, Yu-Chao Chang b,c,⇑ a
Department of Pharmacology, Chung Shan Medical University, Taichung, Taiwan Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan c School of Dentistry, Chung Shan Medical University, Taichung, Taiwan b
a r t i c l e
i n f o
Article history: Received 10 March 2012 Accepted 9 August 2012 Available online 24 August 2012 Keywords: BisGMA Macrophage NO and ROS Cytokines NFjB PI3K/Akt
a b s t r a c t Aim: Bisphenol A-glycidyl-methacrylate (BisGMA), a dental composite resin and dentin bonding agent, might prompt inflammatory effects to adjacent tissues. Macrophages are a major cellular component of the inflammatory sites. Little is known about the mechanisms of BisGMA on macrophages activation. The aim of this study was to evaluate BisGMA on proinflammatory mediators generation of murine macrophage RAW264.7 cells. Methods: IL-1b and IL-6 were analyzed by enzyme-linked immunosorbent assay. Nitric oxide, extracellular superoxide anion, and intracellular reaction oxygen species were measured by Griess assay, ferricytochrome c, and 20 ,70 -dichlorofluorescein assay, respectively. Expression of iNOS, p-p65, IjB, and p-Akt was analyzed by Western blotting. Results: BisGMA augmented the generation of IL-1b, IL-6, nitric oxide and the expression of iNOS in a time- and dose-dependent manner (p < 0.05). BisGMA enhanced the generation of intracellular and extracellular ROS in a dose-dependent manner (p < 0.05). The levels of p65 phosphorylation, IjB degradation, and Akt phosphorylation were found to be increased in a time- and dose-dependent manner (p < 0.05). Conclusions: These results indicate that BisGMA could induce nitric oxide, ROS, and inflammatory cytokines in macrophages. In addition, BisGMA may active macrophage via NF-jB activation, IjB degradation, and p-Akt activation. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction Various composite materials are frequently used for the restoration of dental cavity due to excellent adhesion to tooth surface, rapid polymerization, and esthetic appearance. Bisphenol Aglycidyl-methacrylate (BisGMA) is one of the resin-based dental composite resins and dentin bonding agents. However, BisGMA was found to leach from light-cured dental composite resins after 24 h polymerization (Yap et al., 2004). BisGMA could be rapidly incorporated from stomach and intestine and then are widely distributed anywhere in the human body (Reichl et al., 2008). The released BisGMA may induce inflammatory or immunologic responses. Recent studies have shown that BisGMA could induce cytotoxicity, genotoxicity, and inflammatory reactions in human dental pulp cells (Chang et al., 2009, 2010, 2012), lymphocytes (Drozdz et al., 2011), and macrophages (Li et al., 2012a; Kuan et al., 2012) in vitro. Despite the above evidences, ⇑ Corresponding author at: School of Dentistry, Chung Shan Medical University, 110, Sec. 1, Chien-Kuo N. Rd., Taichung, Taiwan. Tel.: +886 4 24718668x55011; fax: +886 4 24759065. E-mail address: [email protected] (Y.-C. Chang).
the mechanisms of BisGMA-induced inflammation still remain to be elucidated. In the early stages of inflammation, the functions of macrophages are to destroy pathogens, remove apoptotic neutrophils, and present antigens to T lymphocytes (Wells et al., 2005). Macrophages can initiate an immune response by the secretion of proinflammatory cytokines, such as interleukin (IL)-1b and IL-6 (Kopf et al., 2010). The production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) in macrophages is important for the killing of intracellular infectious agents. In addition, reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, and hydroxyl radical also play a crucial role against microenvironment changes (Billack, 2006). However, excess NO and ROS generation can damage cells and tissues then resulting in the development of inflammation (Billack, 2006). Nuclear factor kappa B (NF-jB) activation is an important transcription factor for the expression of iNOS and cytokines in macrophages (Kim et al., 2007). NF-jB activation is regulated by inhibitory kappa B (IjB) degradation and its upstream phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) pathway (Karin and Ben-Neriah, 2000; Park et al., 2009). Recently, we have demonstrated that BisGMA could upregulate of TNF-a and surface antigens (Kuan et al., 2012). In the present
0278-6915/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.08.019
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study, the effects of BisGMA on murine macrophage cell line RAW264.7 were determined through measuring the production of proinflammatory mediators, such as IL-1b, IL-6, NO, and ROS. Furthermore, it is interesting to know whether BisGMA may induce proinflammatory mediators therefore resulting in the upregulation of PI3K/Akt, the degradation of IjB, and the activation of NFjB.
to reduce nonspecific binding, washed with PBS containing 0.1% Tween-20 (PBST), then probed with antibodies including iNOS, p65, IjB, NFjB, Akt, b-actin, p-p65 (Ser536), and p-Akt (Ser473) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After the membranes were washed again with PBST, a 1:10,000 (v/v) dilution of horseradish peroxidase-labeled IgG (Jackson Immunoresearch, West. Grove, PA, USA) was added at room temperature for 1 h, and the blots were developed using ECL Western blotting reagents (Kuo et al., 2011).
2. Materials and methods
2.7. Statistical analysis
2.1. Cell culture of RAW264.7
At least three independent experiments were performed as indicated in the figure legends. All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by using ANOVA followed by the Bonferroni’s t-test for multigroup comparisons; p < 0.05 was considered significantly for each test.
The murine macrophage cell line RAW264.7 (Bioresource Collection and Research Center, Hsinchu, Taiwan) was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories, Grand Island, NY, USA) at 37 °C in 5% CO2-enriched air. The culture medium contained 10% heat-inactivated fetal bovine serum (FBS; Gibco Laboratories, Grand Island, NY, USA) supplemented with 100 lg/ml streptomycin and 100 U/ml penicillin (Gibco Laboratories, Grand Island, NY, USA).
3. Results 3.1. Effects of BisGMA on inflammatory cytokines secretion
2.2. Measurement of inflammatory cytokines The protein concentrations of IL-1b and IL-6 were measured by using enzymelinked immunosorbent assay (ELISA) (Li et al., 2012b). Briefly, 5 104 cells were incubated with or without 3 lM BisGMA for the indicated periods. The cells were also incubated with or without different concentrations of BisGMA for 2 h. The cytokine levels in culture medium were measured by ELISA kit (eBiosciences, Diego, CA, USA) according to the manufacturer’s instructions.
2.3. Measurement of extracellular O2 generation
The generation of extracellular O2 level was accorded by the superoxide dismutase-inhibitable reduction of ferricytochrome c (Lee et al., 2010). Briefly, 5 104 cells were incubated with 40 lM of ferricytochrome c (Sigma–Aldrich, St. Louis, MO, USA) in 24-well plates for 5 min. The reference well contained 17.5 U/ ml superoxide dismutase (Sigma–Aldrich, St. Louis, MO, USA). After the addition of various concentrations BisGMA or vehicle, the absorbance changes in the reduction of ferricytochrome c were continuously monitored at 550 nm every 1 min and were determined with a microplate reader. The amount of O2 in the reaction mix ture was calculated from the formula: O2 (nmol) = 71.55 absorbance.
The effect of BisGMA on the expression of IL-1b and IL-6 in RAW264.7 macrophages by ELISA assay was shown in Fig. 1. The secretion of IL-6 and IL-1b was demonstrated in a time-dependent manner (p < 0.05). The levels of IL-6 were 581.49, 817.39, and 1476.50 pg/ml incubated with 3 lM BisGMA for 0, 60, and 120 min, respectively. The levels of IL-1b were 185.06, 323.06, and 654.13 pg/ml incubated with 3 lM BisGMA for 0, 60, and 120 min, respectively (Fig. 1A). The secretion of IL-6 and IL-1b was also demonstrated in a concentration-dependent manner (p < 0.05). The levels of IL-6 were 611.50, 1449.38, and 1430.15 pg/ml at concentrations of 0, 1, and 3 lM BisGMA for
2.4. Measurement of intracellular ROS generation The semi-quantitative 20 ,70 -dichlorfluorescein-diacetate (DCFH-DA) fluorescence technique was used to detect the intracellular level of ROS (Cannito et al., 2008). Briefly, 2 104 cells were grown in 96-well plates for 24 h, and then incubated with 20 lM DCFH-DA for 30 min in the dark. DCFH-DA is a non-fluorescent dye easily permeating into cells and then being hydrolyzed by intracellular esterase to DCFH for storage in the cells. At the end of the DCFH-DA incubation, cells were washed with PBS. Cells were then treated with different concentrations of BisGMA for 2 h. After washing, the formation of fluorescence dichlorofluoroscein (DCF), which is the oxidized product of DCFH-DA in the presence of several ROS primarily hydroperoxide, was measured by using the fluorescence microplate reader (Molecular Devices, CA, USA) at excitation/emission wavelengths of 400/505 nm. Results were expressed as the fluorescence intensity. 2.5. Nitrite assay The nitrite concentration in the supernatant was measured as an indicator of NO production detected by Griess reaction (Chen et al., 2011). Briefly, 5 104 cells were seeded in 24-well plates for 24 h, treated with different concentrations of BisGMA or vehicle for an additional 2 h. In addition, the cells were incubated with or without 3 lM BisGMA for the indicated periods. 50 lL of culture media was mixed with an equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl-ethylenediamine dihydrochloride in distilled water; Sigma–Aldrich, St. Louis, MO, USA) and then shaken for 10 min at room temperature. The absorbance at 540 nm was determined with a microplate reader. The nitrite concentration was compared with a standard curve generated with known concentrations of sodium nitrite. 2.6. Western blotting Briefly, cells were mixed with RIPA buffer (50 mmol/L Tris–HCl pH 7.4, 150 mmol/L NaCl, 0.5% deoxycholate, 0.1% SDS, 1% Protease inhibitor cocktail, and 1% NP-40) by incubating on ice for 30 min. After centrifugation at 12,000g (4 °C, 30 min), the protein concentration in the supernatant was determined by Bradford assay. Each well was loaded with 50 lg of protein, separated by SDS–PAGE, and electrophoretically transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% (w/v) nonfat dried milk for 1 h at room temperature
Fig. 1. Effects of BisGMA on the expression of IL-1b and IL-6. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. (B) RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. Values are expressed as mean ± SD (n = 5). Represents significant difference from control values with p < 0.05.
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To investigate whether BisGMA may induce NO production via the induction of the corresponding gene expression, we determined iNOS expression by Western blot. As shown in Fig. 3C, RAW 264.7 macrophages expressed extremely low levels of iNOS protein in an un-stimulated condition. However, iNOS protein expression was demonstrated in a time-dependent manner (p < 0.05). The levels of iNOS was significantly increased about 1.36 and 1.72-fold after exposure to 3 lM BisGMA, incubated for 60 and 120 min, respectively. As shown in Fig. 3D, iNOS protein expression was also demonstrated in a concentration-dependent manner (p < 0.05). The levels of iNOS was significantly increased about 1.41 and 1.53-fold after exposure to 1 and 3 lM BisGMA for 2 h, respectively. 3.4. Effect of BisGMA on NF-jB activation To investigate BisGMA on the stimulation of NF-jB activity in RAW264.7 macrophages, we determined the phosphorylation of NF-jB p65 at Ser536 by Western blot. As shown in Fig. 4A and B, the treatment of RAW264.7 macrophages with or without BisGMA was demonstrated to augment p65 phosphorylation at Ser536 in a time- and dose-dependent manner (p < 0.05). The phosphorylation of p65 was significantly increased about 1.38, 1.94, and 1.91-fold after exposure to 3 lM BisGMA for 30, 60, and 120 min, respectively (Fig. 4B). The phosphorylation of p65 was significantly increased about 1.6 and 1.4-fold after exposure to 1 and 3 lM BisGMA for 2 h, respectively. 3.5. Effect of BisGMA on IjB degradation Fig. 2. Effects of BisGMA on (A) extracellular and (B) intracellular ROS generation. RAW264.7 cells were stimulated with the indicated concentrations of BisGMA. Values are expressed as mean ± SD (n = 5). Represents significant difference from control values with p < 0.05.
2 h, respectively. The levels of IL-1b were 177.62, 634.19, and 724.67 pg/ml at concentrations of 0, 1, and 3 lM BisGMA for 2 h, respectively (Fig. 1B). 3.2. Effects of BisGMA on intracellular and extracellular ROS generation As shown in Fig. 2, the exposure of RAW264.7 macrophages to BisGMA evoked both extracellular O2 and intracellular ROS generation in a concentration-dependent manner (p < 0.05) by ferricytochrome c and DCFH-DA, respectively. The concentration of BisGMA higher than 1 lM significantly upregulated the intracellular and extracellular ROS generation (p < 0.05). The levels of extracellular O2 were 0.21, 1.02, and 2.18 nmol at the concentrations of 0, 1, and 3 lM BisGMA, respectively. The levels of intracellular ROS were 233.62, 450.46, and 660.34 RFU at the concentrations of 0, 1, and 3 lM BisGMA, respectively. 3.3. Effects of BisGMA on NO production and iNOS expression To evaluate the effects of BisGMA on NO production in RAW 264.7 macrophages, nitrite accumulation was examined by the Griess assay. As shown in Fig. 3A, BisGMA was found to induce nitrite generation in a time-dependent manner (p < 0.05). The levels of NO were 5.57, 6.29, and 7.16 lM incubated with 3 lM BisGMA for 0, 60, 120 min, respectively. After treatment with BisGMA for 2 h, BisGMA was found to induce nitrite generation in a concentrationdependent manner (p < 0.05) (Fig. 3B). The levels of NO were 5.81, 6.27, and 7.07 lM at the concentrations of 0, 1, and 3 lM BisGMA, respectively.
To clarify the promoting mechanism of BisGMA for NF-jB, phosphorylation of IjB degradation was also examined. As shown in Fig. 5A and B, the degradation of IjB by BisGMA was increased in a time- and dose-dependent manner (p < 0.05). The degradation of IjB was significantly increased about 0.79, 0.34, and 0.38-fold after exposure to 3 lM BisGMA for 30, 60, and 120 min, respectively (Fig. 5A). The degradation of IjB was significantly increased about 0.51, 0.36-fold after exposure to 1 and 3 lM BisGMA for 2 h, respectively (Fig. 5B). 3.6. Effect of BisGMA on Akt phosphorylation Akt is an upstream factor for NFjB activation. Therefore, the effect of BisGMA on Akt phosphorylation at Ser473 was assessed by Western blot. As shown in Fig. 6A and B, the addition of BisGMA was demonstrated to upregulate Akt phosphorylation in a timeand dose-dependent manner (p < 0.05). The amount of p-Akt was elevated about 2.97 and 2.55-fold after exposure to 3 lM BisGMA for 5 and 15 min, respectively (Fig. 6A). The amount of p-Akt was elevated about 1.99 and 2.94-fold after exposure to 1 and 3 lM BisGMA for 2 h, respectively (Fig. 6B). 3.7. Effects of LY294002 on BisGMA-induced NO, IL-1b, and IL-6 generation To address the relevance of the PI3K signal pathway activated by BisGMA, PI3K inhibitor LY294002 was added to evaluate the effect of NO, IL-1b, and IL-6 generation by BisGMA. As shown in Fig. 7 and 20 lM LY294002 was found to significantly downregulate the BisGMA-induced NO, IL-1b, and IL-6 generation (p < 0.05). 4. Discussion Dental resin materials release residual monomers and other components into the oral environment even after polymerization.
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Fig. 3. Effects of BisGMA on NO generation and iNOS expression. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. The supernatant was harvested and detected by Griess reaction. Values are expressed as mean ± SD (n = 5). Represents significant difference from control values with p < 0.05. (C) Cells were harvested and protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against iNOS and b-actin. RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. (B) The supernatant was harvested and detected by Griess reaction. Values are expressed as mean ± SD (n = 5). Represents significant difference from control values with p < 0.05. (D) Cells were harvested and protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against iNOS and b-actin.
Fig. 4. Effect of BisGMA on p65 phosphorylation. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. (B) RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. Cells were harvested and protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against phosphorylated and total p65. The ratio of immunointensity between the phosphorylation and total protein was calculated. Values are expressed as means ± SD (n = 3 in each group). ⁄p < 0.05 versus control group.
Previously, Kim and Chung (2005) have reported that BisGMA could be eluted form composite based dental resins at the concentration of 175 lM. Moreover, 5.8 lM of unbound monomers was detected in water. Tabatabaee et al. (2009) have showed that BisGMA could be eluted from light cured resin at the concentrations from 0.98 to 9.75 nM. However, it is difficult, if not impossible, to determine how much BisGMA acts on immune cells clinically. Lower concentrations of BisGMA might be more suitable to elucidate BisGMA-induced inflammatory reactions. Recently, our studies Li et al. (2012a) and Kuan et al. (2012) demonstrated that the
concentration level of 3 lM BisGMA could easily reach the effective cytotoxic and genotoxic levels to cause adverse effects. Therefore, the concentrations of BisGMA lower than 3 lM were chosen for the activation of macrophages in this study. Inflammation is a complex process mediated by proinflammatory cytokines and free radicals produced by macrophages. The release of IL-1b and IL-6 can active macrophages during infection, injury, and inflammation (Kim et al., 2007; Li et al., 2012a). In this study, BisGMA was found to induce proinflammatory cytokines IL-1b and IL-6 in RAW264.7 macrophages. Similar results were
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Fig. 5. Effect of BisGMA on IjB degradation. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. Cells were harvested and 100 lg protein extracts were subjected to SDS–PAGE. (B) RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. Cells were harvested and 50 lg protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against IjB and b-actin. The ratio of immunointensity between the IjB and b-actin was calculated. Values are expressed as means ± S.D. (n = 3 in each group). ⁄p < 0.05 versus control group.
Fig. 6. Effect of BisGMA on Akt phosphorylation. (A) RAW264.7 cells were stimulated with 3 lM BisGMA for the indicated treatment periods. (B) RAW264.7 cells were stimulated with the indicated concentrations of BisGMA for 2 h. Cells were harvested and protein extracts were subjected to SDS–PAGE Western blot analysis using antibodies against phosphorylated and total Akt. The ratio of immunointensity between the phosphorylation and total protein was calculated. Values are expressed as means ± S.D. (n = 3 in each group). ⁄p < 0.05 versus control group.
reported by Bailey et al. (2006) who found the upregulation of IL1b by BisGMA in macrophages. Consistently, one of our recent reports demonstrated that TNF-a was augmented by BisGMA in RAW264.7 macrophages (Kuan et al., 2012). Taken together, BisGMA could activate various proinflammatory cytokines in macrophages. Dysregulated production of these proinflammatory cytokines may lead to an excessive local amplification of immune response which may be partly responsible for tissue destruction. One important function of activated macrophages is to kill foreign invasive bacteria through the secretion of ROS and NO (Billack, 2006). However, overexpression of ROS and NO can cause various illnesses by injuring surrounding cells resulting in acute or chronic inflammatory diseases (Johar et al., 2004). The ROS include O2, hydroxyl radical, and hydrogen peroxide. O2, the main species of ROS and the precursor of other ROS, is generated by oxygen consumption through the activity of nicotinamide adenine dinucleotide phosphate oxidase (Roberts et al., 2010). In this study, the exposure of RAW264.7 macrophages to BisGMA could evoke both extracellular O2 and intracellular ROS generation by ferricytochrome c and DCFH-DA, respectively. Recently, BisGMA was demonstrated to generate cellular ROS by labeling with DCFH-DA in human pulp
cells (Chang et al., 2009, 2010). Consistently, BisGMA could decrease intracellular glutathione in human gingival fibroblasts (Engelmann et al., 2004). NO is generated by iNOS conversion of L-arginine to L-citrulline (Wink and Mitchell, 1998). To the best of our knowledge, we first found that BisGMA could stimulate NO secretion and iNOS expression in RAW264.7 macrophages. Consistently, dental adhesive systems were found to produce NO generation in primary murine macrophages (Porto et al., 2011). Taken together, macrophage activated by BisGMA may lead to generate ROS/NO and result in inflammatory reaction. Expression of iNOS, IL-1b, and IL-6 in macrophages is regulated by NF-jB activation (Kim et al., 2007; Baeuerle and Henkel, 1994). NF-jB is composed of homo- and hetero-dimers of a family of proteins which include p65, p50, p52, c-Rel, p65 (RelA), and RelB (Karin and Ben-Neriah, 2000). The p65 subunit is a major component associated with transcription activation. In un-stimulated state, p65 is presented in the cytoplasm and complexed with IjB. After stimulation, phosphorylation and translocation to the nucleus of p65 is caused by IjB degradation (Karin and Ben-Neriah, 2000). In addition, the phosphorylation of p65 at Ser536 following LPS stimulation could raise the p65 transcriptional activity (Hsieh
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kinase and a major downstream factor of PI3K, can promote IjB kinase phosphorylation (Ozes et al., 1999) and then result in IjB degradation (Park et al., 2009). The treatment of RAW264.7 macrophages with BisGMA was found to induce Akt phosphorylation. Taken together, it is imply that NF-jB activation by BisGMA may be via PI3K/Akt pathway. In the present study, we demonstrated that the activation of RAW264.7 macrophages by BisGMA may involve NFjB via PI3K/ Akt pathway (Fig. 8). BisGMA was demonstrated to significantly upregulate the secretion of IL-1b and IL-6, the expression of iNOS and NO, the activation of NF-jB, the degradation of IjB, and the phosphorylation of Akt. Further studies on the other signaling pathways responsible for the induction of ROS, cytokines, and NO generation by BisGMA should be done to further punctuate our comprehension of the macrophages response to inflammation of dental composites. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This study was supported by NSC 100-2314-B-040-006. Microplate reader was performed in the Instrument Center of Chung Shan Medical University, which is supported by National Science Council, Ministry of Education and Chung Shan Medical University. References
Fig. 7. Effects of LY294002 on BisGMA-induced NO, IL-1b, and IL-6 generation. RAW264.7 cells were pretreated with or without 20 lM LY294002 for 30 min before stimulated the 3 lM BisGMA for 2 h. The supernatant was harvested and detected by Griess reaction and ELISA assay. Values are expressed as mean ± SD (n = 5). represents significant difference from control values with p < 0.05. #represents significant difference from BisGMA values with p < 0.05.
et al., 2011). After exposure to BisGMA, RAW264.7 macrophages trigger p65 phosphorylation at Ser536 and IjB degradation. These results indicated that BisGMA could induce iNOS, IL-1b, and IL-6 generation via NFjB activation. On the basis of our results, BisGMA could induce NF-jB activation via IjB degradation. The investigation of upstream signaling pathway is worth further assessment. Akt, a serine/threonine
Fig. 8. Schematic diagram illustrating the signaling pathways involved in BisGMAinduced inflammation associated proteins in RAW264.7 macrophages.
Baeuerle, P.A., Henkel, T., 1994. Function and activation of NF-jB in the immune system. Annu. Rev. Immunol. 12, 141–179. Bailey, L.Q., Weir, M.D., Washburn, N.R., 2006. Quantification of macrophage viability and inflammatory response to dental bonding resins. J. Bioact. Compat. Pol. 21, 185–206. Billack, B., 2006. Macrophage activation: role of toll-like receptors, nitric oxide, and nuclear factor kappa B. Am. J. Pharm. Educ. 70, 102. Cannito, S., Novo, E., Compagnone, A., Valfrè di Bonzo, L., Busletta, C., Zamara, E., et al., 2008. Redox mechanisms switch on hypoxia-dependent epithelialmesenchymal transition in cancer cells. Carcinogenesis 29, 2267–2278. Chang, M.C., Chen, L.I., Chan, C.P., Lee, J.J., Wang, T.M., Yang, T.T., et al., 2010. The role of reactive oxygen species and hemeoxygenase-1 expression in the cytotoxicity, cell cycle alteration and apoptosis of dental pulp cells induced by BisGMA. Biomaterials 31, 8164–8171. Chang, M.C., Lin, L.D., Chan, C.P., Chang, H.H., Chen, L.I., Lin, H.J., et al., 2009. The effect of BisGMA on cyclooxygenase-2 expression, PGE2 production and cytotoxicity via reactive oxygen species- and MEK/ERK-dependent and independent pathways. Biomaterials 30, 4070–4077. Chang, M.C., Lin, L.D., Chuang, F.H., Chan, C.P., Wang, T.M., Lee, J.J., et al., 2012. Carboxylesterase expression in human dental pulp cells: role in regulation of BisGMA-induced prostanoid production and cytotoxicity. Acta Biomater. 8, 1380–1387. Chen, C.J., Ou, Y.C., Chang, C.Y., Pan, H.C., Liao, S.L., Raung, S.L., et al., 2011. TNF-a and IL-1b mediate Japanese encephalitis virus-induced RANTES gene expression in astrocytes. Neurochem. Int. 58, 234–242. Drozdz, K., Wysokinski, D., Krupa, R., Wozniak, K., 2011. Bisphenol A-glycidyl methacrylate induces a broad spectrum of DNA damage in human lymphocytes. Arch. Toxicol. 85, 1453–1461. Engelmann, J., Janke, V., Volk, J., Leyhausen, G., von Neuhoff, N., Schlegelberger, B., et al., 2004. Effects of BisGMA on glutathione metabolism and apoptosis in human gingival fibroblasts in vitro. Biomaterials 25, 4573–4580. Hsieh, C.Y., Hsu, M.J., Hsiao, G., Wang, Y.H., Huang, C.W., Chen, S.W., et al., 2011. Andrographolide enhances nuclear factor-kappaB subunit p65 Ser536 dephosphorylation through activation of protein phosphatase 2A in vascular smooth muscle cells. J. Biol. Chem. 286, 5942–5955. Johar, D., Roth, J.C., Bay, G.H., Walker, J.N., Kroczak, T.J., Los, M., 2004. Inflammatory response, reactive oxygen species, programmed (necrotic-like and apoptotic) cell death and cancer. Rocz. Akad. Med. Bialymst. 49, 31–39. Karin, M., Ben-Neriah, Y., 2000. Phosphorylation meets ubiquitination: the control of NF-jB activity. Annu. Rev. Immunol. 18, 621–663. Kim, J.B., Han, A.R., Park, E.Y., Kim, J.Y., Cho, W., Lee, J., et al., 2007. Inhibition of LPSinduced iNOS, COX-2 and cytokines expression by poncirin through the NFkappa B inactivation in RAW264.7 macrophage cells. Biol. Pharm. Bull. 30, 2345–2351. Kim, J.G., Chung, C.M., 2005. Elution from light-cured dental composites: comparison of trimethacrylate and dimethacrylate as base monomers. J. Biomed. Mater. Res. B Appl. Biomater. 72, 328–333.
Y.-H. Kuan et al. / Food and Chemical Toxicology 50 (2012) 4003–4009 Kopf, M., Bachmann, M.F., Marsland, B.J., 2010. Averting inflammation by targeting the cytokine environment. Nat. Rev. Drug Discov. 9, 703–718. Kuan, Y.H., Li, Y.C., Huang, F.M., Chang, Y.C., 2012. The upregulation of tumour necrosis factor-a and surface antigens expression on macrophages by bisphenol A- glycidyl-methacrylate. Int. Endod. J. 45, 619–626. Kuo, M.Y., Liao, M.F., Chen, F.L., Li, Y.C., Yang, M.L., Lin, R.H., et al., 2011. Luteolin attenuates the pulmonary inflammatory response involves abilities of antioxidation and inhibition of MAPK and NFjB pathways in mice with endotoxin-induced acute lung injury. Food Chem. Toxicol. 49, 2660–2666. Lee, J.P., Li, Y.C., Chen, H.Y., Lin, R.H., Huang, S.S., Chen, H.L., et al., 2010. Protective effects of luteolin against lipopolysaccharide-induced acute lung injury involves inhibition of MEK/ERK and PI3K/Akt pathways in neutrophils. Acta Pharmacol. Sin. 31, 831–838. Li, Y.C., Kuan, Y.H., Huang, F.M., Chang, Y.C., 2012a. The role of DNA damage and caspase activation in cytotoxicity and genotoxicity of macrophages induced by bisphenol-A-glycidyldimethacrylate. Int. Endodon. J. 45, 499–507. Li, Y.C., Yeh, C.H., Yang, M.L., Kuan, Y.H., 2012b. Luteolin suppresses inflammatory mediator expression by blocking the Akt/NFjB pathway in acute lung injury induced by lipopolysaccharide in mice. Evid. Based Complement Alternat. Med. 2012, 383608. Ozes, O.N., Mayo, L.D., Gustin, J.A., Pfeffer, S.R., Pfeffer, L.M., Donner, D.B., 1999. NFjB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401, 82–85.
4009
Park, S.Y., Park, G.Y., Ko, W.S., Kim, Y., 2009. Dichroa febrifuga Lour. inhibits the production of IL-1beta and IL-6 through blocking NF-kappaB, MAPK and Akt activation in macrophages. J. Ethnopharmacol. 125, 246–251. Porto, I.C., Oliveira, D.C., Raele, R.A., Ribas, K.H., Montes, M.A., De Castro, C.M., 2011. Cytotoxicity of current adhesive systems: in vitro testing on cell cultures of primary murine macrophages. Dent. Mater. 27, 221–228. Reichl, F.X., Seiss, M., Kleinsasser, N., Kehe, K., Kunzelmann, K.H., Thomas, P., Spahl, W., Hickel, R., 2008. Distribution and excretion of BisGMA in guinea pigs. J. Dent. Res. 87, 378–380. Roberts, R.A., Smith, R.A., Safe, S., Szabo, C., Tjalkens, R.B., Robertson, F.M., 2010. Toxicological and pathophysiological roles of reactive oxygen and nitrogen species. Toxicology 276, 85–94. Tabatabaee, M.H., Mahdavi, H., Zandi, S., Kharrazi, M.J., 2009. HPLC analysis of eluted monomers from two composite resins cured with LED and Halogen curing lights. J. Biomed. Mater. Res. B Appl. Biomater. 88B, 191–196. Wells, C.A., Ravasi, T., Hume, D.A., 2005. Inflammation suppressor genes: please switch out all the lights. J. Leukoc. Biol. 78, 9–13. Wink, D.A., Mitchell, J.B., 1998. Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med. 25, 434–456. Yap, A.U., Han, V.T., Soh, M.S., Siow, K.S., 2004. Elution of leachable components from composites after LED and halogen light irradiation. Oper. Dent. 29, 448– 453.