Flavin-containing enzymes as a source of reactive oxygen species in HEMA-induced apoptosis

Oxidative stress induced by compounds of dental composites like 2-hydroxyethyl methacrylate (HEMA) due to excess formation of reactive oxygen species (ROS) disturbs vital cell functions leading to apoptosis. The sources of ROS in cells exposed to resin monomers are unknown. The present study investigates functions of flavin-containing ROS and RNS (reactive nitrogen species) producing enzymes in cells exposed to HEMA.

The formation of oxidative stress in RAW264.7 mouse macrophages exposed to HEMA (0-6-8mM) was determined by flow cytometry (FACS) after staining of cells with 2'7'-dichlorodihydrofluorescin diacetate (H₂DCF-DA), dihydroethidium (DHE) or dihydrorhodamine 123 (DHR123). Cells in apoptosis or necrosis were identified by annexin-V-FITC/propidium iodide labeling followed by FACS analysis. Expression of ROS/RNS producing enzymes was analyzed by Western blotting.

DCF fluorescence increased in cells exposed to HEMA for 1h suggesting the production of hydroxyl radicals, H₂O₂, or nitric oxide and superoxide anions which form peroxynitrite (ONOO-). Increased DHR123 fluorescence after 24h indicated the formation of mostly H₂O₂. The induction of apoptosis in the presence of HEMA was decreased by low concentrations of diphenylene iodonium (DPI), an inhibitor of flavin-containing enzymes. Expression of p47^phox, a regulatory subunit of the superoxide producing Nox2, was downregulated, and the expression of NOS which produces nitric oxide (NO) was possibly inhibited by feedback loop mechanisms in HEMA-exposed cultures. Inhibition of HEMA-induced apoptosis by VAS2870 or apocynin further suggested a crucial function of Nox2.

The present findings show the physiological relevance of flavin-containing enzymes in monomer-induced oxidative stress and apoptosis.