Effects of Ion Beam Radiation on the Expression of Glutathione Peroxidase in Cultured Human Retinal Vascular Endothelial Cells Exposed to L-Dopa

Abstract

Purpose: : Damage to retinal endothelial cells (RE) is a characteristic feature of the pathogenesis of senile macular degeneration and diabetic retinopathy. Glutathione peroxidase (GPX) reduces lipid peroxides and causes the concomitant oxidation of glutathione. Faucher et al. measured the expression of bax and bcl-2 genes in human endothelial-like cell-line overexpressing GPX. We found that gamma-ray irradiation prevented programmed cell death regulated by the p53 and bcl-2 genes in organ-cultured ciliary bodies, and was useful in the preservation of organs by protecting against the effects of inflammatory reactions. Deficiency of L-dopa, used in the treatment of Parkinson’s disease, causes degeneration of the substantia nigra in the brain. We demonstrated that L-dopa induced the production of NO and superoxide, had cytotoxic effects on RPE cells, and that oxidative stress, such as hyperoxia, augmented the L-dopa-induced cytotoxicity in aortic endothelial cells. Therefore, to investigate how L-dopa influences GPX in preventing damage to membrane phopholipids of RE, we used ion beams to induce the oxidative stress that influences GPX and measured the expression GPX with a LightCycler system by using a real time reverse transcriptase polymerase chain reaction (RT-PCR).

Methods: : Human RE incubated in vitro with 250 µM L-dopa were exposed to three different ion beams with different linear energy transfer (LET): ⁴He (16.2 KeV/µm), ¹²C (108 KeV/µm), and ²⁰Ne (321 KeV/µm). The total radiation dose was 20 Gy for every irradiation, but the property of spatial distribution of energy deposition to the cells were different according to LET. We collected RE after 0, 4, 8, 24 hrs of irradiation, extracted total cellular RNA, and then using designed primers. The expression of 18S RNA and GPX was measured using the LightCycler system.

Results: : Exposure to L-dopa inhibited the expression of GPX in RE. The expression of GPX in RE incubated with L-dopa decreased significantly after exposure to ⁴He and ¹²C. In contrast, ²⁰Ne-ion irradiation significantly increased the expression of GPX in RE incubated with L-dopa.

Conclusions: : We demonstrated that the expression GPX in RE incubated with L-dopa varied with different distribution of energy deposition by different ion beam radiations. The result may become a clue for elucidating how L-dopa influences GPX gene expression.

Keywords: antioxidants • retinal degenerations: cell biology • radiation damage: light/UV