Mustard Gas Induced Corneal Injury Involves Ferroptosis and p38 MAPK Signaling.

Purpose: Sulfur mustard gas (SM) exposure to eyes causes multiple corneal injuries including stromal cell loss in vivo. However, mechanisms mediating stromal cell loss/death remains elusive. This study sought to test the novel hypothesis that SM-induced toxicity to human corneal stromal fibroblasts involves ferroptosis mechanism via p38 MAPK signaling. Methods: New Zealand white rabbit corneas, naïve and SM exposed (200 mg-min/m3 for eight minutes and collected after three days) were used to examine the levels of cell death and reactive oxygen species (ROS) for in vivo studies. Donor human corneas were used to generate primary human corneal stromal fibroblasts (hCSF) for in vitro studies. The hCSFs were exposed to nitrogen mustard (NM; SM analogue) at various timepoints (30 minutes, eight hours, and 24 hours). A p38 MAPK specific inhibitor, SB202190, was also used. Quantitative reverse transcription polymerase chain reaction, Western blotting, reactive oxygen species (ROS), lipid peroxidation, live/dead assay, and RNASeq were used in various investigations. Results: SM caused a significant increase in cell death and ROS production three days after SM exposure in rabbit corneas. NM exposure to hCSF demonstrated a significant increase in ROS, lipid peroxidation, and ferroptosis biomarkers ACSL4 (inducer) and significant decrease in reducer (SLC7A11 and GPX4) compared to controls in a time-dependent manner. The inhibition of p38 MAPK promoted cell survival and reduced ROS production following mustard gas exposure. Conclusions: The results of in vivo and in vitro investigations uncovered a novel mechanism that mustard gas toxicity to the cornea involves ferroptosis pathway and p38 MAPK activation.