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S. P. Epstein, Y. Wei, P. A. Asbell; Evaluation of Toxicity and Apoptosis Induced by Commercial Ophthalmic Fluoroquinolone Antibiotics. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1953.
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Topical fluoroquinolones are the most commonly used ocular antibiotics. In addition to antimicrobial activity, selection of antibiotic is dependent upon toxicity. Frequent use of ocular antimicrobial agents has been shown to be associated with toxicity, damage to the ocular surface and interference with wound healing. This project sought to evaluate both the toxicity and apoptosis induced by topical ocular fluoroquinolone antibiotics utilizing a cell culture model.
Preconfluent immortalized conjunctival (CCC) and human corneal (HCE) epithelial cells were incubated (37oC, 5% CO2) for 1 hour with 100uls of one of the following commercial preparations: 1) IQ (levofloxacin 1.5%); 2) VI (moxifloxacin 0.5%); 3) ZY (gatifloxacin 0.3%, BAK 0.005%); one of the following components: 4) LE (levofloxacin 1.5% in appropriate medium); 5) BAK (benzakonium chloride 0.005% in appropriate medium); and/or one of the following controls: 6) medium alone (100% viable control); 7) formalin (100% toxic control); 8) DNAse (100% apoptosis control).MTT Cytotoxicity Assay: Solutions were then removed and 150uls of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoniumbromide) added for 4 hours. Precipitates were decanted, then dissolved in 100uls of acid isopropanol.TUNEL Apoptosis Assay: After the incubation, the solutions were removed and commercial TUNEL assay performed as per manufacturers’ directions.Absorbance was determined at 572nm. Use of medium alone was equated with 0% toxicity (MTT) or 0% apoptosis (TUNEL), while formalin was equated with 100% toxicity and DNAse with 100% apoptosis.
IQ (levofloxacin 1.5%) induced the least toxicity (CCC: 39.29%; HCE: 41.63%) and the lowest apoptosis (CCC: 11.66%; HCE: 15.19%). The order of both toxicity and apoptosis was (averaging results of cell lines): IQ (tox: 41%, apop: 12%) < VI (tox: 48%, apop: 30%) < ZY (tox: 96%, apop: 58%).
Use of cell culture assays demonstrated some toxicity as well as elevated induction of apoptosis by each of the topical ocular antibiotics tested. Toxicity as determined in these tissue culture models was not equivalent among the fluoroquinolones tested and, in fact, was least for IQ and greatest for ZY (IQ < VI < ZY). The preservative benzalkonium chloride appeared to add to the observed toxicity non-synergistically, but active agents seem to have an effect even in the absence of BAK. Tissue culture models provide a rapid and cost effective method to screen for surface toxicity of topical agents; however, these results need to be correlated with in vivo wound healing and clinical findings.
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