Abstract
Purpose: :
To study effects of Ketorolac Tromethamine (KT) ophthalmic solution 0.4% (Acular LS, Allergan, Irvine, CA) in human ARPE-19 and rat neurosensory (R28) cells in vitro.
Methods: :
ARPE-19 & R28 cells were treated for 24 hours with 400µg/ml(4X), 200µg/ml(2X),100µg/ml(X), 50µg/ml(1/2X) and 25µg/ml(1/4X) of KT. Cell viability (CV) was measured using the trypan blue dye-exclusion assay. Mitochondrial membrane potential (ΔΨm) was measured using JC-1 assay kit. Mitochondrial dehydrogenase (MD) activity was determined using WST-1 colorimetric assay. Activity of caspase 3-7 was measured by fluorescence caspase kit.
Results: :
The mean CV of ARPE-19 and R28 cells cells was reduced after 24 hour exposure to 4X dose of KT, (38.3±11.2%,[p<0.001] versus 96.2±1.5% for the untreated cells for ARPE-19 ) while for the R28 cells it was 64.4±12.5,[p<0.001] versus 89.4±5.1% for the untreated cells. KT reduced the ΔΨm of ARPE-19 cells at the 4X dose, (0.69±0.04, [p<0.001] versus 1.1±0.1 for untreated controls). There was no significant change in ΔΨm of R28 at any of doses. The MD activity was reduced in the ARPE-19 cells at the 4X dose only, (0.046±0.002, [p<0.01] versus 0.65±0.1 for untreated controls) while the MD activities of R28 cells were reduced in the 4X, 2X and X doses of KT, (0.046±0.02,[p<0.001], 0.07±0.02,[p<0.001] and 0.43±0.05,[p<0.001] respectively versus 0.6±0.1 for the untreated controls). There was no significant increase in caspase 3-7 activity in either cell line at any dose of KT.
Conclusions: :
Ketorolac tromethamine decreases the cell viability of ARPE-19 and R28 cells at highest tested doses. As seen with the JC-1 and MD assays, KT appears to have variable toxicity to the mitochondria .
Keywords: apoptosis/cell death • age-related macular degeneration • mitochondria