Cytotoxic, Apoptotic, and Oxidative Effects of Preserved and Preservative-Free Brimonidine in a Corneal Epithelial Cell Line

Abstract

Purpose: This study aims to compare the cytotoxic, apoptotic, and oxidative effects of preserved and preservative-free forms of brimonidine 0.15% on the human corneal epithelial cell (HCEC) line. Methods: Time-dependent cytotoxicity studies were performed with the Alamar Blue method. For apoptotic studies, PE Annexin V and 7-amino-actinomycin (7-AAD) staining and flow cytometry were performed. Messenger RNA (mRNA) expressions of Bax, Bcl-2, and caspase-3, -9, -12, and protein expressions of Bax and Bcl-2 were evaluated by quantitative real-time polymerase chain reaction and Western blot method, respectively. Results: Cell viability was 76.4% with the preserved solution and 36.05% with the preservative-free solution at the fifth minute. No significant difference was observed with either solution at the 15-min mark, whereas cell viability did not change significantly after 1 h. In the apoptosis evaluation, it was observed that the preservativefree solution increased the early apoptotic activity to a greater degree (P < 0.05). Preservative-free solution also induced gene expression of proapoptotic Bax, caspase-9 and -12, and protein expression of Bax while reducing the protein expression of anti-apoptotic Bcl-2 (P < 0.0001). Preserved solution induced only the gene expression of caspase-12, and reduced the protein expression of Bcl-2 (P < 0.0001). No significant difference was observed in the reactive oxygen species (ROS) levels of either solution compared with the control group (P > 0.05). Conclusion: It was demonstrated that the preserved solution is less cytotoxic to the HCEC line in the early period, has less early apoptotic activity, and does not significantly increase ROS levels.

Impact of Antiglaucoma Drug Number and Class on Corneal Epithelial Thickness Measured by OCT

“…However, despite its favorable tolerability profile, in vitro studies have indicated that brimonidine may exert cytotoxic effects on corneal epithelial cells. Kucukoduk et al demonstrated that preservative-free brimonidine induced significantly stronger early apoptosis than preserved formulations [ 39 ], and Fisenko et al reported that high-concentration brimonidine (1:10 dilution), even without BAK, caused morphological damage and reduced cell viability [ 40 ]. Similarly, timolol at comparable dilutions exhibited cytotoxic effects only when preserved with BAK, while preservative-free timolol showed minimal toxicity.…”

Impact of Antiglaucoma Drug Number and Class on Corneal Epithelial Thickness Measured by OCT

“…However, despite its favorable tolerability profile, in vitro studies have indicated that brimonidine may exert cytotoxic effects on corneal epithelial cells. Kucukoduk et al demonstrated that preservative-free brimonidine induced significantly stronger early apoptosis than preserved formulations [ 39 ], and Fisenko et al reported that high-concentration brimonidine (1:10 dilution), even without BAK, caused morphological damage and reduced cell viability [ 40 ]. Similarly, timolol at comparable dilutions exhibited cytotoxic effects only when preserved with BAK, while preservative-free timolol showed minimal toxicity.…”

Impact of Antiglaucoma Drug Number and Class on Corneal Epithelial Thickness Measured by OCT

“…However, despite its favorable tolerability profile, in vitro studies have indicated that brimonidine may exert cytotoxic effects on corneal epithelial cells. Kucukoduk et al demonstrated that preservative-free brimonidine induced significantly stronger early apoptosis than preserved formulations [ 39 ], and Fisenko et al reported that high-concentration brimonidine (1:10 dilution), even without BAK, caused morphological damage and reduced cell viability [ 40 ]. Similarly, timolol at comparable dilutions exhibited cytotoxic effects only when preserved with BAK, while preservative-free timolol showed minimal toxicity.…”

Impact of Antiglaucoma Drug Number and Class on Corneal Epithelial Thickness Measured by OCT

“…However, despite its favorable tolerability profile, in vitro studies have indicated that brimonidine may exert cytotoxic effects on corneal epithelial cells. Kucukoduk et al demonstrated that preservative-free brimonidine induced significantly stronger early apoptosis than preserved formulations [ 39 ], and Fisenko et al reported that high-concentration brimonidine (1:10 dilution), even without BAK, caused morphological damage and reduced cell viability [ 40 ]. Similarly, timolol at comparable dilutions exhibited cytotoxic effects only when preserved with BAK, while preservative-free timolol showed minimal toxicity.…”