Protective Effects of Donepezil Against Alcohol-Induced Toxicity in Cell Culture: Role of Caspase-3

Abstract

Ethanol (EtOH) is one of the most frequently abused drugs with heavy health, economic, and societal burdens. Although moderate to low EtOH may have some neuroprotective effects, heavy EtOH consumption associated with high blood alcohol level (BAL) can be quite detrimental. The brain is particularly vulnerable to the damaging effects of high BAL, leading to neuronal loss, cognitive, and behavioral deficits. Although the exact causes of these detriments are not fully elucidated, it is believed that damage to the cholinergic system is at least partially responsible for the cognitive impairment. Thus, high BAL may result in selective apoptotic damage to the cholinergic neurons. Donepezil (DON), a centrally acting, reversible and non-competitive acetylcholinesterase (AChE) inhibitor, approved for use in Alzheimer's disease (AD), may also attenuate EtOH-induced cognitive impairment. Cognitive effects of DON might be due to an anti-apoptotic activity as some AChE inhibitors have been shown to have this property. The aim of this study was to determine whether DON might protect against EtOH-induced toxicity and whether such protection might be apoptotically mediated. We exposed the human neuroblastoma-derived, SH-SY5Y cells to a relatively high concentration of EtOH (500 mM) for 24 h and evaluated the effects of two concentrations of DON (0.1 and 1.0 μM) on alcohol-induced toxicity and caspase-3, an apoptotic marker. We found a dose-dependent protection of DON against EtOH-induced toxicity as well as dose-dependent attenuation of EtOH-induced increases in caspase-3 levels. Thus, DON may inhibit apoptosis as well as alcohol-induced toxicity.

Keywords: Acetylcholinesterase inhibitor; Apoptosis; Caspase-3; Neuroprotection; Neurotoxicity; SH-SY5Y cells.

Figure 1

Effect of different concentrations of ethanol (EtOH) on cell viability as determined by MTT assay in SH-SY5Y cells. Cells were incubated for 24 h. Values are mean ± SEM of 3 independent experiments. In every experiment, each sample was run in replicates of 6. *P<0.05, **P<0.01 compared to control.

Figure 2

Effects of donepezil (DON), 0.1 and 1 μM on EtOH-induced cell death in SH-SY5Y cells. DON was applied 2 h before EtOH (500 mM) and cells were incubated for 24 h. Values are mean ± SEM of 5 independent experiments. In every experiment, each sample was run in triplicate. **P<0.01 compared to control, ^##P<0.01 compared to EtOH.

Figure 3

Effects of ethanol (EtOH) 500 mM and donepezil (DON 0.1 and 1 μM) alone or in combination on caspase-3 levels in SH-SY5Y cells. DON was applied 2 h before EtOH and cells were incubated for 24 h. Immunnoblots of beta-actin and total caspase-3 are included. Caspase-3 values were normalized per beta-actin protein. Values are mean ± SEM of 4 independent experiments. In every experiment, each sample was run in duplicate. **P<0.01 compared to control, ^#P<0.05, ^##P<0.01 compared to EtOH.