Assessment of initial damage and recovery following exposure of MDCK cellsto an irritant

The ability of Madin-Darby canine kidney cells (MDCK) to form cell tight-ju nctions and orientate correctly on porous membranes has been exploited to m odel corneal barrier function. While the ability to monitor recovery profil es has not yet been included in a prevalidation trial, its inclusion facili tates the prediction of potential adverse reactions. Combining a viability assay (Alamar blue reduction) with the fluorescein leakage assay, the chemi cal effects on cell membrane and adhesion molecules can be distinguished. T he dose-response curves obtained with Tween 20, isopropanol and benzalkoniu m chloride indicate that Tween 20 at 200 mg/ml caused a 50% decline in Alam ar blue reduction and fluorescein retention, while 100 mg isopropanol:ml an d 0.1 mg benzalkonium chloride/ml; cause similar effects. The mure severe t he initial damage the longer the recovery period. If a 20% increase in fluo rescein leakage gave recovery in 48 hours, 50% gave marginal recovery in 96 hours. Comparable effects were noted for the restoration of the ability to reduce the Alamar blue dye. Resolution of the damaging effects of benzalko nium chloride and isopropanol took longer than for Tween 20, as is the case in the rabbit in vivo. (C) 1999 Elsevier Science Ltd. All rights reserved.