TEGDMA and filler particles from dental composites additively attenuate LPS-induced cytokine release from the macrophage cell line RAW 264.7.

OBJECTIVES: Due to incomplete curing and material degradation, cells in the oral cavity may be exposed to monomers and filler particles from dental composite fillings. The objective of the present study was to investigate if combined exposures to particles and a methacrylate monomer from composite fillings resulted in additive effects on the macrophage immune response. MATERIAL AND METHODS: Two filler particles, Nanosilica (12 nm) and Quartz (1 µm), were studied at concentrations 0.5-4 µg/cm(2), while the methacrylate monomer triethyleneglycol dimethacrylate (TEGDMA) was applied at 5 and 50 µM. RAW 264.7 macrophages were exposed to monomers and/or particles for 24 h, with a subsequent 24 h combined exposure to monomers and/or particles and the bacterial factor lipopolysaccharide (LPS) to stimulate an immune response. Release of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were measured as well as the cellular viability. RESULTS: Co-exposure to Nanosilica and Quartz resulted in an additive attenuation of the LPS-induced IL-1ß release. Moreover, co-exposure to TEGDMA and both types of filler particles also resulted in an additive attenuation, although with a weak synergistic trend. The cellular viability and TNF-α release were not significantly affected by the exposures. CONCLUSION: The present findings emphasize the necessity of considering effects of combined exposure to dental degradation products in future risk assessments. CLINICAL RELEVANCE: Attenuated cytokine release could have implications for the macrophage immune response and result in impaired bacterial clearance. Further studies are necessary to determine implications for formation of dental biofilms and caries development.

Protocol for designing INVITES-IN, a tool for assessing the internal validity of in vitro studies.

This protocol describes the design and development of a tool for evaluation of the internal validity of in vitro studies, which is needed to include the data as evidence in systematic reviews and chemical risk assessments. The tool will be designed specifically to be applied to cell culture studies, including, but not restricted to, studies meeting the new approach methodology (NAM) definition. The tool is called INVITES-IN (IN VITro Experimental Studies INternal validity). In this protocol, three of the four studies that will be performed to create the release version of INVITES-IN are described. In the first study, evaluation of existing assessment tools will be combined with focus group discussions to identify how characteristics of the design or conduct of an in vitro study can affect its internal validity. Bias domains and items considered to be of relevance for in vitro studies will be identified. In the second study, group agreement on internal validity domains and items of importance for in vitro studies will be identified via a modified Delphi methodology. In the third study, the draft version of the tool will be created, based on the data on relevance and importance of bias domains and items collected in Studies 1 and 2. A separate protocol will be prepared for the fourth study, which includes the user testing and validation of the tool, and collection of users' experience.

Delayed translocation of NGFI-B/RXR in glutamate stimulated neurons allows late protection by 9-cis retinoic acid.

Nuclear receptor and apoptosis inducer NGFI-B translocates out of the nucleus as a heterodimer with RXR in response to different apoptosis stimuli, and therefore represents a potential pharmacological target. We found that the cytosolic levels of NGFI-B and RXRα were increased in cultures of cerebellar granule neurons 2h after treatment with glutamate (excitatory neurotransmitter in the brain, involved in stroke). To find a time-window for potential intervention the neurons were transfected with gfp-tagged expressor plasmids for NGFI-B and RXR. The default localization of NGFI-Bgfp and RXRgfp was nuclear, however, translocation out of the nucleus was observed 2-3h after glutamate treatment. We therefore hypothesized that the time-window between treatment and translocation would allow late protection against neuronal death. The RXR ligand 9-cis retinoic acid was used to arrest NGFI-B and RXR in the nucleus. Addition of 9-cis retinoic acid 1h after treatment with glutamate reduced the cytosolic translocation of NGFI-B and RXRα, the cytosolic translocation of NGFI-Bgfp observed in live neurons, as well as the neuronal death. However, the reduced translocation and the reduced cell death were not observed when 9-cis retinoic acid was added after 3h. Thus, late protection from glutamate induced death by addition of 9-cis retinoic acid is possible in a time-window after apoptosis induction.

Secretory PLA2-IIA and ROS generation in peripheral mitochondria are critical for neuronal death.

In this study the role of mitochondrial secretory PLA2-IIA in glutamate-induced cell death in cultured cerebellar granule neurons has been investigated. Inhibition of secretory PLA2-IIA blocked glutamate-induced cell death. Since PLA2 may generate reactive oxygen species (ROS), we have investigated ROS production, detected as dihydrorhodamine 123 oxidation and nitrotyrosine modifications of proteins, following glutamate treatment in the absence or presence of an inhibitor of secretory PLA2-IIA. There was an increased generation of ROS in both glutamate- and buffer-treated neurons compared to untreated neurons. Scavenging with dihydrorhodamine 123 reduced glutamate-induced death (60%), showing that ROS detected in glutamate-treated neurons were associated with cell death. However, ROS detected in buffer-treated neurons were not associated with toxicity. Glutamate treatment led to ROS production predominantly in peripheral mitochondria, whereas buffer treatment led to ROS production in somal mitochondria. Inhibition of secretory PLA2-IIA (i) reduced the generation of ROS after glutamate treatment, (ii) reduced the ROS production in peripheral mitochondria in glutamate-treated neurons, consistent with the fact that calcium entry through glutamate (NMDA) receptors has a privileged access to peripheral mitochondria, and (iii) did not reduce the generation of ROS after buffer treatment. In conclusion, activation of NMDA receptors induces ROS, which is critical for neuronal death, due to secretory PLA2-IIA associated with peripheral mitochondria.

Chicken cerebellar granule neurons rapidly develop excitotoxicity in culture.

Rat cerebellar granule cell culture is widely used as a model to study factors that control neuronal differentiation and death (e.g. excitotoxicity). However, a main drawback of this model is its dependence on depolarizing culture condition (25 mM potassium). In addition, it is quite expensive to maintain and requires animal facilities. Here we report that cerebellar granule neuron cultures from chicken may be used as an alternative model to study excitotoxicity. Surprisingly, fetal chicken cells may be grown in a physiological potassium concentration (5 mM potassium). They develop excitotoxicity rapidly in culture (fully developed at 3 days in vitro), and respond to glutamate excitotoxicity similar to rat cultures (ROS production and activation of caspase-3).

Calcium-induced apoptosis of developing cerebellar granule neurons depends causally on NGFI-B.

Immediate early gene nerve growth factor-induced clone B (NGFI-B), a nuclear receptor important for differentiation and apoptosis, is expressed in mice and rat cerebellum from an early stage of postnatal development. Following apoptotic stimuli NGFI-B translocates to mitochondria to initiate cell death processes. Controlled cell death is critical for correct cerebellar development. Immunohistochemical analysis of NGFI-B in sections of mice cerebella showed NGFI-B to be expressed in granule neurons in vivo at a time (P8-11) when apoptosis is known to occur. The importance of NGFI-B for apoptosis of cultured rat cerebellar granule neurons was investigated by inducing apoptosis with calcium ionophore A23187 (CaI, 0.1µM). Imaging studies of gfp-tagged NGFI-B confirmed that mitochondrial translocation of NGFI-B occurred following treatment with CaI and was reduced by addition of 9-cis-retinoic acid (1µM), a retinoid X receptor (RXR) agonist that prevents dimerization of RXR and NGFI-B that is known to occur before translocation. Consequently, 9-cis-retinoic acid partly reduced cell death. To address the causality of NGFI-B in apoptosis further, knock-down by siRNA was performed and it removed 85% of the NGFI-B protein. This resulted in a complete inhibition of apoptosis after CaI exposure. Together these findings suggest that NGFI-B plays a role in controlling correct cerebellar development.

Combretastatin A-4 and structurally related triazole analogues induce caspase-3 and reactive oxygen species-dependent cell death in PC12 cells.

Cancer cells are more sensitive to oxidative stress due to higher levels of reactive oxygen species. Therefore, the ability of anti-cancer agent combretastatin A-4 (CA-4) and triazole analogues to induce reactive oxygen species may be important for selectivity against cancer cells. The purpose of the present study was to investigate the structural requirements for reactive oxygen species production by CA-4 and the triazole analogues Ana-2, Ana-3 and Ana-4. Ana-2 and Ana-3 mimic the cis configuration in CA-4; Ana-3 lacks the phenolic hydroxyl group, while Ana-4 mimics a trans configuration. The rat pheochromocytoma cancer cell line PC12 was used as model system. CA-4 and Ana-2 were highly toxic; Ana-3 was less toxic, whereas Ana-4 was non-toxic. The probe dihydroethidium detected reactive oxygen species production from CA-4, Ana-2, and Ana-3. CA-4 and Ana-2 also induced oxidation of the reactive oxygen species probe dihydrorhodamine and activation of caspase-3. Thus, the phenolic hydroxyl group in CA-4 and Ana-2 was necessary for dihydrorhodamine oxidation, caspase-3 activation, and increased cytotoxicity.

Prenatal exposure to bisphenol A interferes with the development of cerebellar granule neurons in mice and chicken.

In mice, prenatal exposure to low doses of bisphenol A has been shown to affect neurogenesis and neuronal migration in cortex, resulting in disturbance of both neuronal positioning and the network formation between thalamus and cortex in the offspring brain. In the present study we investigated whether prenatal exposure to bisphenol A disturbs the neurodevelopment of the cerebellum. Two different model systems were used; offspring from two strains of mice from mothers receiving bisphenol A in the drinking water before mating, during gestation and lactation, and chicken embryos exposed to bisphenol A (in the egg) on embryonic day 16 for 24h before preparation of cerebellar granule cell cultures. In the cerebellum, tight regulation of the level of transcription factor Pax6 is critical for correct development of granule neurons. During the development, the Pax6 level in granule neurons is high when these cells are located in the external granule layer and during their migration to the internal granule layer, and it is then reduced. We report that bisphenol A induced an increase in the thickness of the external granule layer and also an increase in the total cerebellar Pax6 level in 11 days old mice offspring. In cultured chicken cerebellar granule neurons from bisphenol A injected eggs the Pax6 level was increased day 6 in vitro. Together, these findings indicate that bisphenol A may affect the granule neurons in the developing cerebellum and thereby may disturb the correct development of the cerebellum.

Differences in NGFI-B, Nurr1, and NOR-1 expression and nucleocytoplasmic translocation in glutamate-treated neurons.

NGFI-B (NR4A1, Nur77 or TR3) together with Nurr1 (NR4A2) and NOR-1 (NR4A3) constitute the NR4A subgroup of orphan nuclear receptors. They play critical roles in proliferation, differentiation, survival and apoptosis in different cell types, including neurons, immature T-cells, and different cancer cells. As ligand-independent and constitutively active receptors, the diverse biological activities of NGFI-B, Nurr1 and NOR-1 depend on their levels of expression, post-translational modifications and subcellular localization. Nuclear localization of the NR4A proteins leads to transcriptional activity, whereas NGFI-B and recently also NOR-1 have been shown to induce apoptosis by a more direct mechanism when localized at mitochondria. In the present study we investigated mRNA expression and subcellular translocation of the NR4A proteins during glutamate excitotoxicity in rat cerebellar granule neurons. NGFI-B and Nurr1 mRNA, but not NOR-1 mRNA, were induced by treatments associated with calcium influx, although their regulation seemed to be different. NR4A(gfp) fusion proteins showed a predominant nuclear localization in untreated cells. After glutamate treatment NGFI-B(gfp) translocated to cytosol and mitochondria within a few hours, whereas Nurr1(gfp) translocation was delayed, and NOR-1(gfp) mainly stayed in the nucleus. Subcellular targeting of NGFI-B seems to be tightly regulated, as a single mutation of threonine 142 altered NGFI-B(gfp) localization. Differences in expression and subcellular translocation of NGFI-B, Nurr1, and NOR-1 may reflect different functions in neurons in glutamate excitotoxicity.

Mono-2-ethylhexylphthalate (MEHP) induces TNF-α release and macrophage differentiation through different signalling pathways in RAW264.7 cells.

Epidemiological studies have associated indoor phthalate exposure with increased incidences and severity of asthma in children and adults, and inflammatory effects have been suggested as a possible mechanism. Recent studies report that phthalates may activate mitogen-activated protein (MAP) kinase p38 and various peroxisome proliferator-activated receptor (PPAR) isoforms. Here we confirm and extend these findings by investigating possible signalling pathways activated in the murine monocyte-macrophage cell line RAW264.7, using mono-2-ethylhexylphthalate (MEHP) as a model compound. MEHP exposure (0.3-1.0 mM) for 3h increased tumour necrosis factor (TNF)-α release and changed the cellular morphology into elongated spindle-like appearance, resembling more differentiated anti-inflammatory macrophages (M2). This was accompanied by increased expression of the macrophage differentiation marker CD163. Western analysis showed phosphorylation of p38 and Akt after 30 min exposure. Experiments using specific inhibitors suggested that MEHP-induced activation of both p38 and the phosphoinositide-3 (PI3) kinase/Akt pathway were involved in the release of TNF-α; whereas only PI3kinase seemed to be involved in differentiation. In contrast, inhibitors of PPARα and γ reduced differentiation, but did not affect TNF-α release. In conclusion, MEHP induced cytokine release and triggered differentiation of RAW264.7 cells, possibly into M2-like macrophages, but different signalling pathways appear to be involved in these responses.

Transfection of chicken cerebellar granule neurons used to study glucocorticoid receptor regulation by nuclear receptor 4A (NR4A).

Transfection is a useful tool for studying molecular signalling pathways. However, neurons have proven hard to transfect. In the present paper we have optimized a new electroporation procedure using the Cellaxess(®) system for transient transfection of adherent primary neurons from chicken (Gallus gallus) and compared it to a liposome based procedure using Metafectene(®) Pro. In order to evaluate the two methods, glucocorticoid receptor (GR) function was chosen as a test. GRs are expressed in high amounts in the cerebellum. GR is regulated by another nuclear receptor (NGFI-B, the first member found in the NR4A family). We first showed that forskolin and phorbol ester activated an NR4A-dependent reporter gene indicating that members of the NR4A nuclear receptor family are present endogenously and upregulated by external stimuli. Then, transfected NGFI-B was shown to antagonize the dexamethasone-activated transcriptional activation by endogenous GR, leading to the conclusion that NR4A-family members are important modulators of GR mediated regulatory processes in the cerebellum, as in other cell types. Both transfection methods proved useful. While the electroporation technique yielded small rings with many transfected cells optimal for microscopy studies, the liposome based method resulted in transfected cells evenly distributed in the dish rendering this method well suited for biochemical studies.

Preconditioning with estradiol abolishes its neuroprotection in cerebellar neurons.

Certain steroids are neuroprotective because they are modulators of neuronal activity or ROS scavengers. We examined neuroprotection following glutamate-induced excitotoxicity in cerebellar granule neuron cultures. 17beta-Estradiol, 17alpha-estradiol (nonestrogenic), or vitamins C+E were equally neuroprotective when coadministered with glutamate, consistent with protection by ROS scavenging. Progesterone protected mainly by an action on GABA-A receptors. Since exogenous antioxidants may influence the level of glutathione, the main endogenous antioxidant in neurons, we investigated if a preconditioning period with the neuroprotectors changed their efficacy as protectors. The neuroprotection by 17beta-estradiol and 17alpha-estradiol, but not progesterone or vitamins C+E, was almost abolished following a preconditioning period of 24h. This reduction was accompanied by an inhibition of the gamma-glutamylcysteine synthetase promoter, and a reduced level of glutathione when preconditioning was combined with the subsequent glutamate exposure. Thus, vitamins C+E and progesterone were more effective long-term neuroprotectors, since preconditioning did not reduce glutathione.