Determination of reactive oxygen species generated by phorbol 12-myristate 13-acetate-stimulated oral polymorphonuclear cells from healthy human volunteers without any dental problems

Abstract

Human oral polymorphonuclear cells (OPMNs) play an important role in the defence of oral cavity from bacteria by releasing reactive oxygen species (ROS). The purpose of the study is to determine ROS generated by OPMNs collected from healthy volunteers without any dental problems by applying a luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt-dependent chemiluminescence (CL) response in combination with radical and ROS scavengers. In the CL response induced by OPMNs primed with phorbol 12-myristate 13-acetate, 0.23M 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, 12.5U/ml of super oxide dismutase (SOD) as a superoxide anion (O2−) scavenger, and 0.96M dimethyl sulfoxide (DMSO) as a hydroxyl radical (OH) scavenger inhibited the response by approximately 90%, 70%, and 60%, respectively. The inhibitory effects were obtained in a range of concentrations where viability of the OPMNs exposed to DMPO, SOD, and DMSO were more than 70%. Electron spin resonance-spin trapping analysis confirmed that at least O2− and OH were generated via primed OPMNs. Furthermore, the addition of both 12.5U/ml of SOD and 0.96M DMSO inhibited the CL response by more than 90%, which was in accordance with the inhibition rate obtained by the addition of DMPO. Therefore, it is suggested that around 90% of the CL response is induced by free radicals, and at least around 70% of the radicals are SOD-inhibitable, meaning that they are originally derived from O2−. In addition, some of the OH are generated independently of O2− because if all of the OH were formed through dismutation of O2−, only 70% of the CL response would be inhibited by the combination of SOD and DMSO as was inhibited by SOD alone.The present study suggests that OPMNs in health individuals have an ability to generate free radicals, which consist mainly of O2−, OH and possibly an intermediate ROS derived from O2− and OH.