Nitrogen mustard induces DNA damage and activates signaling cascades that lead to inflammation, epithelial-stromal separation, cell death and neovascularization in corneal tissue

Abstract

Purpose: Investigation of mechanistic aspects of ocular injuries, mainly to the cornea, inflicted upon exposure to vesicating chemical warfare agents is essential to develop effective therapies against these injuries. Consequently, toxic effects and associated mechanisms were examined in corneal tissue treated with nitrogen mustard (NM), an analog of sulfur mustard (SM).

Methods: Freshly exposed rabbit corneas were exposed to 100 nmol NM for 2 h (washed and cultured for 24 h), and histopathological as well as molecular analyses were carried out. Since vesicating agents mainly target the corneal epithelial cells, we also employed human corneal epithelial (HCE) cells to study and compare the mechanism/s of action of NM-induced injury in these cells with rabbit cornea.

Results: Analysis of NM-exposed rabbit corneas showed increases in epithelial thickness, apoptotic cell death, epithelial-stromal separation, increase in the levels of angiogenic regulator VEGF, and induction of COX-2 and MMP-9. NM (50 and 100 µM) exposure caused a dose-dependent decrease in HCE cell viability and proliferation. These toxic effects could be related to NM-induced DNA damage (p53 phospho ser15, total p53 and H2A.X phospho ser139) in HCE cells. In addition to p53 phosphorylation, NM also induced caspase-3 and PARP cleavage, suggesting their involvement in NM-related HCE cell death and apoptotic cell death in rabbit cornea. Similar to rabbit cornea, NM exposure also caused an increase in COX-2, MMP-9 and VEGF levels in HCE cells, indicating a role of these molecules and related pathways in NM-induced corneal inflammation, epithelial-stromal separation and neovascularization. NM also induced activation of AP-1 transcription factor proteins and upstream signaling pathways including MAPKs and Akt, suggesting that these could be key factors involved in NM-induced injuries.

Conclusions: These molecular targets and pathways involved in NM-induced corneal injuries could be supportive in development of therapeutics against corneal injuries from vesicant exposure.

Keywords: 482 cornea: epithelium • 620 ocular irritancy/toxicity testing • 714 signal transduction