Cellular and molecular changes in human corneal fibroblasts following hydrogen sulfide exposure in vitro

Abstract

Purpose : Hydrogen sulfide (H₂S) is a common environmental pollutant and a chemical warfare agent. H₂S exposure to the eye leads to ocular irritation, pain, and vision loss, however, underlying molecular mechanisms driving its toxicity to the cornea is still unclear. We sought to study changes in cellular and molecular parameters in primary human corneal stromal fibroblasts following H₂S exposure in vitro.

Methods : A total of 30 healthy human donor corneas obtained from the eye bank were used in this study. Primary human corneal stromal fibroblasts (hCSFs) were generated from healthy donor corneas following our standard protocol and passages two to five were used for in vitro experiments. Sodium hydrosulfide (NaSH) was used as a source of H₂S and hCSF cells were exposed to H₂S for up to 72h and the changes in mitochondrial function were assessed using Complex IV (cytochrome c oxidase) Human Specific Activity Microplate Assay Kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s guidelines. The mRNA expression of cell death genes - Receptor-interacting serine/threonine-protein kinase 1 (RIP1), Tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD), Caspase 8, and Mixed lineage kinase domain-like protein (MLKL) was studied using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, CA, USA).

Results : H₂S exposure showed dose and duration dependent cytotoxicity to hCSFs in cytotoxicity assays and IC₅₀ of H₂S was determined to be 5.35 mM. H₂S IC₅₀ exposed hCSFs showed significantly decreased levels of cytochrome c oxidase enzyme activity (p<0.01) and quantity (p<0.0001) compared to healthy cells. The mRNA expression of RIP1, TRADD, Caspase 8, and MLKL genes were significantly altered in H₂S IC₅₀ exposed hCSFs compared to control (p≤0.05).

Conclusions : H₂S exposure to human corneal fibroblasts activates cell death pathways and damages mitochondrial function in vitro.

This is a 2021 ARVO Annual Meeting abstract.