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Published on: December 28, 2021
Cytotoxic effect of spermine on retinal pigment epithelial cells
Shiho Kaneko1, Mami Ueda-Yamada, Akira Ando
1Department of Ophthalmology, Kansai Medical University, Osaka, Japan.
Purpose:
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.
Insights
Excessive spermine, a metabolite of ornithine, is cytotoxic to retinal pigment epithelial (RPE) cells. This finding suggests spermine may contribute to RPE degeneration in gyrate atrophy (GA) of the choroid and retina.
Area of Science:
- Ophthalmology
- Cell Biology
- Biochemistry
Background:
- Gyrate atrophy (GA) is an inherited chorioretinal disease.
- Previous studies indicated ornithine-delta-aminotransferase (OAT) deficiency leads to RPE cell death.
- The precise mechanism of ornithine-induced RPE cell toxicity in GA remains unclear.
Purpose of the Study:
- To investigate the cytotoxicity of ornithine metabolites, particularly spermine, in retinal pigment epithelial (RPE) cells.
- To elucidate the role of spermine in RPE cell degeneration relevant to gyrate atrophy (GA).
Main Methods:
- RPE cells were exposed to ornithine and its metabolic pathway compounds.
- Cell viability and proliferation were assessed using MTT and [3H]thymidine incorporation assays.
- Spermine uptake, localization, and apoptosis induction were analyzed via radiolabeling, microscopy, and flow cytometry.
Main Results:
- Spermine (10 mM) significantly inhibited [3H]thymidine incorporation in RPE cells in a dose- and time-dependent manner.
- Spermine was incorporated into and accumulated within the perinuclear region of RPE cells.
- Spermine induced dose-dependent apoptotic RPE cell death.
Conclusions:
- Elevated levels of spermine exhibit significant cytotoxicity towards RPE cells.
- Spermine, as a key ornithine metabolite, is implicated in the pathogenesis of RPE degeneration in gyrate atrophy (GA).
