Ocular Surface Toxicity as Assesed on Immortalized Conjunctival and Corneal Cells in Tissue Culture

Abstract

Abstract: : Purpose: To evaluate the toxicity of a variety of topical agents, including the newer fluoroquinolone antibiotics, on the ocular surface, a tissue culture model of corneal and conjunctival epithelial cells was used. This technique may prove to be a cost effective method to evaluate toxicity. Methods: Immortalized Chang=s conjunctival cells (CCC) and human corneal epithelial (HCE) cells were seeded into 96 well tissue culture plates and grown at 37^oC, 5% CO₂. When the cells reached confluency, the medium was replaced with 100 Φls of one of the following test solutions: 1) Vigamox [moxifloxacin (0.5%: MX)]; 2) Zymar [gatifloxacin (0.3%: GA)]; 3) Iquix [levofloxacin (1.5%: LE₁₅)]; 4) Quixin [levofloxacin (0.5%: LE₅)]; 5) Ocuflox [ofloxacin (0.3%: OF)]; 6) ciprofloxacin (0.3%: CP)]; 7) medium (viable control); 8) vehicle (dead control). A variety of other commercially available topical ocular agents were also evaluated. After 1 hour, the solution was removed and the cells allowed to dry for 1 hour. One hundred fifty (150) Φls of MTT (3–[4,5–dimethylthiazol–2–yl]–2,5–diphenyl tetrazonium bromide) was then added to each well and incubated at 37^oC for 4 hours. After carefully decanting the solution, 150 Φls of acid isopropanol was added to dissolve the precipitate. The absorbance of each was then determined at 570 nm by a fluorescence reader. Results: The lowest amount of cell death was associated with the viable control. All ophthalmic preparations showed some degree of both epithelial and conjunctival cell toxicity as compared to viable controls. Of the topical ocular antibiotics tested, MX showed the least amount of toxicity (mean percentage of cell death approx 78%). All of the other antibiotics tested were statistically indistinguishable from each other [GA approx 91%; LE₁₅/LE₅ approx 97%; OF approx 89%; CP approx 91%]. Of course, the mean percentage of cell death of the dead control was 100%. Additional data with other topical ocular agents will also be presented. Conclusions: All of the topical ocular antibiotics tested showed evidence of both corneal and conjunctival toxicity (MX < OF < GA = CP < LE), although only the MX was statistically significant. Whether or not this reflects upon in vivo wound healing remains to be determined. This model provides a rapid and cost effective method to screen for surface toxicity of topical agents.

Keywords: drug toxicity/drug effects • antibiotics/antifungals/antiparasitics • wound healing