Abstract
Purpose :
Controversy exists regarding the dosage and treatment of homocysteine in ocular cells. Homocysteine is a non-proteinogenic amino acid involved in methionine metabolism. Elevated homocysteine (Hcy) is known as hyperhomocysteinemia and is implicated in several ocular disorders, including age related macular degeneration (AMD). Retinal pigmented cells (RPE) are the primary cellular target for pathologic changes in AMD. Hhcy is known to induce reactive oxygen species production leading to oxidative stress. Melatonin (Mel) is a hormone important for circadian rhythms but is also an antioxidant. Limited literature exists regarding the role of Mel as a potential treatment for elevated Hcy. The purpose of the current study was to evaluate the therapeutic potential of Mel in elevated Hcy using the RPE cell culture model system.
Methods :
ARPE-19 cells were grown in DMEM F-12 media supplemented with 10% FBS and 1% Penicillin/Streptomycin. MTT assay, Apoptosis/Necrosis Assay, and oxidative stress assays were used to determine manor of cell death of ARPE-19 cells after treatment with Hcy (500 μM) and Mel (5-10 μM). Changes in cellular morphology were determined by phase-contrast microscope images taken pre- and post-treatment, and immunocytochemical analysis of RPE specific proteins. H2O2 acted as the positive control for cellular death. Data was analyzed using a one-way ANOVA. Statistics were analyzed using GraphPad Prism 9 software.
Results :
As concentrations of Hcy increased, instances of cell viability decreased with significant values (p<0.05) at 50, 500, and 1000 μM Hcy. Cells treated with Mel or co-treated with Hcy and Mel resulted in increased cell viability. Cells treated with Hcy resulted in cellular death via apoptosis, however, cells co-treated with Mel showed a decrease in cellular death via apoptosis. Cells treated with Mel showed a decrease in oxidative stress when compared to the positive control. Abnormal cellular morphology was observed in cells treated with Hcy at concentrations over 50 μM. Conditions imporoved with co-treament with Mel.
Conclusions :
We found that cells treated with Hcy led to cell death via apoptosis at concentrations above 50 μM. We also found that cells co-treated with Hcy and Mel resulted in an increase in cell proliferation, and that Mel shows protective effects against oxidative stress. This demonstrates that Mel has potential protective effects against elevated Hcy.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.