To study the initial changes in the proteome in a human monocyte cell line after exposure to methacrylate monomer.
The study was performed using the SILAC (Stable Isotope Labelling with Amino Acids in Cell culture) method. Human THP-1 monocyte cells (ECACC, the European Tissue Type Culture Collection) were cultured in either heavy medium (medium containing 13C labeled L-Arginine and 13C labelled L-Lysine) or light medium containing same concentration of unlabeled (12C) L-Arginine and L-Lysine. After eight cell divisions, cells cultured in heavy medium were exposed to the methacrylate monomer 2-hydroxymethacrylate (HEMA; CAS. 868-77-9, Fluka Chemie AG, Buchs, Switzerland) for 24 h and mixed with the same amount of control cells cultured in light medium. To correct for experimental errors, a label-swap replication was performed (exposed cells in light medium mixed with control cells in heavy medium). The ratio of cellular proteins containing heavy and light arginine and lysine were determined by shotgun proteomic analyses. Tryptic peptides of the samples were analyzed with LC/MS-MS (liquid chromatography/double mass spectrometry). Proteins were identified and quantified using Max Quant and Proteome Discoverer software. Cell viability was assessed by the MTT assay.
The MTT assay did not reveal any effects of HEMA at the concentrations used. Increased levels of several proteins indicative of adaptation to oxidative stress (e.g. pirin, glutathione reductase and thioredoxin reductase 1) were measured in the HEMA exposed cells. Increased levels of Hsp70 family proteins indicate protein damage after HEMA exposure, possibly as a result of increased oxidative stress. The effects increased with increasing HEMA concentration.
It has been suggested that reactive oxygen species are involved in HEMA toxicity. The upregulated levels of antioxidant enzymes and Hsp70 family proteins showed that HEMA exposure lead to cellular stress at concentrations that had no effect on cell viability.
Proteome Changes Indicate Oxidative Stress and Protein Damage after Methacrylate Exposure in Human Monocyte Cell Line
Dahl JE, Michelsen VB, Bruun JA, Jensen E, Örtengren U, Samuelsen JT.
Toxicologist 2016; 150: 341 (abstract 2445)