EFFECTS OF ION BEAM IRRADIATION ON THE EXPRESSION OF GLUTATHIONE PEROXIDASE IN CULTURED HUMAN RETINAL VASCULAR ENDOTHELIAL CELLS EXPOSED TO L-DOPA

Abstract

Purpose: We previously showed that L-3,4-dihydroxyphenylalanine (L-dopa) induces the production of nitric oxide (NO) and superoxide and has cytotoxic effects on cultured bovine retinal pigment epithelial (RPE) cells. Oxidative stresses such as hyperoxia or visible light augment L-dopa-induced cytotoxicity in aortic endothelial cells. In this study, we examined the influence of L-dopa on the inactivation of reactive oxygen species by glutathione peroxidase (GPx), which protects in human retinal vascular endothelial (RE) cells from free radical damage. We used ion beams to induce oxidative stress and measured GPx expression by means of real-time-reverse-transcriptase- polymerase chain reaction (RT-PCR) analysis.
Methods: Human RE cells incubated in vitro with 250 μM L-dopa were exposed to heavy -ions such as 350 MeV Ne, 220 MeV C, and 50 MeV He. Total cellular RNA was isolated from RE cells after 0, 4, 8, and 24 hr of irradiation and was reverse transcribed using primers specific for GPx and 18S ribosomal RNA (rRNA). Quantitative real-time RT-PCR was performed to compare the effects of different ion beams on rRNA and GPx expression using the LightCycler^® system.
Results: Exposure to L-dopa inhibited GPx expression in human RE cells. GPx expression in RE cells incubated with L-dopa decreased significantly after exposure to 50 MeV He and 220 MeV C, disturbing the defense mechanism against the L-dopa-induced production of reactive oxygen species. In contrast, 350 MeV Ne irradiation significantly increased GPx3 expression.
Conclusions: We showed the influence of ion beams on L-dopa-induced stress rather than the influence of L-dopa on ion beam-induced stress because GPx expression in L-dopa -treated human RE cells depended on the types of heavy-ion used during irradiation. L-dopa and ion beams are concentration-dependent pro-oxidative stressors of RE cells.