DNA cross-linking, double-strand breaks, and apoptosis in corneal endothelial cells after a single exposure to mitomycin C.
OBJECTIVE
To investigate the cellular effects of mitomycin C (MMC) treatment on corneal endothelial (CE) cells at clinically relevant applications and dosages.


METHODS
Radial and posterior diffusion of MMC was determined by an Escherichia coli growth inhibition bioassay. A modified version of the comet assay (single cell gel electrophoresis) was used to detect DNA cross-linking. Immunostaining detected the nuclear phosphorylated histone variant H2AX (gamma-H2AX) indicating DNA double-strand breaks. Apoptosis in MMC-treated cells was detected with annexin V staining.


RESULTS
Topical application of 0.02% MMC to intact goat globes resulted in MMC in the CE at 0.37 microg/mL and produced a significant increase in CE DNA cross-linking with as little as 6 seconds of topical MMC treatment. DNA cross-linking was also demonstrated in cultured CE cells by using MMC exposures similar to those detected in CE of intact eyes. Such MMC treatment of CE produced elevated and persistent gamma-H2AX-positive cells indicative of DNA double-strand breaks. Similarly, there was an increase in the proportion of apoptotic CE cells, evidenced by positive annexin V staining.


CONCLUSIONS
The results demonstrate that exposure to MMC at times and concentrations commonly used in refractive surgery produces cross-linking of corneal endothelial DNA, persistent DNA damage, and endothelial death via apoptosis. Current practices of MMC application during refractive surgeries may increase the potential for long-term and permanent deleterious effects on the health of the corneal endothelium.
Roh
Danny S
DS
Department of Ophthalmology, UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Sciences Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213-2588, USA.
Cook
Amanda L
AL
Rhee
Steven S
SS
Joshi
Amar
A
Kowalski
Regis
R
Dhaliwal
Deepinder K
DK
Funderburgh
James L
JL
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000235:genetics*; D002452:Cell Count; D002478:Cells, Cultured; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D006041:Goats; D006657:Histones / Q000378:metabolism; D016685:Mitomycin / Q000008:administration & dosage* / Q000493:pharmacokinetics; D019384:Nucleic Acid Synthesis Inhibitors / Q000008:administration & dosage / Q000493:pharmacokinetics; D009883:Ophthalmic Solutions; D010750:Phosphoproteins; D010766:Phosphorylation / Q000187:drug effects; D011183:Postoperative Complications / Q000517:prevention & control
D017304:Annexin A5; C468783:H2AX protein, human; D006657:Histones; D019384:Nucleic Acid Synthesis Inhibitors; D009883:Ophthalmic Solutions; D010750:Phosphoproteins; D016685:Mitomycin