Giuseppina Nocca

Optimization of phenylthiohydantoinamino acid separation by micellar electrokinetic capillary chromatography

Optimization of phenylthiohydantion (PTH)-amino acid separation by micellar electrokinetic capillary chromatography was achieved by the use of a weighted variable-size simplex algorithm. The optimization procedure concerned the pH of the aqueous buffer, the sodium dodecyl sulphate concentration and the percentage of organic solvent; the organic solvent used was either methanol or acetonitrile. In both instances the optimization procedure led to very similar final experimental conditions and migration order and times of the PTH-amino acids, showing that the organic solvent probably provides a better polydispersity of the micellar phase. The elution pattern observed in the two instances suggests that ionic interactions and polar repartition play a role in the separation mechanism, but other types of interaction cannot be excluded.

N-acetyl cysteine directed detoxification of 2-hydroxyethyl methacrylate by adduct formation

Cytotoxicity of the dental resin monomer 2-hydroxyethyl methacrylate (HEMA) and the protective effects of N-acetyl cysteine (NAC) on monomer-induced cell damage are well demonstrated. The aim of our study was to analyze the hypothesis that the protection of NAC from HEMA cytotoxicity might be due to direct NAC adduct formation. To this end, using HPLC we first measured the actual intracellular HEMA concentrations able to cause toxic effects on 3T3-fibroblasts and then determined the decrease in intracellular and extracellular HEMA levels in the presence of NAC. In addition, by capillary electrophoresis coupled with mass spectrometry analysis (CE-MS), we evaluated NAC-HEMA adduct formation. HEMA reduced 3T3 cell vitality in a dose- and time-dependent manner. The concentration of HEMA inside the cells was 15-20 times lower than that added to the culture medium for cell treatment (0-8 mmol/L). In the presence of 10 mmol/L NAC, both intracellular and extracellular HEMA concentrations greatly decreased in conjunction with cytotoxicity. NAC-HEMA adducts were detected both in the presence and absence of cells. Our findings suggest that the in vitro detoxification ability of NAC against HEMA-induced cell damage occurs through NAC adduct formation. Moreover, we provide evidence that the actual intracellular concentration of HEMA able to cause cytotoxic effects is at least one magnitude lower than that applied extracellularly.

Identification of glutathione-methacrylates adducts in gingival fibroblasts and erythrocytes by HPLC-MS and capillary electrophoresis

OBJECTIVES Methacrylic monomers are released, from dental composite resins, either into the oral cavity or in pulpal tissues, where they can cause local or systemic adverse effects. The mechanisms of these effects are not well understood, probably because such molecules can act at different levels also inducing a depletion of intracellular glutathione (GSH). GSH can detoxify methacrylates by conjugating their α,β-unsaturated carbon-carbon moiety to the thiol group, with the catalysis of glutathione S-transferases (GST). This reaction determines a GSH cellular depletion and belongs to the metabolism of α,β-unsaturated esters, protecting the body against the toxic effects of electrophiles. On the basis of the above considerations, this work aim is to set up a method for the detection of the adducts formed by methacrylic monomers with GSH in cells using HPLC coupled to mass spectrometry (HPLC-MS) and micellar electrokinetic capillary chromatography (MECK) techniques. METHODS AND RESULTS Adducts of glutathione with triethylene glycol dimethacrylate (TEGDMA) and hydroxyethyl methacrylate (HEMA) were incontrovertibly identified by HPLC-MS and MECK in human gingival fibroblasts and erythrocytes, both outside and inside cells. Molecular docking simulations of HEMA and TEGDMA in the experimental structure of glutathione S-transferase, are also reported to rationalize the effectiveness of such enzyme in the catalysis of the above described reaction. SIGNIFICANCE The setup of a method for the identification of GSH-methacrylate adducts allows to determine when the metabolic pathway involving such compounds is employed by cells for the detoxification of monomers leached from composite resins.

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.

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

Amyloid beta-peptide (AbetaP), the central constituent of senile plaques in Alzheimers 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.

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.

In vitro cellular detoxification of triethylene glycol dimethacrylate by adduct formation with N-acetylcysteine

OBJECTIVE Various protective effects of N-acetylcysteine (NAC) against triethylene glycol dimethacrylate (TEGDMA)-induced cell damage have been demonstrated, but so far there is no evidence on NAC direct monomer detoxification mechanism. Here, we hypothesized that NAC might reduce TEGDMA cytotoxicity due to direct NAC adduct formation. METHODS We measured the cytotoxic effects of TEGDMA in presence and in absence of NAC by MTT test. Then we analyzed the presence of TEGDMA-NAC adduct formation in extracellular and intracellular compartments by capillary electrophoresis-UV detection (CE-UV) and capillary electrophoresis-mass spectrometry (CE-MS) analytical techniques. Moreover, we quantified the effective intracellular and extracellular TEGDMA concentrations through HPLC in the presence and absence of 10 mmol/L NAC. RESULTS TEGDMA reduced 3T3 cell vitality in a dose- and time-dependent manner, while NAC decreased monomer cytotoxicity and extracellular monomer concentrations by a direct reaction with TEGDMA. The adducts between the two molecules were detected both in the presence and absence of cell. Moreover a signal ascribed to the methacrylic acid was present in the CE-UV electropherogram of cellular lysates obtained after incubation with TEGDMA. SIGNIFICANCE Our results suggest that in vitro detoxification capability of NAC against TEGDMA-induced cell damage might occur also through the formation of NAC-TEGDMA adduct.

Chitosan based nanoparticles functionalized with peptidomimetic derivatives for oral drug delivery

The goal of this study was to develop an optimized drug delivery carrier for oral mucosa applications able to release in situ bioactive molecules by using biopolymeric materials. Among them chitosan and poly(lactic-co-glycolic acid) (PLGA) have gained considerable attention as biocompatible carriers able to improve the delivery of active agents. The formulation of such vehicles in the form of nanoparticles (NPs) could permit to exploit the peculiar properties of nanomaterials in order to enhance the efficacy of active agents. Chitosan (CS) and PLGA chlorexidine dihydrochloride (CHX)-loaded NPs were synthesized by ionotropic gelation and osmosis based methodology respectively. In order to facilitate NPs adhesion on human dental surfaces, two different strategies were employed: PLGA particles with an external shell of CS to produce a positive surface charge allowing CHX loaded PLGA NPs to interact with the negative charged dental surfaces, while CS particles were functionalized with peptidomimetic derivative glutathione (GSH). The morphology was investigated by scanning electron microscopy. A sustained release profile of CHX from CS NPs was achieved. CS-based NPs adhered on human tooth surfaces in a simulated brushing and rinsing process and their in vitro toxicity evaluation on Human Gingival Fibroblasts (HGFs) was between 20 and 60% in all experimental conditions. Thanks to their adhesion properties and low cytotoxicity, the synthesized CS-based formulations may be efficiently exploited for therapy purposes or to enhance in vivo dental care (i.e. preparation of toothpastes or other cosmetics for daily oral care).

In vitro evaluation of the cytotoxicity of FotoSan™ light-activated disinfection on human fibroblasts

Summary Background Root canal disinfection needs to be improved because actual techniques are not able to eliminate all microorganisms present in the root canal system. The aim of the present study was to investigate the in vitro cytotoxicity of FotoSan (CMS Dental APS, Copenhagen Denmark), 17% EDTA and 2% chlorhexidine. Material/Methods Fibroblasts of periodontal ligament from healthy patients were cultured. FotoSan (with and without light activation for 30 sec.), 17% EDTA and 2% chlorexidine were used for the cell viability tests. Untreated cells were used as control. The cellular vitality was evaluated by MTT test. The production of reactive oxygen species (ROS) was measured using an oxidation-sensitive fluorescent probe. Results were statistically analyzed by ANOVA, followed by a multiple comparison of means by Student-Newman-Keuls, and the statistical significance was set at p<0.05. Results MTT tests showed that cytotoxic effects of FotoSan (both photocured and uncured) were statistically lower (p<0.05) than that observed using 2% Chlorhexidine, while no significant differences were found in comparison with 17% EDTA. No alterations in ROS production were detectable in any of the tested materials. Conclusions Since the toxicity of the FotoSan photosensitizer, both light-activated and not light-activated, is similar to common endodontic irrigants, it can be clinically used with precautions of use similar to those usually recommended for the above-mentioned irrigating solutions.

Effects of 1,4-butanediol dimethacrylate and urethane dimethacrylate on HL-60 cell metabolism.

The polymerization of methacrylic monomers present in dental composite resins never reaches completion and therefore the leakage of residual monomers into the oral cavity and into biological fluids can cause local and systemic adverse effects. This work was carried out to study the in vitro biochemical interactions of urethane dimethacrylate and 1,4-butanediol dimethacrylate monomers with HL-60 cells, a cell line assumed as an experimental model for simulating granulocyte behaviour. Our main finding was that both monomers induce cell differentiation at toxic concentrations and that cytotoxicity seems to be caused by alterations of glucose metabolism arising from mitochondrial dysfunction rather than from oxidative stress, which could not be altogether verified under our experimental conditions. Our study could be considered as a useful approach to investigate the biochemical mechanisms that contribute to the cytotoxicity of methacrylate compounds and it underlines the importance of assessing such parameters for testing biocompatibility in order to promote the development of better and safer dental materials.