Comparative study on the cytotoxic effects of benzalkonium chloride on the Wong-Kilbourne derivative of Chang conjunctival and IOBA-NHC cell lines

PURPOSE: The Wong-Kilbourne derivative of Chang conjunctiva-derived cell line has been widely used for toxicological and functional in vitro studies on the ocular surface. The common reserve to this cell line is the reported contamination with HeLa cells. Thus, the IOBA-NHC spontaneously immortalized conjunctival epithelial cell line has been recently developed and did not show other cell type contamination. Our purpose was to determine whether both cell lines would be equally suitable for in vitro toxicological studies. Therefore, we compared in these two cell types the toxic effects of the preservative, benzalkonium chloride (BAC); its toxicity has been often reported on conjunctival in vivo and in vitro models. METHODS: The necrotic, apoptotic, and oxidative effects of BAC were evaluated on Chang and IOBA-NHC cell lines using microplate cytofluorometry tests (neutral red, 2,7- dichlorofluorescein diacetate dye [H(2)DCF-DA], hydroethidine, and Yopro-1), flow cytometry (Annexin V/7-AAD and DNA content tests), and standard immunofluorescence stainings. Cells were exposed to five concentrations of BAC (10(−2)%, 5.10(−3)%, 10(−3)%, 10(−4)%, and 10(−5)%) for two incubation times: 15 min of treatment and 15 min of treatment followed by 24 h of cell recovery in complete medium. RESULTS: All parameters of toxicity increased in a BAC dose-dependent manner on both cell lines. CONCLUSIONS: The comparison of BAC toxicity on both cell lines supported the use of IOBA-NHC and Chang cells for toxicological in vitro studies. Drawbacks of both cell lines have to be known and considered in studies performed on these cell lines.