Abstract
Purpose::
Carotenoids are used as therapeutic agents in treating AMD. However, carotenoids get oxidized and form carotenoid-derived aldehydes (CDA) in human and animal ocular tissues. Therefore, we have investigated the cytotoxic effect of CDA on human retinal pigment epithelial cells (ARPE-19).
Methods::
The ARPE cells grown in the DMEM/F-12 media with or without N-acetylcysteine (NAC) were incubated with CDA. The cell viability and apoptosis were determined by MTT assay and ELISA, respectively. Reactive oxygen species (ROS) levels were determined by fluorescent dye DCF-DA. The changes in mitochondrial membrane potential (MMP) were assessed by flow cytometry using JC-1 dye. Electrophoretic gel shift assays were carried out to determine the activation of transcription factors NF-kB and AP-1. Western blots analysis was performed to identify upstream signals of NF-kB.
Results::
There was a dose and time-dependent decline in cell viability and increased apoptosis upon incubation of RPE cells with CDA. The CDA treatments also lead to elevation in ROS levels in a dose-dependent manner. However, when the cells were pretreated with antioxidant NAC, the cytotoxicity of CDA in RPE cells was significantly ameliorated. The early apoptotic changes in RPE cells induced by CDA were associated with change in MMP. Pretreatment with NAC lead to prevention of change in MMP. In cells treated with CDA increased chromatin condensation was also observed. Furthermore, CDA induced the activation of NF-kB and AP-1 which was significantly inhibited by NAC.
Conclusions::
Our results provide evidence that CDA causes increased oxidative stress which activates NF-kB/AP-1 signalling in ARPE-19 cells. Thus prolonged carotenoid supplementation in AMD could generate CDA which could potentially cause imbalance in cellular redox state leading to ocular damage. If CDA have similar effects in patients with AMD, prolonged and excessive use of its precursor carotenoids could be harmful.
Keywords: carotenoids/carotenoid binding proteins • retinal pigment epithelium • age-related macular degeneration