Purpose:
To evaluate mechanisms of benzalkonium chloride toxicity in a human trabecular meshwork cell line and the possible role of gap junctions.
Methods:
A human trabecular meshwork (HTM) cell line was established in culture. Cells were treated with increasing concentrations of benzalkonium chloride (BAK) ranging from 0.002 to 0.01% for 1, 3, 10 and 30 minutes and cell death was measured using the MTT assay. Cells were treated with BAK ranging from 0.0001 to 0.001% for 24 and 48 hours and cell death was measured using the MTT assay. Cells were treated with 0.0004% and 0.0008% BAK for 24 hours to assess apoptosis by Cell Death ELISA assay. HTM cells were up-regulated by retroviral transfection with Cx43-GFP and down-regulated with a dominant negative G138R mutant. Endogenous connexin43 expression was measured with Western Blot after 3 minutes BAK treatment and 24 hours incubation.
Results:
HTM cells exhibited time and dose dependent decrease in cell viability when treated with 0.002 to 0.01% BAK for 1 to 30 minutes (n=3; p<0.001). HTM cells exhibited dose dependent decrease in cell viability when treated with 0.0001 to 0.001% BAK for 24 hours and 0.0001 to 0.0004% BAK for 48 hours (n=3; p<0.001). HTM cells exhibited dose dependent apoptosis when exposed to 0.0004 and 0.0008% BAK for 24 hours (n=3; p<0.05). Furthermore, endogenous connexin43 is upregulated upon BAK exposure.
Conclusions:
BAK induces time and dose dependent HTM cell cytotoxicity through necrosis and apoptosis. Connexin43 is affected by the cytotoxic effects of BAK in this cell type.
Keywords: trabecular meshwork • apoptosis/cell death • gap junctions/coupling