DNA cross-linking, double-strand breaks, and apoptosis in corneal endothelial cells after a single exposure to mitomycin C.

Mitomycin C (MMC), an antibiotic isolated from Streptomyces caespitosus, is classified as an alkylating agent capable of covalently binding DNA and inducing inter- and intrastrand cross-links. The presence of such cross-links results in inhibition of DNA synthesis primarily during late G1 and S phases, although it is not cell-cycle specific. In addition, MMC inhibits RNA and protein synthesis and interacts with molecular oxygen, generating free radical damage to DNA and protein. 1 MMC is predominantly used as a systemic chemotherapeutic agent; however, off-label usage of MMC is common during ophthalmic procedures, such as glaucoma filtering surgery, lacrimal surgery, treatment of ocular neoplasia, ocular cicatrization surgery, and corneal refractive surgery.2 Currently, the use of adjuvant MMC as an intraoperative prophylactic for reducing haze has become increasingly widespread in photorefractive keratectomy (PRK).2–9 As a topical adjunct during refractive surgery, MMC is exclusively used to modulate corneal wound healing and, through its effects on stromal keratocytes, to prevent active fibrosis, abnormal collagen deposition, and haze formation. The induction of apoptosis of keratocytes by MMC seems to play a central role in preventing the formation of clinically significant haze.10 –12 MC applied topically has been shown to penetrate the cornea and can be detected in corneal tissue and the aqueous humor well after initial application13–15; thus, treatment with MMC during refractive surgery has the potential to impact ocular tissues other than the target keratocytes. Of particular interest is the corneal endothelium, a monolayer of nonregenerating cells essential to the clarity of the cornea. Initially, several clinical studies regarding corneal endothelial cell density and morphology indicate few short-term complications with the use of adjuvant MMC during refractive surgeries16–18; however, recent studies have suggested potential adverse effects of such treatments. The corneal endothelium appears to exhibit significant vulnerability to the toxicity of MMC. Single intraoperative MMC applications over a denuded stroma result in dose-dependent corneal edema and endothelial apoptosis.19 Similarly, significant endothelial cell loss has been reported in MMC-treated eyes within a few months after photorefractive keratectomy.20 In addition, numerous case reports have suggested that a single intraoperative application of MMC during other ocular procedures may be responsible for corneal endothelial damage and loss.21–24 Thus, as the surgical use of MMC increases, the need to define its cellular effects at relevant clinical applications and dosages on corneal endothelial cells is of high importance. In this study, we assessed the effects of a single MMC application on corneal endothelial cells, using concentrations and times relevant to current clinical practice. We used a modified version of the highly sensitive comet assay (single cell gel electrophoresis) to detect DNA cross-linking and examined the phosphorylated histone variant H2AX (γ-H2AX) to demonstrate DNA double-strand breaks. Finally, we assessed MMC-treated cells for annexin V staining to detect apoptosis. Our results clearly demonstrate that exposure to MMC at the times and concentrations commonly used in refractive surgery produced cross-linking of corneal endothelial DNA, persistent DNA damage, and endothelial death via apoptosis.