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Previous studies revealed that primarily small and relatively hydrophilic comonomers, such as TEGDMA, leach out of resin-based restorative materials into aqueous media. Subsequently, these compounds may cause detrimental reactions with intracellular metabolic systems. The present experiments attempted to elucidate the interactions of TEGDMA with the important intracellular reducing agent glutathione (GSH). The influence of various concentrations of TEGDMA (0.5-7.5 mM) on viability and intracellular GSH concentration of primary human gingival fibroblasts was determined by means of a fluorescence assay (monobromobimane) performed in microtiter plates. Cells were treated with TEDGMA between 2 and 24 h. The incubation of fibroblasts with TEGDMA even at subtoxic concentrations quickly decreased the intracellular glutathione level to 30-50% of controls within the first 2-6 hours. However, no simultaneous adverse effect on cell viability was found. Longer incubation periods up to 24 h caused a regulatory reincrease at TEGDMA concentrations

Earlier studies have shown that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) and the photostabilizer 2-hydroxy-4-methoxybenzophenone (HMBP) are cytotoxic and inhibit cell growth. It was the aim of this study to elucidate the underlying metabolic effects of TEGDMA and HMBP on immortal contact-inhibited Swiss albino mouse embryo cells (3T3 fibroblasts) by nuclear magnetic resonance (NMR) spectroscopy. Cell extracts and culture media were analyzed by NMR spectroscopy for metabolic changes after incubation for 24 hours with ED20-concentrations of TEGDMA and HMBP. TEGDMA could be detected in all fractions (cytosol, lipid fractions, and culture media) of 3T3 cells, while HMBP was found only in the lipid fraction accumulated at a maximum rate (51 nmol/mg DNA) compared with TEGDMA (27 nmol/mg DNA). TEGDMA increased the concentration of phosphomonoesters to 180+/-36% and decreased the phosphodiesters to 65+/-5% of controls (control = 100%). Thus, the turnover of phospholipids was enhanced, whereas content and composition of phospholipids of membranes did not alter markedly. Additionally, TEGDMA changed the metabolic state of cells, indicated by slight decreases of nucleoside triphosphates and an increase in the ratio of nucleoside diphosphates to nucleoside triphosphates, while HMBP had no effect. The most remarkable effect of TEGDMA was a nearly complete decline of the intracellular glutathione levels. Analysis of our data shows that NMR spectroscopy of cell-material interactions may reveal metabolic effects of organic test substances which are not detectable by standard in vitro assays. The comonomer TEGDMA affected the metabolism of the cells on different levels, while HMBP accumulated in the lipid fraction and induced significantly fewer effects on cell metabolism.

Glutathione (GSH) is important for the self-protection of cells against oxidative stress and toxic xenobiotics, whereas reactive oxygen species (ROS) at elevated concentrations may cause detrimental alterations of cell membranes, DNA, and other cellular structures. The present investigation addressed the effects of triethylene-glycoldimethacrylate (TEGDMA) and camphorquinone (CQ) on glutathione metabolism and the formation of ROS in oral cells. Primary human pulp fibroblasts were exposed to various concentrations of TEGDMA and CQ (0.1-5 mM). Subsequently, GSH concentration and ROS formation were analyzed with the use of the monobromobimane assay (GSH) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) (ROS). The endogenous ROS hydrogen peroxide (H2O2) was used as a positive control (0.02-2 mM). TEGDMA significantly decreased GSH at concentrations between 0.5 and 5 mM (p<0.05), but did not elevate ROS levels. Contrary, CQ increased ROS formation at concentrations>or=1 mM, but had only a moderate effect on GSH at the highest test concentration. Hydrogen peroxide increased ROS and simultaneously decreased GSH at concentrations of >or=0.2 mM. These data show that the investigated substances may cause cell damage due to various mechanisms, GSH decrease and/or ROS increase. As a consequence, TEGDMA and CQ released into an aqueous environment from resinous materials might interact, thus generating significant cytotoxic effects even at low concentrations.

Responsive or smart contrast agents (SCAs) represent a promising direction for development of novel functional MRI (fMRI) methods for the eventual noninvasive assessment of brain function. In particular, SCAs that respond to Ca 2+ may allow tracking neuronal activity independent of brain vasculature, thus avoiding the characteristic limitations of current fMRI techniques. Here we report an in vitro proof-ofprinciple study with a Ca 2+ -sensitive, Gd 3+ -based SCA in an attempt to validate its potential use as a functional in vivo marker. First, we quantified its relaxometric response in a complex 3D cell culture model. Subsequently, we examined potential changes in the functionality of primary glial cells following administration of this SCA. Monitoring intracellular Ca 2+ showed that, despite a reduction in the Ca 2+ level, transport of Ca 2+ through the plasma membrane remained unaffected, while stimulation with ATP induced Ca 2+ -transients suggested normal cellular signaling in the presence of low millimolar SCA concentrations. SCAs merely lowered the intracellular Ca 2+ level. Finally, we estimated the longitudinal relaxation times (T 1 ) for an idealized in vivo fMRI experiment with SCA, for extracellular Ca 2+ concentration level changes expected during intense neuronal activity which takes place upon repetitive stimulation. The values we obtained indicate changes in T 1 of around 1−6%, sufficient to be robustly detectable using modern MRI methods in high field scanners. Our results encourage further attempts to develop even more potent SCAs and appropriate fMRI protocols. This would result in novel methods that allow monitoring of essential physiological processes at the cellular and molecular level.

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A series of bimodal metabotropic glutamate-receptor targeted MRI contrast agents has been developed and evaluated, based on established competitive metabotropic Glu receptor subtype 5 (mGluR 5 ) antagonists. In order to directly visualize mGluR 5 binding of these agents on the surface of live astrocytes, variations in the core structure were made. A set of gadolinium conjugates containing either a cyanine dye or a fluorescein moiety was accordingly prepared, to allow visualization by optical microscopy in cellulo. In each case, surface receptor binding was compromised and cell internalization observed. Another approach, examining the location of a terbium analogue via sensitized emission, also exhibited nonspecific cell uptake in neuronal cell line models. Finally, biotin derivatives of two lead compounds were prepared, and the specificity of binding to the mGluR 5 cell surface receptors was demonstrated with the aid of their fluorescently labeled avidin conjugates, using both total internal reflection fluorescence (TIRF) and confocal microscopy.