Toxicity of Branched-Chain Fatty Acids in Conjunctival Cell Line

Abstract

Purpose:: Tear film lipid composition is modified in patients suffering from chronic blepharitis. We previously found that branched chain fatty acids (BCFA) were significantly increased in tears of these patients, suggesting that BCFA may be involved in the disease. The toxicity of different BCFA as potent inducers of apoptosis has already been established in various cell lines but not in the conjunctiva. The aim of this work was to assess the toxic effects of BCFA in cultures of Chang conjunctival cells.

Methods:: Chang’s human conjunctival cells were cultured during 4 or 24h with various concentrations of IsoC16 and IsoC20 as BCFA (from 50 to 200µM), by comparison with phytanic acid as a positive control for cytotoxicity or with palmitic acid as a corresponding straight chain fatty acid. The toxicity of fatty acids was assessed by measurement of mitochondrial dehydrogenase activity (MTT test), cell growth, free radical production, cell permeability (propidium iodure), mitochondrial depolarization (Mitotracker) and chromatin condensation (Hoechst). In addition, incorporation of fatty acids in the cells was measured after analysis by gas chromatography of the cells.

Results:: IsoC20 and palmitic acid transiently impaired cell viability, as illustrated by the decrease in mitochondrial dehydrogenase activity observed after 4 hours of exposure (p<0.0001). Cell viability remained affected by IsoC20, C16 and phytanic acid (100-200µM) after 24 hours of exposure (p<0.0001). This effect was dependent on fatty acid concentration (p<0.001). On the contrary, cell growth, free radical production, membrane permeability, mitochondrial depolarization and chromatin condensation were not affected. Fatty acids were positively incorporated in the cells.

Conclusions:: IsoC20 is the main BCFA present in tears of blepharitis patients (3.5% of total fatty acids) and was shown to modify mitochondrial dehydrogenase activity, thereby may be suspected to alter cell viability. Nevertheless, this effect was not associated with any reduction of cellular growth or with apoptosis. Consequently, the presence of IsoC20 in tears of blepharitis patients may not be linked to a toxic effect but might mirror the adaptative response of the ocular surface to inflammation.

Keywords: lipids • conjunctiva • apoptosis/cell death