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Sub-lethal Concentrations of HEMA Alter Protein Expression in Human Monocytes



Objective: Several in vitro studies have shown increased intracellular level of reactive oxygen species (ROS) after exposure to 2-hydroxyethyl methacrylate (HEMA), a commonly used monomer in resin-based dental materials. This study aimed to investigate the initial changes of the proteome in a human monocyte cell line after exposure to HEMA by using Stable Isotope Labelling with Amino Acids in Cell culture (SILAC). This approach has the potential to reveal key information of primary cellular targets in HEMA exposed cells. Method: Two parallel cell cultures (A and B) of the human monocyte leukaemia cell line, THP-1 (ECACC, the European Tissue Type Culture Collection) were grown in RPMI media for SILAC. Culture A was labelled with C13 L-Arginine and L-Lysine (R6K6 medium), and culture B with unlabelled L-Arginine and L-Lysine (R0K0 medium). After complete labelling (6 cell divisions), the cells in culture A were exposed to sub-lethal concentrations of HEMA (0.5 - 2 mM), culture B was added medium only. Samples for shotgun proteomic analyses were prepared by mixing HEMA exposed cell culture A with unexposed cell culture B. The experiment was performed again (label-swap) exposing culture B for HEMA. The proteins were analysed by an Orbitrap Q Exactive mass spectrometer after tryptic digestion. Protein identification and SILAC quantitation (ratio of R6K6-labelled: R0K0-labelled peptides) were done using the MaxQuant and Proteome Discoverer software. Result: Approximately 3900 proteins were detected and more than 2800 were quantified. The levels of several proteins were altered after HEMA exposure. Changes in proteins associated with endoplasmic reticulum (ER)-stress seem to be an early response to the HEMA concentrations tested. Conclusion: HEMA affects protein regulation in human monocytes at sub-lethal concentrations. The identified changes may be linked to HEMA effects in ER.
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