Abstract
Purpose: :
To evaluate the effect of solubilized triamcinolone acetonide (sTA) on human retinal pigment epithelial (ARPE- 19) and rat neurosensory (R28) cells in culture.
Methods: :
Triamcinolone acetonide (Kenalog®, Bristol Myers Squibb, Princeton, NJ) was centrifuged at 5000 rpm for 1 minute and the supernatant was discarded. The pellet of TA was re-suspended in an equivalent amount of DMSO to achieve the same concentration of TA found in the commercial suspension. ARPE-19 cells (ATCC, Manassas, VA) and R28 cells (courtesy of Gail Seigel) were treated with 100, 200, 500 and 1000 (clinical dose) µg/ml of TA re-suspended in DMSO for 24 hours. Toxicity was evaluated by trypan blue dye-exclusion cell viability assay, WST-1 mitochondrial dehydrogenase assay, and caspase-3/7 apoptosis related assay.
Results: :
The mean cell viabilities of ARPE-19 cells treated with sTA (95.7 ± 2.0 %; 94.9 ± 1.8 %; 93.0 ± 2.1%; 88.4 ± 14.1 for 100, 200, 500, 1000 µg/ml, respectively) were not significantly different from control cells treated with DMSO concentration equivalent to 1000 µg/ml (95.2 ± 1.5%, p>0.05). In contrast, R28 cells showed a significant reduction in cell viability at the highest doses after 24 hour exposure to sTA compared to corresponding DMSO-treated controls (68.7 ± 8.4% vs 88.1 ± 2.1% and 61.6 ± 12.1% vs 82.4 ± 2.42 %, for 500 and 1000 µg/ml respectively, p<0.05). sTA did not affect the mitochondrial dehydrogenase activity in either cell line at any concentration. Caspase-3/7 was not activated by treatment with sTA in R28 cells A significant difference in Caspase-3/7 activity was found in ARPE-19 cells treated for 24 hours with 200, 500, and 1000µg/ml sTA compared to DMSO-treated controls (2663 ± 2066 msi vs. -370 ± 1433 msi, p<0.001; 5191 ± 3383 msi vs. 908 ± 1682 msi, p<0.001; and 10008 ± 5071 msi vs. 423 ± 1553 msi, p<0.001 for 200, 500 and 1000µg/ml respectively), suggestive of apoptotic up regulation.
Conclusions: :
Our data suggest that sTA is toxic to retinal cells in culture, in part via caspase dependent apoptosis. However, the toxic effect observed with solubilized TA is less than seen in the commercial crystalline preparation.
Keywords: corticosteroids • apoptosis/cell death • retinal culture