Abstract
Purpose: :
Cigarette smoking is a major risk factor for developing age-related macular degeneration (AMD). We recently showed that cigarette smoke extract (CSE) damages human retinal pigment epithelial cells (ARPE-19) in vitro (Bertram et al. Am J Physiol Cell 2009). In these studies, heme oxygenase-1 (HO-1), an enzyme induced under conditions of oxidative stress, was shown to be important for ARPE-19 cell viability after CSE exposure. Here, we tested the hypothesis that antioxidants would prevent cell death and cigarette smoke-induced HO-1 expression in human RPE cells.
Methods: :
ARPE-19 cells were pretreated with 1mM N-acetyl-cysteine (NAC), exposed to CSE, and HO-1 protein expression determined by western blot. The MTT viability assay was utilized to determine if antioxidant pretreatment could prevent cell death in ARPE-19 cells exposed to CSE. All experiments were repeated at least three times.
Results: :
A non-lethal dose of CSE (0.1% CSE, 104 ± 7.9% cell survival compared to unexposed cells) caused increased HO-1 protein expression compared with HO-1 levels in media-only treated cells (8.8 ± 0.9 fold increase; P<0.05). NAC treatment did not prevent HO-1 upregulation (3.2 ± 0.7 fold increase compared to media-only treated cells; P>0.05); however, the upregulation was significantly less than the increase in HO-1 without NAC treatment (P<0.001). The viability of ARPE-19 cells was significantly reduced after exposure to 1% CSE (51.5 ± 3.2% compared to unexposed cells; P<0.05). Antioxidant pretreatment with NAC prevented the cell loss caused by 1% CSE exposure (106 ± 12.7% compared to unexposed cells).
Conclusions: :
Antioxidant treatment prevented CSE induced cytotoxicity, but was unable to fully prevent HO-1 upregulation. These data highlight the complex regulation of HO-1 in human RPE cells exposed to cigarette smoke and suggest that components of CSE besides oxidizing agents might contribute to HO-1 upregulation after exposure to CSE. Further investigations are warranted to determine how HO-1 is regulated in RPE cells.
Keywords: age-related macular degeneration • oxidation/oxidative or free radical damage • retinal pigment epithelium