Reactive oxygen species generation and photo-cytotoxicity of eugenol in solutions of various pH.

In order to clarify the mechanism of photo-damage caused by eugenol (4-allyl-2-methoxyphenol), we measured cell survival in the presence of eugenol at concentrations of 10(-3) - 10(-7) M, with and without VL (visible light) irradiation by a VL dental lamp and at various pHs (7.2, 7.8 and 8.2) using two different cells (HSG, a human submandibular gland tumor cell line; HGF, a human gingival fibroblast in primary culture). Also, ROS (reactive oxygen species) generation in the above adherent single cells was measured by ACAS laser cytometry combined with CDFH-DA, a peroxide probe. The survival of both HSG and HGF cells treated with eugenol was significantly decreased as the VL irradiation time and/or the pH of the medium was increased. The amount of ROS generated from eugenol was also enhanced by increasing the VL irradiation time and elevating the pH of the medium. Cytotoxicity and ROS generation of HGF cells were significantly lower than that of HSG cells. Glutathione (1 mM) or cysteine (1 mM) protected the photo damages. We conclude that the cytotoxicity of VL-irradiated eugenol possibly was caused by the generation of eugenol radicals and additionally by ROS, both of which were produced dependent on the dose of eugenol, length of irradiation time, and pH of the medium.