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Cytostatic and Cytotoxic Effects of 5-Fluorouracil on Human Corneal Epithelial Cells and Keratocytes

Midena, Edoardo MD, PhD; Lazzarini, Daniela PhD; Catania, Anton Giulio MD; Moretto, Erika MD; Fregona, Iva ScD, PhD; Parrozzani, Raffaele MD

doi: 10.1097/ICO.0b013e31825d56c1
Basic Investigation

Purpose: To investigate the effects of various 5-fluorouracil (5-FU) concentrations, exposure times, and application techniques on in vitro–cultured human corneal cells.

Methods: Human corneal epithelial cell (HCEC) and human corneal keratocyte (HCK) cultures were exposed to different 5-FU concentrations (0.025%–1%) and incubation durations (5 minutes to 2 hours). The cytostatic effect was evaluated as the percentage of inhibition of migration relative to the control. The evaluation of cytotoxic effect included both phase contrast microscopic observations and viability measures performed using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] colorimetric assay. The results are expressed as ratio of optical density (OD) reduction 24 hours after exposure.

Results: The cytostatic effect was time and dose dependent. The 50% inhibiting dose was 0.55% after 1 hour of incubation for HCECs and was 0.5% after 2 hours of incubation for HCKs. A 100% inhibitory effect was never observed at any concentration or incubation duration. No cytotoxic changes were observed using an 5-FU concentration of <1%; 1% 5-FU showed time-dependent cytotoxic changes in HCEC cultures only. MTT analysis showed no OD reduction at 5-FU concentrations of <1%, whereas 1% 5-FU showed OD reduction <50% at any tested exposure time. HCECs showed higher reduction in OD than HCKs.

Conclusions: 5-FU formulations topically used in clinical practice showed limited toxicity in normal cultured corneal epithelial cells and keratocytes.

*Department of Ophthalmology, University of Padova, Padova, Italy

G.B. Bietti Eye Foundation, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.

Reprints: Edoardo Midena, Department of Ophthalmology, University of Padova, Via Giustiniani 2, Padova 35128, Italy (e-mail:

The authors state that they have no financial or conflicts of interest to disclose.

Received November 25, 2011

Accepted May 2, 2012

Copyright © 2013 Wolters Kluwer Health, Inc. All rights reserved.