Evaluation of the toxicity of benzalkonium chloride on the ocular surface

Benzalkonium chloride (BAC), the most widely used preservative in ophthalmi c solutions, acts on the ocular surface through toxic or immunoallergic rea ctions. Due to its surfactant properties, this quaternary ammonium strongly decreases lachrymal fluid stability. It also causes toxic effects to epith elial cells and increases corneal permeability. In vivo, strong histopathol ogical changes are observed after topical treatments with preservatives, in cluding infiltration by inflammatory cells, similar to those induced in hum ans by long-term topical treatments. We designed a series of in vitro exper iments confirming the toxicity of BAC, even at very low concentrations. In vitro, BAC induces cell necrosis at the concentrations found in most eye dr ops after a few minutes, and apoptosis when applied at lower concentrations . This apoptotic phenomenon is confirmed by 4',6-diamidimo-2-phenylindole, dilactate (DAPI) coloration and with the use of how cytometry. A decrease i n cell size is observed with BAC at 0.001% and is confirmed by morphologica l assay. An overexpression of Apo 2.7 associated with an increase of sub GI phase cells is also shown. BAC induces irreversible cytotoxic damages with some characteristics of apoptosis in a concentration-dependent manner.