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M. Vallabhajosyula, S. P. Epstein, D. Chen, P. A. Asbell; Evaluation of Biomarkers of Inflammation in Response to Benzalkonium Chloride on Ocular Surface Epithelia. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2384. doi: https://doi.org/.
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Most eye drops contain preservatives, the most common of which is benzalkonium chloride (BAK), used in concentrations from 0.004% to 0.05% and known to be degraded into hydrogen peroxide (H2O2), a known ophthalmic irritant. C-reactive protein (CRP), interleukins (IL)-1, 10 and 12 and tumor necrosis factor (TNF) have long been described as biomarkers of inflammation. These inflammatory biomarkers were quantified via enzyme linked immunosorbant assays (ELISAs) following BAK administration to evaluate the resulting inflammatory response.
Human conjunctival (CCC: P61 HCK) and corneal epithelial (HCE: CEPI 17) cells at confluency were incubated with 100 uL of: BAK from 0.001 to 0.1%; H2O2 from 0.01 to 0.1%; pure cell media and lipopolysaccharide (LPS) at 100 and 10 ng/mL as controls. After 1 hour, solutions were removed (cytokine release) and replaced with pure cell media for an additional 23 hours (cytokine production). Inflammatory biomarkers (CRP, IL-1, IL-10, IL12, TNF and H2O2) of both supernatents were quantified via enzyme-linked immunosorbent assays [ELISAs; results in pg/mL (H2O2: mg/mL)].
One hour exposure to BAK induced significant levels of IL-1 and TNF in both CCC and HCE as compared to media controls. BAK induced only moderate amounts of CRP and ILs 10 and 12 under the same conditions. H2O2 was produced in moderately high amounts after BAK exposure. The addition of H2O2 induced increased quantities of all cytokines studied. Lower concentrations of BAK and/or H2O2 induced the elaboration of proportionally lower amounts of all studied biomarkers in a dose-dependent fashion. Replacing the testing solution with media and providing 23 hours for cytokine elaboration significantly increased the elaboration/release of only TNF. LPS positive controls at 100 and 10 ng/mL induced substantial elaboration/release of IL-1 and TNF. In general, HCE cells were more sensitive than the conjunctival.
After 1 hour of exposure, BAK and H2O2 induced, in a dose dependent fashion, increased quantities of all biomarkers studied. The biomarkers in decreasing order of induction/upregulation were: TNF ≥ IL-1 ≥ IL-12 ≥ IL-10 ≥ CRP. The addition of benzalkonium chloride at concentrations in the range commonly utilized in ophthalmic pharmaceuticals evoked an increase of inflammatory biomarkers in this cell culture model of surface epithelium. Even low concentrations caused some degree of inflammatory response. IL-1 and TNF was significantly induced secondary to BAK and/or H2O2 administration.
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