Protein S-glutathionylation (PSSG) is the reversible formation of mixed disulfides between glutathione and protein cysteinyl residues. This process is suggested to be a physiologically central redox signaling mechanism. Several methacrylates that are known to leak from resin based biomaterials, form adducts spontaneously with the cysteine-thiol of glutathione (GSH) and causes increased cellular levels of reactive oxygen species (ROS) in vitro. Both these changes could potentially influence PSSG. Increased oxidative stress may shift the state of protein thiols against an oxidized state (PSSG), while a reduced level of PSSG could be a result of GSH-depletion. In this study we aim to examine if methacrylate exposure affect the cellular PSSG state. The human bronchial epithelial cell line BEAS-2B grown in Lechner and LaVeck (LHC9) medium was used as a model system. The cells were exposed to methylmethacrylate (MMA), 2-hydroxyethylmethacrylate (HEMA), triethyleneglycol-dimethacrylate (TEGDMA) and glycerol-dimethacrylate (GDMA). The MTT assay was used to determine cell viability after 24 h exposure. Cellular GSH and ROS levels were measured by flow cytometry after 4 h exposure using the fluorescent probes monobromobimane (MBBr) and dichloro-dihydro-fluorescein diacetate (DCFH-DA), respectively. After 4 h exposure, the cellular PSSG state was visualized by western blotting using a PSSG specific antibody. No change in viability was detected by the MTT assay after exposure to <16 mM MMA, <8 mM HEMA, <2 mM TEGDMA and <1 mM GDMA. Using these concentrations, significant glutathione depletion and increased cellular ROS were measured in cells exposed to HEMA, TEGDMA and GDMA. No changes were observed in MMA exposed cells. Western blotting showed one prominent protein-band at approximately 40 kDa. Incubating the filter with antibodies to both β-actin and PSSG showed identical migration in the gel of the detected proteins. The intensity of the PSSG band decreased after exposure to all methacrylates, although not significantly in MMA exposed cells. In summary, this study shows that protein-S-glutathionylation of at least on protein, probably β-actin, is reduced in BEAS-2B cells after exposure to methacrylates. The decrease was most evident after exposure to methacrylates with higher thiol-reactivity (higher GSH-depletion observed).
Effect of Components in Resin-Based Dental Restoratives on Protein-S-Glutathionylation
Samuelsen JT, Uvsløkk S, Morisbak E, Becher R, Dahl JE.
Toxicologist 2016; 150: 340 (abstract 2443).