In vitro effects of fluoroquinolone and aminoglycoside antibiotics on human keratocytes.
OBJECTIVE
The purpose of this study was to assess the cytotoxic effects of the fluoquinolone ofloxacin and the aminoglycoside netilmicin on stromal human keratocytes in vitro.


METHODS
Cultured human keratocytes were exposed to various concentrations of ofloxacin or netilmicin (0.16-5.0 mg/mL). Both cell proliferation (MTT assay) and cell morphology (phase-contrast microscopy) were evaluated after 1, 4, 12, and 24 hours of incubation. Measurement of annexin V binding performed in association with the dye exclusion test using propidium iodide (PI) was also performed by FACS analysis after 4 hours of exposure.


RESULTS
Both antimicrobials induced dose- and time-dependent morphologic changes in keratocytes, yet the effects of netilmicin were minimal. After 24 hours of exposure, both drugs induced a dose-dependent inhibition of cell proliferation; however, ofloxacin demonstrated significantly more toxic effects than netilmicin (t test for ED50 values, P < 0.0001). Statistical differences between 2 antibiotics start at concentrations above 1.25 mg/mL (ANOVA with post-hoc test, P < 0.01). Expression of the apoptotic marker annexin V was unaffected by antibiotic exposure, whereas the uptake of the necrotic marker PI was increased by ofloxacin (5 mg/mL) but not by netilmicin (ofloxacin versus netilmicin, ANOVA, P < 0.05).


CONCLUSIONS
Relative effects of aminoglycosides and fluoroquinolones on stromal keratocytes appear to be different: netilmicin was shown to be significantly less toxic than ofloxacin. This finding is particularly relevant in deciding the optimal antibiotic to be applied in clinical situations in which the epithelium is absent or compromised, as after photorefractive keratectomy, alkali burns, or ulcerative keratitis.
Leonardi
Andrea
A
Department of Neuroscience, Ophthalmology Unit, University of Padova, Italy.
Papa
Vincenzo
V
Fregona
Iva
I
Russo
Pietro
P
De Franchis
Giuseppe
G
Milazzo
Giovanni
G
D000328:Adult; D017304:Annexin A5 / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000633:toxicity*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D009428:Netilmicin / Q000633:toxicity*; D015242:Ofloxacin / Q000633:toxicity*; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors
D017304:Annexin A5; D000900:Anti-Bacterial Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D009428:Netilmicin; D015242:Ofloxacin; C022616:thiazolyl blue