April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Effects of Sub-Lethal Levels of Omega-3 and -6 Polyunsaturated Fatty Acid Oxidation End Products in RPE Cell Culture
Author Affiliations & Notes
  • A. R. Wielgus
    Duke University, Durham, North Carolina
  • S. W. Cousins
    Duke University, Durham, North Carolina
  • G. Malek
    Ophthalmology and Pathology,
    Duke University, Durham, North Carolina
  • Footnotes
    Commercial Relationships  A.R. Wielgus, None; S.W. Cousins, None; G. Malek, None.
  • Footnotes
    Support  International Retinal Research Foundation and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 6001. doi:
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      A. R. Wielgus, S. W. Cousins, G. Malek; Effects of Sub-Lethal Levels of Omega-3 and -6 Polyunsaturated Fatty Acid Oxidation End Products in RPE Cell Culture. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6001.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Age-related macular degeneration (AMD) is the leading cause of vision loss. Studies on the relationship between dietary fat and AMD have concluded that a higher intake of omega-6 polyunsaturated fatty acids (PUFAs), considered pro-inflammatory, may be associated with a greater risk, while diets rich in omega-3 PUFAs, considered anti-inflammatory, are associated with a reduced risk for AMD. Given that PUFAs are vulnerable to oxidation and may differentially modulate mitochondrial function, as seen in cardiovascular disease, we investigated the affect PUFAs and their oxidation products on mitochondrial function and morphology of RPE cells in vitro.

Methods: : Confluent ARPE19 cells were fed 0-30 µM omega-6 (linoleic and arachidonic), omega-3 (α-linolenic and docosahexanoic) PUFAs, or one of their oxidation end products (4-HNE and 4-HHE) for 7 days. Morphological changes of the cells during the incubation process were monitored through bright field microscopy. Integrity of tight junctions and cytoskeleton alterations in the cells were evaluated using immunocytochemical staining with antibodies to ZO-1 and cytokeratin 18. Transepithelial resistance of cells grown in transwells was monitored with Millicell-ERS ohmometer (Millipore). Cell metabolism and cytoplasmic membrane permeability were determined with MTS and LDH assays, respectively. The effect of PUFAs and their oxidation end products on mitochondria (mitochondria number, membrane potential, and reactive oxygen species production) was monitored with appropriate fluorescence probes.

Results: : We found that ARPE19 cells chronically treated with 20 µM native PUFAs or oxidized omega-3 (HHE) appeared morphologically normal, while cells treated with 20 µM oxidized omega-6 PUFA (HNE), showed changes at both the cellular and mitochondrial level. These changes included decay of tight junctions, cytoskeletal changes, decreased metabolic activity and decrease in mitochondrial membrane potential. Treatment with 10 µM concentration of native and oxidized PUFAs did not affect ARPE19 cell morphology, metabolism, cytoplasmic membrane integrity and mitochondria number or membrane potential, while incubation with 30 µM 4-HNE and 4-HHE was toxic and resulted in cell death.

Conclusions: : 4-HNE as an end-product of omega-6 PUFA oxidation shows higher sub-lethal toxicity in ARPE19 cells than 4-HHE - the product of omega-3 PUFA oxidation. Thus, diets rich in omega-6 fatty acids such as the Western diet may impair mitochondrial functioning in RPE cells and contribute to AMD progression.

Keywords: retinal pigment epithelium • lipids • mitochondria 

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