Multiple endpoint analysis of the 3D-reconstituted corneal epithelium after treatment with benzalkonium chloride: early detection of toxic damage.

Purpose: To investigate the effects of benzalkonium chloride (BAK) on the human reconstituted corneal epithelial model (HCE) and to optimize the operating potential of this model in the field of ophthalmic toxicology.
Methods: The HCEs were treated with 0.001% to 0.5% BAK for 24 hours followed or not by a 24-hour postincubation period. To complete the histologic analysis, the authors designed a new MTT procedure to assess cellular viability. Frozen sections were analyzed by using fluorescence confocal microscopy for the presence of TUNEL, activated caspase-3, Ki67, ICAM-1, HLA-DR, E-cadherin, and occludin. Occludin gene expression was also investigated by using quantitative RT-PCR.
Results: The MTT test revealed a dose-dependent response of BAK with significant toxic effects for concentrations as low as 0.005%. Increasing BAK concentrations induced an increased number of apoptotic cells, found from the superficial to the deeper layers, with the activation of caspase-3 at 0.01% and 0.02% concentrations. The number of Ki67- and ICAM-1-positive cells increased with 0.01% BAK and with 0.001% to 0.01% BAK, respectively. BAK induced the dose-dependent disappearance of occludin in the superficial layers while increasing its gene expression up to the 0.02% BAK concentration.
Conclusions: Fluorescence techniques conjugated with confocal microscopy on 3D-reconstructed corneal epithelia were well suited for the investigation of toxicological markers such as cell junction alteration, apoptosis, cell activation, and proliferation and gave relevant results compared with the known human data. They complement the new sensitive MTT test and improve the operating potential of this new, valuable 3D model in ophthalmic toxicology.