In vitro modulation of preservative toxicity: High molecular weight hyaluronan decreases apoptosis and oxidative stress induced by benzalkonium chloride

Abstract

Objective Benzalkonium chloride (BAK) is one of the most often used preservative in pharmaceutical products and it is known to induce toxic effects. Hyaluronan (HA), a linear biopolymer, is involved in several biological processes. The aim of this work is to in vitro investigate if HA is able to decrease BAK toxicity. Methods Two human epithelial cell lines were treated with different incubation time protocol with BAK and three different molecular weights HA (HA 20 kDa, HA 100 kDa and HA 1000 kDa, 0.2%, w/v). Flow cytometry, fluorescence microscopy, microplate cytofluorometry and confocal microscopy were performed to evaluate expression of CD44 receptor, cell viability, oxidative stress, mitochondrial mass, chromatin condensation, plasma-membrane permeability, DNA fragmentation and cytoskeleton morphology. Results The three HAs studied induce neither oxidative stress nor apoptosis. HA 1000 kDa significantly decreases oxidative stress, apoptosis and necrosis induced by BAK. Experiments with HA 20 kDa or HA 100 kDa did not show the same effects. For instance, the more molecular weight decreases, the more protection decreases. Moreover, we suggest that HA interacts with cell plasma-membrane and inhibits cell death receptors. Conclusion High molecular weight HA (1000 kDa, 0.2%) is an effective protective agent against BAK.