Nitrogen Mustard-Induced Corneal Injury Involves DNA Damage and Pathways Related to Inflammation, Epithelial-Stromal Separation, and Neovascularization

Abstract

Purpose To evaluate the toxic effects and associated mechanisms in corneal tissue exposed to vesicating agent, nitrogen mustard (NM), a bi-functional alkylating analog of chemical warfare agent sulfur mustard (SM). Methods Toxic effects and associated mechanisms were examined in maximal affected corneal tissue employing corneal cultures and human corneal epithelial (HCE) cells exposed to nitrogen mustard (NM). Results Analysis of ex vivo rabbit corneas showed that NM exposure increased apoptotic cell death, epithelial thickness, epithelial-stromal separation and levels of VEGF, COX-2 and MMP-9. In HCE cells, NM exposure resulted in a dose-dependent decrease in cell viability and proliferation, which was associated with DNA damage in terms of an increase in p53 ser15, total p53 and H2A.X ser139 levels. NM exposure also induced caspase-3 and PARP cleavage, suggesting their involvement in NM-induced apoptotic death in rabbit cornea and HCE cells. Similar to rabbit cornea, NM exposure 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 exposure 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 corneal injury. Conclusion Results from this study provide insight into the molecular targets and pathways that could be involved in NM-induced corneal injuries laying the background for further investigation of these pathways in vesicant–induced ocular injuries, which could be helpful in the development of targeted therapies.

Corneal toxicity induced by vesicating agents and effective treatment options

“…In addition, NM exposure is also reported to cause an increase in the expression of cyclooxygenase‐2 (COX‐2, a key molecule involved in the prostaglandin synthesis and inflammatory and cytotoxic responses), matrix metalloproteinase‐9 (MMP‐9, plays a major role in the degradation of the basement membrane components), and angiogenic factors, namely VEGF (plays an important role in neovascularization (Fig. )) . Further elucidation of mechanisms and pathways in HCE cells related to these lesions in rabbit corneal tissue reveal that DNA damage is an early event, involving p53 and H2A.X phosphorylation, that leads to decreased cell viability and proliferation.…”

“…The ex vivo rabbit corneal organ cultures are reported to be beneficial in primary screening and identification of therapies against vesicant‐induced ocular injuries . Furthermore, this culture system has also been useful to study corneal wound healing, and the injuries have been found to be similar to those occurring in vivo in the rabbit cornea . The rabbit corneas are obtained from Pel‐freez, a company that sells eyes of rabbits that are used for other purposes, which makes it cost effective and reduces the animal use in our studies.…”

“…The rabbit corneas are obtained from Pel‐freez, a company that sells eyes of rabbits that are used for other purposes, which makes it cost effective and reduces the animal use in our studies. This rabbit corneal injury model with NM could serve as an ideal ex vivo system in the laboratory settings to establish injury and to evaluate and optimize therapeutic agents before testing them in in vivo ocular injury rabbit models with vesicating agents like SM and LEW . In addition, since the corneal epithelial layer is the outermost layer of the eye and is highly susceptible to vesicant‐induced injuries, human corneal epithelial (HCE) cells are a useful model system to assess the mechanisms of pathogenesis of corneal injury .…”

“…Also, NM induces activation of activator protein 1 (AP‐1) transcription factor proteins and upstream signaling pathways, including mitogen‐activated protein kinases (MAPKs; phosphorylation of ERK1/2, p38, and JNK1/2) and Akt protein kinase, signifying that these could be important factors in NM‐induced corneal injury (Fig. ) . Notably, the MAPK/Akt/AP‐1 pathway is activated by growth factors, inflammatory cytokines, stress stimuli, and oxidative stress; oxidative stress is a key initiating event after vesicant exposure.…”

“…Owing to the limitation of access and use of SM in laboratory settings, NM has been employed to study vesicant‐induced ocular injuries and related mechanism of action to identify targeted therapies. This is of importance since the countermeasures currently available are mainly symptomatic and supportive, accentuating an immediate need for the development of therapies …”