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Resin based biomaterials, such as dental composites, are based on methacrylate monomers that are polymerized in situ. Due to lacement before curing and incomplete polymerization, patients are exposed to unreacted monomers that leak from these materials. Several methacrylates spontaneously adduct with the cysteine-thiol of glutathione (GSH), and it is commonly suggested that increased cellular levels of reactive oxygen species (ROS) caused by GSH depletion is the main mechanism of observed methacrylate toxicity in vitro. However, studies verifying this hypothesis are lacking. In the present study we aimed to further map the suggested relation between GSH-depletion and methacrylate toxicity by comparing the molecular effects of methacrylate exposure and GSH-depletion by inhibiting GSH-synthesis. Human bronchial epithelial cell line BEAS-2B grown in Lechner and LaVeck (LHC9) medium was used as a model system. Cells were exposed to 2-hydroxyethylmethacrylate (HEMA) and buthionine sulfoximine (BSO), an inhibitor of glutamatecysteine ligase (GCL; the rate limiting enzyme in cellular GSH synthesis). 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 and 8 h exposure using the fluorescent probes  monobromobimane (MBBr) and dichloro-dihydro-fluorescein diacetate (DCFHDA), respectively. Western blotting was used to determine altered protein-expression in unexposed cells and cells exposed to HEMA and BSO. Exposure to both HEMA and BSO resulted in GSH-depletion and increased ROS-levels. However, only HEMA-exposure reduced cell viability in a concentration dependent manner. In the additional comparison of BSO and HEMA, only HEMA concentrations that had no effect on cell viability were used. Confirming previous studies, HEMA exposure resulted in increased cellular levels of proteins associated with increased ROS-handling capacity, increased thiol-reducing capacity, increased protein degradation and increased capacity to detoxify lipid peroxidation products. In BSO-exposed cells, only increased levels of proteins with ROS-handling capacity was observed. In summary, this study does not support the hypothesis suggesting the main mechanism of methacrylate toxicity to involve GSH-depletion and increased oxidative load. The results from our study suggest that methacrylate toxicity involves direct interactions with proteins and lipids.

Reference
J. T. Samuelsen ¹, R. Becher ², H. Valen ¹, and J. E. Dahl ¹.
¹ NIOM, Oslo, Norway; and
² NIPH, Oslo, Norway.