Abstract
Purpose: :
To study the protective effects of 0.1% brimonidine tartrate on ARPE-19 and Müller Cells (MIO-M1) exposed to hydroquinone (HQ), a toxic component found in cigarette smoke.
Methods: :
ARPE-19 and MIO-M1 cells were initially pre-treated for 6 hours with one of four different concentrations (1/2x, 1x, 5x and 10x) of 0.1% brimonidine tartrate ophthalmic solution. The clinical dose (1x, 1mg/mL) is 0.1 ml of 0.1% brimonidine if injected into the 4ml human vitreous. After 6 hours of brimonidine pre-treatment, cells were exposed to 100 µM HQ dissolved in DMSO for 24 hours while brimonidine was still present. The production of reactive oxygen species and nitrogen species (ROS/RNS) were determined with 2`, 7`-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay. The lactate dehydrogenase (LDH) levels were measured using LDH cytotoxicity kit II.
Results: :
ROS/RNS levels for ARPE-19 cells treated with 100µM HQ were 22420±610.6 compared to DMSO alone 5259±223. ARPE-19 cells pre-treated with 5x (19120±417.1,p=0.01) and 10x (5739 ±73.33, p<0.0001) doses of brimonidine showed significant lower levels of ROS/RNS. The 1/2x (21950±265.6,p>0.5) and 1x (21690±1031,p>0.5) brimonidine pre-treated cultures were similar to HQ alone. In MIO-M1 cells pre-treated with brimonidine the ROS/RNS levels were significantly lower at the 10x dose compared to 100µM HQ alone treated cells (15390±162.6 vs. 26950±542.7, p<0.0001). There are no significant changes in 1/2x , 1x and 5x doses.ARPE-19 cells pre-treated with 10x brimonidine showed significant lower LDH levels compared to 100µM HQ alone (1.6±0.1 vs. 2.633±0.1453,p=0.0042). There were no protective effects for the 1/2x (2.6 ±0.05774, p>0.8), 1x (2.5 ±0.05773, p>0.4) and 5x (2.527 ±0.06489, p>0.5) brimonidine doses. In the MIO-M1 cells, LDH levels did not decrease at any of the doses,: 2.516±0.06549,p>0.3; 2.554±0.04631,p>0.4; 2.494±0.07394,p>0.2 and 2.479±0.03371,p>0.1 for 1/2x, 1x, 5x and 10x doses respectively compared to 2.614±0.05744 for 100µM HQ alone treated cells.
Conclusions: :
Brimonidine 0.1% has an in vitro protective effect against HQ induced toxicity, reducing the ROS/RNS levels at 5x and 10x doses in ARPE-19 cells and at 10x on MIO-M1 HQ treated cells. Pre-treatment with10x brimonidine also decreases the LDH levels in ARPE-19, but didn’t show any change in HQ treated MIO-M1 cells.
Keywords: drug toxicity/drug effects • retinal pigment epithelium • stress response