title: In vitro effects of fluoroquinolone and aminoglycoside antibiotics on human keratocytes.
abstract: 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.
authors.affiliation: Department of Neuroscience, Ophthalmology Unit, University of Padova, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Leonardi
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Papa
authors.forename: Iva
authors.initials: I
authors.lastname: Fregona
authors.forename: Pietro
authors.initials: P
authors.lastname: Russo
authors.forename: Giuseppe
authors.initials: G
authors.lastname: De Franchis
authors.forename: Giovanni
authors.initials: G
authors.lastname: Milazzo
mesh_terms: 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
chemical_list: D017304:Annexin A5; D000900:Anti-Bacterial Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D009428:Netilmicin; D015242:Ofloxacin; C022616:thiazolyl blue