Cytotoxic effect of spermine on retinal pigment epithelial cells.
OBJECTIVE
A prior study showed inactivation of ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells by a specific irreversible inhibitor (5-fluoromethylornithine; 5-FMO) leading to cell death, in an in vitro model of gyrate atrophy (GA) of the choroid and retina. In the present study, the cytotoxicity of metabolites of ornithine, especially spermine, in RPE cells was investigated, to clarify the mechanism of ornithine cytotoxicity in RPE cells.


METHODS
RPE cells were incubated with ornithine or compounds involved in ornithine metabolic pathways. The effects on RPE cell viability and proliferative activity were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric and [(3)H]thymidine incorporation assays. Incorporation of spermine into RPE cells was examined by using [(14)C]spermine and dansyl-spermine. To assess spermine-induced RPE cell death, cells were double stained with annexin V and propidium iodide and subjected to flow cytometry.


RESULTS
Ornithine, arginine, glutamate, proline, creatine, glycine, and putrescine exhibited no effects on the viability and proliferative activities of RPE cells, whereas spermidine and spermine (10 mM) inhibited [(3)H]thymidine incorporation by 13% and 89%, respectively. The inhibition of [(3)H]thymidine incorporation by spermine was dose dependent and was observed as early as 4 hours after addition. Further, spermine was incorporated and accumulated in the perinuclear region of RPE cells. Apoptotic RPE cell death was induced by spermine in a dose-dependent manner.


CONCLUSIONS
The present results demonstrated that excessive spermine is cytotoxic to RPE cells and suggest that metabolites of ornithine, especially spermine, may be involved in the mechanism of RPE degeneration in GA.
Kaneko
Shiho
S
Department of Ophthalmology, Kansai Medical University, Osaka, Japan.
Ueda-Yamada
Mami
M
Ando
Akira
A
Matsumura
Shinji
S
Okuda-Ashitaka
Emiko
E
Matsumura
Miyo
M
Uyama
Masanobu
M
Ito
Seiji
S
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002417:Cattle; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D003124:Colorimetry; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D018613:Microscopy, Confocal; D009952:Ornithine / Q000633:toxicity; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000473:pathology; D011419:Propidium / Q000378:metabolism; D013096:Spermine / Q000633:toxicity*; D013778:Tetrazolium Salts; D013844:Thiazoles; D013936:Thymidine / Q000378:metabolism
D017304:Annexin A5; D013778:Tetrazolium Salts; D013844:Thiazoles; D013096:Spermine; D011419:Propidium; D004247:DNA; D009952:Ornithine; C022616:thiazolyl blue; D013936:Thymidine