Mechanism of corneal toxicity from chloropicrin exposure

Abstract

Purpose : Lack of research efforts to evaluate the mechanisms of corneal toxicity from chloropicrin (Trichloronitromethane; PS, CP) exposure has hampered the development of effective treatments against ocular surface injury from this fumigant and pesticide, which is a potential agent of warfare and terrorism. This study tested our hypothesis that CP-induced oxidative stress-related 4-hydroxynonenal (4-HNE) production and subsequent protein carbonylation could play an important role in the mechanism of corneal toxicity from CP.

Methods : Toxic effects of CP (0-100 µM) were studied in primary human corneal epithelial (HCE) cells. Cell viability and apoptotic cell death was determined using MTT and Hoechst assays, respectively. Western blot analysis was carried out at 24 h post-exposure to examine molecular markers of CP-induced toxicity, which could contribute to the CP (50 µM)-induced cellular toxicity. Proteomic analysis was carried out to identify carbonylated proteins via biotin-hydrazide enrichment and nUltra Performance Liquid Chromatography (nUPLC)-LC-MS/MS.

Results : CP (50 µM) exposure in HCE cells caused increases in Heme oxygenase-1 (3-fold), H2A.X (7-fold), and P53 (2-fold) phosphorylation, demonstrating oxidative stress and DNA damage. Up-regulation of cleaved caspase 3 (32-fold) and PARP (9-fold) following CP exposure indicated the contribution of this pathway in CP-induced apoptotic cell death. MAPK- JNK was maximally (5-fold) activated after CP exposure as compared to other MAPKs, and a CP-induced increase in inflammatory mediator COX-2 (3-fold), 4-HNE adduct formation (3-fold), and protein carbonylation (8-fold) was also observed. Proteomic analysis revealed an increase in the carbonylation of 179 proteins following CP exposure and further bioinformatics analysis found enrichment of the proteasome pathway (8-fold), catabolic process (2.4-fold), intracellular protein transport (2 fold), and tRNA aminoacylation for protein translation (18-fold).

Conclusions : The study outcomes in HCE cells indicate that CP-induced oxidative stress signals and lipid peroxidation can cause protein carbonylation, prompting alterations in corneal epithelial proteins as well as activating signaling pathways resulting in toxic effects in the corneal epithelium. Major pathways and processes identified in proteomic analysis following CP exposure could be lead-hit targets for further biochemical characterization and therapeutic intervention.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.