title: Nitrogen Mustard-Induced Corneal Injury Involves DNA Damage and Pathways Related to Inflammation, Epithelial-Stromal Separation, and Neovascularization.
abstract: OBJECTIVE
  To evaluate the toxic effects and associated mechanisms in corneal tissue exposed to the vesicating agent, nitrogen mustard (NM), a bifunctional alkylating analog of the chemical warfare agent sulfur mustard.
  
  
  METHODS
  Toxic effects and associated mechanisms were examined in maximally affected corneal tissue using corneal cultures and human corneal epithelial (HCE) cells exposed to NM.
  
  
  RESULTS
  Analysis of ex vivo rabbit corneas showed that NM exposure increased apoptotic cell death, epithelial thickness, epithelial-stromal separation, and levels of vascular endothelial growth factor, cyclooxygenase 2, and matrix metalloproteinase-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 poly ADP ribose polymerase cleavage, suggesting their involvement in NM-induced apoptotic death in the rabbit cornea and HCE cells. Similar to rabbit cornea, NM exposure caused an increase in cyclooxygenase 2, matrix metalloproteinase-9, and vascular endothelial growth factor 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 activator protein 1 transcription factor proteins and upstream signaling pathways including mitogen-activated protein kinases and Akt protein kinase, suggesting that these could be key factors involved in NM-induced corneal injury.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Departments of *Pharmaceutical Sciences; and †Ophthalmology, University of Colorado Anschutz Medical Campus, Aurora, CO.
authors.forename: Dinesh G
authors.initials: DG
authors.lastname: Goswami
authors.forename: Neera
authors.initials: N
authors.lastname: Tewari-Singh
authors.forename: Deepanshi
authors.initials: D
authors.lastname: Dhar
authors.forename: Dileep
authors.initials: D
authors.lastname: Kumar
authors.forename: Chapla
authors.initials: C
authors.lastname: Agarwal
authors.forename: David A
authors.initials: DA
authors.lastname: Ammar
authors.forename: Rama
authors.initials: R
authors.lastname: Kant
authors.forename: Robert W
authors.initials: RW
authors.lastname: Enzenauer
authors.forename: J Mark
authors.initials: JM
authors.lastname: Petrash
authors.forename: Rajesh
authors.initials: R
authors.lastname: Agarwal
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002619:Chemical Warfare Agents / Q000633:toxicity*; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016510:Corneal Neovascularization / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D003319:Corneal Stroma / Q000473:pathology*; D051546:Cyclooxygenase 2 / Q000378:metabolism; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000473:pathology*; D007150:Immunohistochemistry; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008466:Mechlorethamine / Q000633:toxicity*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009924:Organ Culture Techniques; D011817:Rabbits; D012421:Rupture; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D015415:Biomarkers; D002619:Chemical Warfare Agents; D042461:Vascular Endothelial Growth Factor A; D008466:Mechlorethamine; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D020780:Matrix Metalloproteinase 9