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

• Published in: Dental materials 2011-05, Vol.27 (5), p.e87-e98
• Main Authors: Nocca, Giuseppina, Ragno, Rino, Carbone, Virginia, Martorana, Giuseppe E, Rossetti, Diana V, Gambarini, Gianluca, Giardina, Bruno, Lupi, Alessandro
• Format: Article
• Language: English
• Subjects: Advanced Basic Science; Biocatalysis; Capillary electrophoresis; Cells, Cultured; Chromatography, High Pressure Liquid; Chromatography, Micellar Electrokinetic Capillary; Cysteine - metabolism; Cytoplasm - metabolism; Dentistry; Erythrocytes - enzymology; Erythrocytes - metabolism; Ethacrynic Acid - metabolism; Extracellular Space - metabolism; Fibroblasts - enzymology; Fibroblasts - metabolism; Gingiva - cytology; Gingiva - enzymology; Gingiva - metabolism; Glutathione - analysis; Glutathione - metabolism; Glutathione Transferase - metabolism; GSH; HPLC–MS; Humans; Materials Testing; Metabolism; Methacrylates - analysis; Methacrylates - metabolism; Methacrylic monomers; Molecular Conformation; Polyethylene Glycols - analysis; Polyethylene Glycols - metabolism; Polymethacrylic Acids - analysis; Polymethacrylic Acids - metabolism; Tandem Mass Spectrometry
• ISSN: 0109-5641; 1879-0097
• DOI: 10.1016/j.dental.2011.01.002

Abstract 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.