Relation between conversion degree and cytotoxicity of a flowable bulk-fill and three conventional flowable resin-composites.

OBJECTIVE: The aim of this study is to evaluate if the cytotoxic effects of the Surefil SDR flow, bulk fill flowable composite resin and three conventional flowable materials (Venus Diamond Flow, Filtex Supreme XTE Flowable and Enamel plus HRi Flow) correlated with the conversion degree (DC); hardness and depth of cure are also assessed. MATERIALS AND METHODS: Disks of each materials--cured using LED lamp--are utilized to evaluate DC (by FT-IR technique), amount of leached monomers (by HPLC technique), hardness (by Vickers hardness tester) and cytotoxicity (by MTT test). RESULTS: All tested materials show light cytotoxic effects, independently from DC values. Both the latter parameter and the hardness, in fact, change in function of thickness and type of material. HPLC results show that the monomers amount leached from each specimen is influenced by thickness but it is always very low which justifies the absence of any cytotoxic effect. CONCLUSIONS: Our findings suggest that there are not statistically significant differences in cytotoxicity in all experimental conditions, notwithstanding the differences in hardness and in degree of conversion.

Mapping viscoelastic properties of healthy and pathological red blood cells at the nanoscale level.

In order to pass through the microcirculation, red blood cells (RBCs) need to undergo extensive deformations and to recover the original shape. This extreme deformability is altered by various pathological conditions. On the other hand, an altered RBC deformability can have major effects on blood flow and can lead to pathological implications. The study of the viscoelastic response of red blood cells to mechanical stimuli is crucial to fully understand deformability changes under pathological conditions. However, the typical erythrocyte biconcave shape hints to a complex and intrinsically heterogeneous mechanical response that must be investigated by using probes at the nanoscale level. In this work, the local viscoelastic behaviour of healthy and pathological red blood cells was probed by Atomic Force Microscopy (AFM). Our results clearly show that the RBC stiffness is not spatially homogeneous, suggesting a strong correlation with the erythrocyte biconcave shape. Moreover, our nanoscale mapping highlights the key role played by viscous forces, demonstrating that RBCs do not behave as pure elastic bodies. The fundamental role played by viscous forces is further strengthened by the comparison between healthy and pathological (diabetes mellitus) RBCs. It is well known that pathological RBCs are usually stiffer than the healthy ones. Our measures unveil a more complex scenario according to which the difference between normal and pathological red blood cells does not merely lie in their stiffness but also in a different dynamical response to external stimuli that is governed by viscous forces.

Structural and functional characterization of the porcine proline-rich antifungal peptide SP-B isolated from salivary gland granules.

A 1905-Da cationic proline-rich peptide, named SP-B, was recently isolated by our group as the main component of salivary gland granules, and its primary sequence fully characterized by means of automated Edman sequencing and LC-MS/MS tools. In the present study SP-B is shown to possess antifungal activity when challenged with strains of Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, while only negligible antibacterial activity was detected. Furthermore, SP-B was found to be non-cytotoxic when tested on fibroblast cell lines. To obtain information regarding its structure affinity, capillary electrophoresis (CE), circular dichroism (CD) and attenuated total reflection (ATR)-FT/IR experiments were performed. CE revealed a pH dependence of the hydrodynamic radial dimensions both in aqueous and 2,2,2-trifluoroethanol solutions. CD and ATR-FT/IR measurements confirmed the structure-pH relationship, revealing a secondary structure composed of mixed proportions of polyproline-II, unordered and turn motifs, the last being more evident in the zwitterionic form of the peptide. From these findings SP-B peptide could be classified as a new member of the proline-rich antimicrobial peptide family.

Alterations of energy metabolism and glutathione levels of HL-60 cells induced by methacrylates present in composite resins.

OBJECTIVES: Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and bisphenol A glycerolate (1 glycerol/phenol) dimethacrylate (Bis-GMA) are largely present in auto- or photopolymerizable composite resins. Since the polymerization reaction is never complete, these molecules are released into the oral cavity tissues and biological fluids where they could cause local adverse effects. The aim of this work was to verify the hypothesis that the biological effects of HEMA, TEGDMA and Bis-GMA - at a non-cytotoxic concentration - depend on the interaction with mitochondria and exert consequent alterations of energy metabolism, GSH levels and the related pathways in human promyelocytic cell line (HL-60). METHODS: The biological effects of methacrylic monomers were determined by analyzing the following parameters: GSH concentration, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activity, oxygen and glucose consumption and lactate production along with cell differentiation and proliferation. RESULTS: All monomers induced both cellular differentiation and decrease in oxygen consumption. Cells treated with TEGDMA and Bis-GMA showed a significant enhancement of glucose consumption and lactate production. TEGDMA and HEMA induced GSH depletion stimulating G6PDH and GR activity. CONCLUSIONS: All the monomers under study affect the metabolism of HL-60 cells and show differentiating activity. Since alterations in cellular metabolism occurred at compound concentrations well below cytotoxic levels, the changes in energy metabolism and glutathione redox balance could be considered as potential mechanisms for inducing clinical and sub-clinical adverse effects and thus providing useful parameters when testing biocompatibility of dental materials.

In vitro comparison of the cytotoxicity of two orthodontic composite resins.

AIM: Several studies have reported that dental resin-based materials release substances with biological activity: for this reason in this study we evaluated the in vitro cytopathic effects of a self-curing and a light-curing orthodontic composite resins by a cytotoxicity test. METHODS: The cytotoxic potential of specimens, prepared according to the manufacturer instructions, was evaluated using the thiazolyl blue tetrazolium bromide (MTT) assay on the mouse fibroblast cell line (3T3 Swiss) with 2 cells-material contact systems: the 24 h extracts method and the indirect toxicity method. RESULTS: The results obtained in this study elicit a close agreement between the 2 procedures; from the data obtained in the reported experimental conditions, it was possible to establish that the examined chemical-cured material is more cytotoxic than the light-cured one. CONCLUSIONS: From a clinical point of view, the photo-polymerizable resins are undoubtedly more useful in the daily practice, because of the larger precision of the bonding obtainable by the greater period available for setting the brackets before their lock. The results obtained in this study, even considering the limits of the in vitro tests, represent a further favourable feature of the light-curing composite resins. However, further investigations about the influence of polymerization methods of the materials on the biological effects are suggested to contribute to the determination of the best clinical operative conditions.

Methionine 35 oxidation reduces toxic and pro-apoptotic effects of the amyloid beta-protein fragment (31-35) on isolated brain mitochondria.

Amyloid beta-peptide (AbetaP), the central constituent of senile plaques in Alzheimer's disease (AD) brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In particular, it is well known that oxidation of methionine 35, is strongly related to the pathogenesis of AD, since it represents the residue in AbetaP most susceptible to oxidation in vivo. In the present study, we used the fragment 31-35 of AbetaP, which has a single methionine at residue 35, in order to investigate the influence of the oxidation state of methionine-35 on the toxic and pro-apoptotic effects induced by Abeta(31-35) on isolated brain mitochondria. The obtained results show that exposure of isolated mitochondria from rat brain to AbetaP(31-35) determines (i) a large release of cytochrome c (ii) a significant reduction in mitochondrial respiration and (iii) a slight drop in the mitochondrial membrane potential (deltapsi). In contrast, the amplitude of these events resulted attenuated or completely abrogated in isolated brain mitochondria exposed to the AbetaP(31-35)Met35OX, in which methionine 35 was oxidized to methionine sulfoxide. We have further characterized the action of AbetaP(31-35) and Abeta(31-35)Met35OX peptide on PC12 cells. Although these two peptides, compromised mitochondrial function at a different extent as assessed by MTT reduction, neither one of them decreased cell viability as measured by Trypan Blue exclusion assay. The results obtained in this study support the hypothesis that the oxidative state of Met-35 may play a critical role in the mechanisms responsible of neurotoxicity exerted by this peptide.

Bezafibrate induces a mitochondrial derangement in human cell lines: a PPAR-independent mechanism for a peroxisome proliferator.

Bezafibrate is a hypolipidemic drug that belongs to the group of peroxisome proliferators because it binds to peroxisome proliferator-activated receptors type alpha (PPARs). Peroxisome proliferators produce a myriad of extraperoxisomal effects, which are not necessarily dependent on their interaction with PPARs. An investigation on the peculiar activities of bezafibrate could clarify some of the molecular events and the relationship with the biochemical and pharmacological properties of this class of compounds. In this view, the human acute promyelocytic leukemia HL-60 cell line and human rabdomiosarcoma TE-671 cell line were cultured in media containing bezafibrate and a number of observations such as spectrophotometric analysis of mitochondrial respiratory chain enzymes, NMR metabolite determinations, phosphofructokinase enzymatic analysis, and differentiation assays were carried on. Bezafibrate induced a derangement of NADH cytochrome c reductase activity accompanied by metabolic alterations, mainly a shift to anaerobic glycolysis and an increase of fatty acid oxidation, as shown by NMR analysis of culture supernatants where acetate, lactate, and alanine levels increased. On the whole, the present results suggest a biochemical profile and a therapeutic role of this class of PPARs ligands more complex than those previously proposed.

Cytotoxicity of a new endodontic filling material.

In vitro cell cultures have been widely used as a means of evaluating cytotoxicity of root canal filling materials. Following ANSI/ADA spec. no. 41, the aim of the present study was to investigate the biological compatibility of a new sealer (FibreFill) and compare it with some commercially available endodontic sealers (Bioseal and Acroseal). Mouse 3T3 fibroblasts were seeded and cultured and subsequently extracts of the cements were added. After 24 hours incubation, the cellular vitality of fibroblasts was evaluated by the neutral red uptake test (NRU), which measures the membrane permeability. Data were collected and statistically analysed. Results showed that all tested materials exhibited mild cytotoxic effects, which are compatible with normal clinical use, and no statistically significant difference was noted between FibreFill and the other tested materials. Therefore, selection amongst these sealers should be based on other factors.

Bezafibrate as differentiating factor of human myeloid leukemia cells.

Bezafibrate belongs to the class of fibric acid derivatives usually used as antihyperlipidemia agents. From the biochemical point of view, these drugs show intriguing properties which leads one to think they may promote a differentiation process in tumour cells. This new pharmacological activity of fibrates could partially depend on the induction of an oxidative stress. To test this hypothesis, the effect of bezafibrate, as well as of clofibric acid and gemfibrozil, on growth, functional and cytochemical characteristics of human leukaemia-derived cell lines HL-60, U-937 and K-562 has been studied in some details. The results show that bezafibrate, gemfibrozil and clofibric acid, do induce differentiation in human myeloid leukaemia cell lines as indicated by several differentiation markers. Moreover fibrates, in dose dependent manner, significantly alter the cell cycle distributions, mainly leading to G0/G1 phase increment and G2/M phase reduction. The differentiating activity of fibrates could have significant implications both for the pharmacotoxicological profile of this class of compounds and for the pathophysiology of neoplastic disease.

Impaired reactive oxygen metabolism of phagocytic leukocytes in NIDDM patients. A role for non-enzymatic glycosylation of collagen.

Non-enzymatic glycosylation (NEG) of collagen has been previously shown to significantly influence the reactive oxygen metabolism (ROM) of phagocytic cells in healthy subjects. Considering the role of NEG in the pathophysiology of diabetes, we have further analysed the oxidative metabolism of polymorphonuclear cells (PMNs) and monocytes in 23 patients with non-insulin dependent diabetes mellitus in order to better elucidate a possible pathogenic role of NEG of the extracellular matrix in long-term complications of diabetes. Experiments were performed in triplicate on native-collagen and glycated-collagen coated vials, using a chemiluminescence (CL) assay. Results show that PMNs from diabetic patients display a significant increased basal and zymosan-induced CL activity with respect to controls that are not related to the glycation state of the substrate. Conversely, the CL activity of monocytes induced by zymosan shows a decrease in diabetic patients with respect to healthy volunteers (p < 0.05). Moreover, monocyte CL was reduced by the glycated matrix, both in healthy volunteers and in diabetic subjects (p < 0.05 and p < 0.01, respectively). These data highlight a complex role of phagocytic leukocytes in the pathophysiology of extracellular matrix alterations secondary to NEG that are typically present in clinical conditions such as diabetes or ageing.

The priming effect of gemfibrozil on reactive oxygen metabolism of phagocytic leucocytes. An intriguing side effect.

Gemfibrozil is an antihyperlipidaemia agent used in therapy to reduce the occurrence of coronary heart disease. Considering the biochemical and pharmacological peculiarities of this class of drugs, we investigated the influence of a single oral therapeutic dose of gemfibrozil on the reactive oxygen metabolism of phagocytic leucocytes. Analysis was carried out adopting a chemiluminescence assay. Results clearly indicate that gemfibrozil, acting as a primer, significantly enhances the induced reactive-oxygen-species (ROS) production by overall blood phagocytes (increment of Stimulation Index (SI) = +52% with respect to time 0 values; P < 0.01), by polymorphonuclear leucocytes (increment of SI = +28% with respect to control values; P < 0.01) and by monocytes (increment of SI = +83% with respect control values; P < 0.001), when these cells are stimulated by phorbol myristate acetate. This iatrogenic derangement of ROS metabolism could explain, at least in part, the occurrence of some side effects that occur in treatment with fibrates.

Monocyte oxygenation activities are significantly influenced by non-enzymatic glycosylation of collagen.

The pathophysiology of the acquired extracellular matrix alterations, like non-enzymatic glycosylation (NEG) of proteins secondary to diabetes or ageing, is not well characterized, particularly considering its relationship with leukocytes. We have analysed the influence of collagen NEG on the fundamental function of phagocytic cells, i.e. the production of reactive oxygen species (ROS). To this aim, we considered the activity of polymorphonuclear leukocytes and monocytes incubated in the presence of both non-glycated and glycated collagen. ROS production was monitored by chemi-luminescence (CL), a standardized and sensitive assay of phagocytes oxidative metabolism. All experiments were performed in triplicate on collagen-coated and glycated collage-coated vials. Results showed that PMNs ROS metabolism appeared unrelated to the glycation state of the substrate. Conversely, data regarding zymosan-induced CL by monocytes indicated a significant and intriguing decrease in reactive oxygen metabolism, which appeared greatly compromised by the glycation state of the matrix (monocytes in collagen, 197.4 +/- 31.2 vs. monocytes in glycated collagen, 138.0 +/- 20.4; p < 0.001 expressed as counts/cell/60 min). These data highlight the different role of polymophonuclear and monocytic phagocytes in the pathophysiology of the acquired extracellular matrix alteration secondary to non-enzymatic glycosylation (NEG) of proteins.

Effects of gemfibrozil on the oxygen transport properties of erythrocytes.

1. In the present study we have investigated the effects of the relatively low plasma concentrations of gemfibrozil (GFZ) found in clinical practice on the oxygen dissociation curve (ODC) of erythrocytes. 2. ODCs were measured at 30 degrees C and 37 degrees C and at pH 7.4: a) both on HbA solution and erythrocytes incubated in vitro with gemfibrozil and clofibric acid; b) on erythrocytes from healthy volunteers treated with a single oral dose of gemfibrozil. 3. These experiments showed a significant drug-induced shift of the ODC towards lower O2 affinity values without any significant modification of metabolic parameters of erythrocytes such as intracellular pH and intraerythrocytic levels of ATP and DPG. 4. In our experimental conditions gemfibrozil appears to lower both in vitro and in vivo, the partial pressure of oxygen required to give 50% of the haemes saturated with oxygen (P50) of erythrocytes from the control value of 24 +/- 0.5 mm Hg to 29 +/- 0.5 mm Hg (mean +/- s.d.; P < 0.02 by ANOVA). 5. These data clearly indicate that therapeutic doses of gemfibrozil may influence the oxygen transport properties of red cells. This effect could have relevant pharmacological and toxicological implications.

Capillary zone electrophoresis of peptides: prediction of the electrophoretic mobility and resolution.

The determination of the pKa values of some selected peptides of similar size was performed by microtitration, which makes possible an accurate determination of the peptide charge as a function of the solution pH. Capillary zone electrophoresis separation of these peptides on modified capillaries at acidic pH showed that the electrophoretic mobility correlates with the peptide charge. This observation suggests that when an appropriate charge value is used, the basic electrophoretic equation is respected and, at least at a peptide charge value less than 1, the utilization of alternative semi-empirical predictions is not necessary. As a general rule, a peptide separation at acidic pH values is to be preferred to that at basic pH values. In fact, at basic pH a separation in the absence of both electroosmotic flow and of spurious interactions between the peptides and the inner wall of the capillary is difficult, owing to the instability of capillary modification. Further, from the differences in the peptide charge, a prediction of the best resolution as a function of the pH could be obtained; in fact, the resolution, for peptides of similar size and in the absence of electroosmotic flow, is connected to a simple equation, where the principal term depends on the effective charge of the peptides, which is a function of the pH of the solution and the pKa values of the peptides. The predictions of resolution at acidic pH agreed well with the experimental results; the spatial resolution measured in the separation of met- and leu-enkephalin was virtually coincident with the predicted resolution; in the case of a mixture of four model tetrapeptides of sequence GGNA, GGQA, GGDA and GGEA some anomalous results with respect to the predicted resolutions were observed. Nevertheless, an acceptable prediction can also be made in this case.

Separation of reduced and oxidized glutathione by micellar electrokinetic capillary chromatography.

The separation of reduced and oxidized glutathione at an absolute sensitivity of about 100 pg by micellar electrokinetic capillary chromatography without derivatization is described. The time required for the separation is less than 10 min (the time between two following injections is about 15 min). The separation is characterized by high efficiency and good reliability. A partition mechanism is responsible for the high resolution observed. The method was utilized for the analysis of commercial preparations of glutathione and a good agreement with the expected results was obtained; the oxidation of the commercial glutathione in solution was easily analysed.