Does Preheating Influence the Cytotoxic Potential of Dental Resin Composites?

Resin-based dental composites (RBC) release cytotoxic components, however the extent of the elution from preheated RBCs is barely investigated. The aim was therefore to determine the cytotoxic effect of preheated conventional, bulk, and thermoviscous RBCs of clinically relevant sizes using different cell viability methods in a contact-free model. Samples (6 × 4 mm) were prepared from conventional [Estelite Sigma Quick (ESQ), Filtek Z250 (FZ)] and bulk-filled [Filtek One BulkFill Restorative (FOB), SDR Plus Bulk Flow (SDR), VisCalor Bulk (VCB)] RBCs. The pre-polymerization temperature was set to room temperature (RT) and 55/65 °C. Pulp cells were cultured, followed by a 2-day exposure to monomers released from solid RBC specimens suspended in the culture medium. Cytotoxicity was assessed using a WST-1, MTT, and LDH colorimetric viability assays. Data were analyzed using one-way ANOVA, Tukey's post hoc test, multivariate analysis, and independent t-test. The effect size (ƞp2) of material and temperature factors was also assessed. All the RBCs demonstrated cytotoxic effect upon exposure to pulp cells, but to a varying extent (ESQ >> VCB > FZ = FOB = SDR). The effect of pre-polymerization temperature was insignificant (ƞp2 < 0.03), except for the thermoviscous RBC, which showed inconsistent findings when subjected to distinct viability tests. Cell viability was predominantly dependent on the type of material used (p < 0.001) which showed a large effect size (ƞp2 > 0.90). Irrespective of the pre-polymerization temperature, RBC samples in a clinically relevant size can release monomers to such an extent, which can substantially decrease the cytocompatibility.

Urocortin stimulates the ERK1/2 signaling pathway and the proliferation of HeLa cells via CRF receptor 1.

Corticotropin-releasing factor (CRF) stimulates adrenocorticotropic hormone (ACTH) secretion from the pituitary gland and is an essential regulator of the hypothalamic-pituitary-adrenocortical axis. Isoforms of CRF receptor are known to mediate the effects of urocortin stress ligands on the regulation of stress responses, anxiety, and feeding behavior; however, urocortin stress ligands also influence cell proliferation. In view of the tumor-promoting capacity of prolonged stress, here we investigated (a) the effect of urocortin on cell proliferative signaling via extracellular signal-regulated kinase 1/2, (b) the expression and cellular distribution of the specific CRF receptor isoforms, and (c) the intracellular localization of phosphorylated ERK1/2 in HeLa cells. Stimulation of cell proliferation was observed in the presence of 10 nm urocortin. Our data also suggest that MAP kinase MEK, the transcription factors E2F-1 and p53, and PKB/Akt are involved in this process. These findings may have therapeutic relevance for the targeted treatment of various malignancies.

Prolonged treatment with the proteasome inhibitor MG-132 induces apoptosis in PC12 rat pheochromocytoma cells.

Rat pheochromocytoma (PC12) cells were treated with the proteasome inhibitor MG-132 and morphological changes were recorded. Initially, neuronal differentiation was induced but after 24 h signs of morphological deterioration became apparent. We performed nuclear staining, flow cytometry and WST-1 assay then analyzed signal transduction pathways involving Akt, p38 MAPK (Mitogen-Activated Protein Kinase), JNK (c-Jun N-terminal Kinase), c-Jun and caspase-3. Stress signaling via p38, JNK and c-Jun was active even after 24 h of MG-132 treatment, while the survival-mediating Akt phosphorylation declined and the executor of apoptosis (caspase-3) was activated by that time and apoptosis was also observable. We examined subcellular localization of stress signaling components, applied kinase inhibitors and dominant negative H-Ras mutant-expressing PC12 cells in order to decipher connections of stress-mediating pathways. Our results are suggestive of that treatment with the proteasome inhibitor MG-132 has a biphasic nature in PC12 cells. Initially, it induces neuronal differentiation but prolonged treatments lead to apoptosis.

Urocortin stimulates ERK1/2 phosphorylation and proliferation but reduces ATP production of MCF7 breast cancer cells.

Urocortins are members of the stress-related corticotropin-releasing factor family. Small amounts of them are present in the circulation and they are produced locally in various tissues of higher vertebrates. Aside from regulating circulation, or food uptake they also influence, via auto- and paracrine mechanisms, cell proliferation. In the present study we investigated in MCF7 human breast cancer cells the effect of urocortin onto mitogenic signaling via ERK1/2. Our results revealed that already 10 nM urocortin could stimulate the phosphorylation of these kinases and cell proliferation of MCF7 cells while ATP production was reduced when kept in the presence of the peptide up to two days. We examined the expression and contribution of the specific receptors of urocortin to the activation of ERK1/2 and to cell proliferation, the intracellular distribution of phosphorylated ERK1/2, and the involvement of additional proteins like PKA, PKB/Akt, MEK, p53, Rb and E2F-1 behind the observed phenomena.

Subnormothermic isolated organ perfusion with Nicorandil increased cold ischemic tolerance of liver in experimental model.

BACKGROUND: The cold ischemia -reperfusion injury may lead to microcirculatory disturbances, hepatocellular swelling, inflammation, and organ dysfunction. Nicorandil is an anti-ischemic, ATP-sensitive potassium (KATP) channel opener drug and has proved its effectiveness against hepatic Ischemia/Reperfusion (I/R) injury. OBJECTIVE: This study aimed to investigate the effect of Nicorandil on mitochondrial apoptosis, oxidative stress, inflammation, histopathological changes, and cold ischemic tolerance of the liver in an ex vivo experimental isolated-organ-perfusion model. METHODS: We used an ex vivo isolated rat liver perfusion system for this study. The grafts were retrieved from male Wistar rats (n = 5 in each), preserved in cold storage (CS) for 2 or 4 hours (group 1, 2), or perfused for 2 or 4 hours (group 3, 4) immediately after removal with Krebs Henseleit Buffer (KHB) solution or Nicorandil containing KHB solution under subnormothermic (22-25°C) conditions (group 5, 6). After 15 minutes incubation at room temperature, the livers were reperfused with acellular, oxygenated solution under normothermic condition for 60 minutes. RESULTS: In the Nicorandil perfused groups, significantly decreased liver enzymes, GLDH, TNF-alpha, and IL-1ß were measured from the perfusate. Antioxidant enzymactivity was higher in the perfused groups. Histopathological examination showed ameliorated tissue deterioration, preserved parenchymal structure, decreased apoptosis, and increased Bcl-2 activity in the Nicorandil perfused groups. CONCLUSIONS: Perfusion with Nicorandil containing KHB solution may increase cold ischemic tolerance of the liver via mitochondrial protection which can be a potential therapeutic target to improve graft survival during transplantation.

Characterizing Autophagy in the Cold Ischemic Injury of Small Bowel Grafts: Evidence from Rat Jejunum.

Cold ischemic injury to the intestine during preservation remains an unresolved issue in transplantation medicine. Autophagy, a cytoplasmic protein degradation pathway, is essential for metabolic adaptation to starvation, hypoxia, and ischemia. It has been implicated in the cold ischemia (CI) of other transplantable organs. This study determines the changes in intestinal autophagy evoked by cold storage and explores the effects of autophagy on ischemic grafts. Cold preservation was simulated by placing the small intestines of Wistar rats in an IGL-1 (Institute George Lopez) solution at 4 °C for varying periods (3, 6, 9, and 12 h). The extent of graft preservation injury (mucosal and cellular injury) and changes in autophagy were measured after each CI time. Subsequently, we determined the differences in apoptosis and preservation injury after activating autophagy with rapamycin or inhibiting it with 3-methyladenine. The results revealed that ischemic injury and autophagy were induced by cold storage. Autophagy peaked at 3 h and subsequently declined. After 12 h of storage, autophagic expression was reduced significantly. Additionally, enhanced intestinal autophagy by rapamycin was associated with less tissue, cellular, and apoptotic damage during and after the 12-h long preservation. After reperfusion, grafts with enhanced autophagy still presented with less injury. Inhibiting autophagy exhibited the opposite trend. These findings demonstrate intestinal autophagy changes in cold preservation. Furthermore, enhanced autophagy was protective against cold ischemia-reperfusion damage of the small bowels.

Effect of Pioglitazone on endoplasmic reticulum stress regarding in situ perfusion rat model.

BACKGROUND: Ischemia-reperfusion injury (IRI) can cause insufficient microcirculation of the transplanted organ and results in a diminished and inferior graft survival rate. OBJECTIVE: This study aimed to investigate the effect of different doses of an anti-diabetic drug, Pioglitazone (Pio), on endoplasmic reticulum stress and histopathological changes, using an in situ perfusion rat model. METHODS: Sixty male Wistar rats were used and were divided into six groups, consisting of the control group, vehicle-treated group and four Pio-treated groups (10, 20, 30 and 40 mg/kg Pio was administered). The rats were perfused through vena cava and an outflow on the abdominal aorta occurred. Following the experiment, kidneys and livers were collected. The level of the endoplasmic reticulum stress markers (XBP1 and Caspase 12) was analyzed using Western blot and histopathological changes were evaluated. RESULTS: Histopathological findings were correlated with the Western blot results and depict a protective effect corresponding to the elevated dosage of Pioglitazone regarding in situ perfusion rat model. CONCLUSIONS: In our study, Pioglitazone can reduce the endoplasmic reticulum stress, and the most effective dosage proved to be the 40 mg/kg Pio referencing the kidney and liver samples.

TEGDMA (Triethylene Glycol Dimethacrylate) Induces Both Caspase-Dependent and Caspase-Independent Apoptotic Pathways in Pulp Cells.

Monomers leached from resin-based composites (RBCs) may reach intrapulpal concentrations of the millimolar (mM) range, which could contribute to inflammation. The aim of this investigation was to assess the cytotoxicity of triethylene glycol dimethacrylate (TEGDMA) monomers on pulp cells as well as to identify molecular mechanisms leading to apoptosis. Pulp cells were harvested from molars extracted for orthodontic reasons and cultured through an explant method. To assess cytotoxicity, cells underwent a 5-day exposure to 0.75, 1.5, and 3 mM TEGDMA and were subject to cell counting and WST-1 staining. Based on the findings, cells were subsequently exposed to 0.1, 0.2, 0.75, 1.5, and 3 mM TEGDMA for 24 h to uncover the details of apoptosis. Changes in the production or cleavage of the apoptosis-specific proteins caspase-8, caspase-9, caspase-3, caspase-12, and Apoptosis-Inducing Factor (AIF) were measured by Western blot. The 5-day study showed concentration- and time-dependent cytotoxicity. Significant cell death was detected after 24 h with TEGDMA concentrations of 1.5 and 3 mM. One-day exposure to TEGDMA led to the activation of caspase-8, -9, -3, and -12 and an increased AIF production. Results suggest that relevant concentrations of TEGDMA monomers, leached from RBCs, induce apoptosis in pulp cells through both caspase-dependent as well as caspase-independent mechanisms. Endoplasmic reticulum stress and the activation of caspase-independent apoptotic pathways may be further mechanisms by which monomers induce apoptosis in pulp cells.

Influence of TEGDMA monomer on MMP-2, MMP-8, and MMP-9 production and collagenase activity in pulp cells.

OBJECTIVES: Resin-based composites may leach monomers such as triethylene-glycol dimethacrylate (TEGDMA), which could contribute to intrapulpal inflammation. The aim of this investigation was to examine whether various concentrations of TEGDMA are able to influence dentally relevant Matrix metalloproteinase (MMP)-2, MMP-8, and MMP-9 production, total collagenase/gelatinase activity in pulp cells, and suggest possible signaling mechanisms. MATERIALS AND METHODS: Pulp cells were cultured, followed by a 1-day exposure to sublethal TEGDMA concentrations (0.1, 0.2, and 0.75 mM). Total MMP activity was measured by an EnzCheck total collagenase/gelatinase assay, while the production of specific MMPs and the relative changes of phosphorylated, i.e., activated signaling protein levels of extracellular signal-regulated kinase (ERK)1/2, p38, c-Jun N-terminal kinase (JNK) were identified by western blot. Immunocytochemistry image data was also plotted and analyzed to see whether TEGDMA could possibly alter MMP production. RESULTS: An increase in activated MMP-2, MMP-8, and MMP-9 production as well as total collagenase activity was seen after a 24-h exposure to the abovementioned TEGDMA concentrations. Increase was most substantial at 0.1 (P = 0.002) and 0.2 mM (P = 0.0381). Concurrent p-ERK, p-p38, and p-JNK elevations were also detected. CONCLUSIONS: Results suggest that monomers such as TEGDMA, leached from resin-based restorative materials, activate and induce the production of dentally relevant MMPs in pulp cells. Activation of ERK1/2, p38, or JNK and MMP increase may play a role in and/or can be part of a broader stress response. Clinical relevance Induction of MMP production and activity may further be components in the mechanisms of intrapulpal monomer toxicity.

Prostaglandin E2, a postulated mediator of neurovascular coupling, at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles.

There is considerable controversy regarding the vasoactive action of prostaglandin E2 (PGE2). On the one hand, indirect evidence implicates that astrocytic release of PGE2 contributes to neurovascular coupling responses mediating functional hyperemia in the brain. On the other hand, overproduction of PGE2 was also reported to contribute to cerebral vasospasm associated with subarachnoid hemorrhage. The present study was conducted to resolve this controversy by determining the direct vasoactive effects of PGE2 in resistance-sized human cerebral parenchymal arterioles. To achieve this goal PGE2-induced isotonic vasomotor responses were assessed in parenchymal arterioles isolated from fronto-temporo-parietal cortical tissues surgically removed from patients and expression of PGE2 receptors were examined. In functionally intact parenchymal arterioles lower concentrations of PGE2 (from 10-8 to 10-6 mol/l) caused significant, endothelium-independent vasorelaxation, which was inhibited by the EP4 receptor blocker BGC201531. In contrast, higher concentrations of PGE2 evoked significant EP1-dependent vasoconstriction, which could not be reversed by the EP4 receptor agonist CAY10598. We also confirmed previous observations that PGE2 primarily evokes constriction in intracerebral arterioles isolated from R. norvegicus. Importantly, vascular mRNA and protein expression of vasodilator EP4 receptors was significantly higher than that of vasoconstrictor EP1 receptors in human cerebral arterioles. PGE2 at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles. This bimodal vasomotor response is consistent with both the proposed vasodilator role of PGE2 during functional hyperemia and its putative role in cerebral vasospasm associated with subarachnoid hemorrhage in human patients.

Sodium Pentosan Polysulfate Reduced Renal Ischemia-Reperfusion-Induced Oxidative Stress and Inflammatory Responses in an Experimental Animal Model.

Acute kidney injury (AKI) remains an independent risk factor for mortality and morbidity after vascular surgery (affecting the renal arteries) or aortic surgery (requiring suprarenal aortic clamping). These types of vascular surgery produce renal ischemia/reperfusion (I/R) injury, a common cause of AKI. The present studies aimed at monitoring the course of renal I/R injury at the cellular level and investigating the efficacy of long-term preoperative and single-shot intraoperative administration of sodium pentosan polysulfate (PPS) to protect renal tissue from acute I/R injury both in native and diabetic kidneys in rats. Western blot analyses of the proapoptotic (bax) and antiapoptotic (bcl-2) signaling pathways, as well as the extent of DNA damage (phospho-p53), were performed. Oxidative stress followed upon the termination of malondialdehyde, reduced glutathione, thiol group, and superoxide dismutase plasma levels. Inflammatory changes were measured by the determination of serum tumor necrosis factor-α and interleukin-1 levels. Morphological changes were detected by histological examinations. Our results showed that the long-term administration of PPS has an advantage in reducing I/R kidney injury in diabetic rats, while high-dose, single-shot parenteral administration of PPS prior to revascularization might be useful in nondiabetic rats.

Partial rescue of geldanamycin-induced TrkA depletion by a proteasome inhibitor in PC12 cells.

In this work we tried to identify mechanisms that could explain how chemical inhibition of heat-shock protein 90 reduces nerve growth factor signaling in rat pheochromocytoma PC12 cells. Geldanamycin is an antibiotic originally discovered based on its ability to bind heat-shock protein 90. This interaction can lead to the disruption of heat-shock protein 90-containing multimolecular complexes. It can also induce the inhibition or even degradation of partner proteins dissociated from the 90 kDa chaperone and, eventually, can cause apoptosis, for instance, in PC12 cells. Before the onset of initial apoptotic events, however, a marked decrease in the activity of extracellular signal-regulated kinases ERK 1/2 and protein kinase B/Akt can be observed together with reduced expression of the high affinity nerve growth factor receptor, tropomyosine-related kinase, TrkA, in this cell type. The proteasome inhibitor MG-132 can effectively counteract the geldanamycin-induced reduction of TrkA expression and it can render TrkA and ERK1/2 phosphorylation but not that of protein kinase B/Akt by nerve growth factor again inducible. We have found altered intracellular distribution of TrkA in geldanamycin-treated and proteasome-inhibited PC12 cells that may, at least from the viewpoint of protein localization explain why nerve growth factor remains without effect on protein kinase B/Akt. The lack of protein kinase B/Akt stimulation by nerve growth factor in turn reveals why nerve growth factor treatment cannot save PC12 cells from geldanamycin-induced programmed cell death. Our observations can help to better understand the mechanism of action of geldanamycin, a compound with strong human therapeutical potential.

The effects of a mutant p53 protein on the proliferation and differentiation of PC12 rat phaeochromocytoma cells.

PC12 rat phaeochromocytoma cells show neuronal differentiation upon NGF treatment. NGF induces prolonged activation of the Ras/Raf/MEK/ERK pathway in which the 42/44 kDa mitogen-activated protein kinases (MAPKs), ERK 1 and 2 are thought to be the key mediators of the differentiation signals. Activation of ERKs leads to the increased transcription of early response genes resulting in cell cycle arrest. Upon NGF treatment the p53 protein, the most commonly mutated tumor suppressor in human cancers, translocates to the nucleus and may play a role in the mediation of NGF-induced cell cycle arrest and neuronal differentiation. Here we demonstrate that in PC12 cells expressing both wild-type and V143A mutant p53 proteins (p143p53PC12 cells), p53-mediated biological responses are critically influenced. p143p53PC12 cells are not able to cease their proliferation and begin their neuronal differentiation program upon NGF treatment. The presence of mutant p53 also reduces the DNA-binding activity of endogenous p53 and disturbs the regulatory machinery of p53 including both the phosphorylation of ERK 1/2, p38 and SAPK/JNK MAP kinases and itself.