Effects of preserved and preservative free glaucoma drugs on proteomic expression levels in corneal and conjunctival epithelial cells in vitro

Abstract

Purpose: Benzalkonium chloride (BAC), is the most common preservative used in eye drops and it is known to be cytotoxic to epithelial cells of the ocular surface, but the pathways in this toxicity are largely unknown. In this study the effects of preserved and preservative-free glaucoma drugs were studied in vitro by exposing human corneal and conjunctival epithelial cells (HCE and NHC) to these drugs. The goal was to identify if any of the differentially expressed proteins are linked to inflammatory pathways.

Methods: HCE and NHC cells were exposed to either preservative-free prostaglandin tafluprost, preserved latanoprost or preservative BAC for 24 hours and the proteomic profiles of treated and untreated cells were analyzed with NanoLC-TripleTOFMS mass spectrometry using iTRAQ labeling. A dilution of 1:300 was applied to tafluprost and latanoprost. BAC concentration of 0.000067% was used, which is equal to the BAC concentration in 1:300 diluted latanoprost. Mixed-effects ANOVA model was implemented to normalized data for statistical analysis and q-value adjustment was used to account for multiple testing.

Results: Statistical analysis identified 29 differentially expressed proteins for NHC cells (fold change>1.25 or <0.8, q-value<0.25) and 28 for HCE cells (fold change>1.5 or <0.67, q-value<0.25). For the NHC cell line, interesting proteins include INHA(1.491.07,p=0.0005) and PSMB8(2.521.32,p=0.008), which are both related to apoptosis and were overexpressed in cells receiving BAC. For the HCE cell line, particularly interesting were proteins HSPD1(1.641.13, p=0.008), OAS3(1.821.17,p=0.01) and LAMP1(7.851.81,p=0.01) which were connected to immune system activation. MYL6, MYL12A and MYH9 were all highly over-expressed in preservative-treated samples in HCE cells. Two of these proteins are directly connected to the immune system via leukocyte transendothelial migration (MYL12A) and phagocytosis (MYH9).

Conclusions: Proteins related to the immune response and apoptosis were identified as statistically significant for the BAC induced changes with the induction of apoptotic pathways and inflammation in both corneal and conjunctival epithelial cells. These potential novel proteomic biomarkers will be further analyzed in ongoing clinical studies of glaucoma patients.