The Role Of Oxidative Stress In Acrolein-induced DNA Damage In HepG2 Cells

Objective: Acrolein is found widely in the environment, particularly as a component of smoke. Acrolein can generate endogeneously as a metabolic product of the anticancer-drug cyclophosphamide and during conditions of lipid oxidation.Acrolein is an intense irritant and displays a range of toxic effects. The International Agency for Research on Cancer (IARC) concluded that there was inadequate evidence for its carcinogenicity in experimental animals. In addition, there is increasing evidence that acrolein is genotoxic. Previous studies demonstrated that acrolein was able to induce the generation of reactive oxygen species (ROS) in some cell types. In addition, both in vitro and in vivo studies showed that acrolein caused a significant reduction of intracellular GSH. The genotoxic effects of acrolein have already been demonstrated in many cell lines. However, this is the first evidence of acrolein-induced DNA damage in human hepatoma line (HepG2). HepG2 cells retain the activities of several phase I and II xenobiotic metabolizing enzymes presented in human hepatocytes. It has been shown to be a suitable system for investigation of genotoxicity.The overall object of present study is to explore whether acrolein causes DNA damage in HepG2 cells and to elucidate the underlying mechanism of acrolein-induced DNA damage. Thus it may provide some information for safety assessment to humans on acrolein.Method: DNA damage induced by acrolein was assessed by standard and proteinase K-modified alkaline single cell gel electrophoresis (SCGE) assays. To elucidate the oxidative DNA damage mechanism in HepG2 cells, we used the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH). We analyzed the oxidative DNA damage in acrolein-treated cells by immunocytochemistry staining of 8-hydroxydeoxyguanosine (8-OHdG). To further evaluate the involvements of ROS and GSH in the formation of acrolein-induced DNA strand breaks, we studied the protective effect of NAC.Result: Using the standard SCGE assay, a significant dose-dependent increment in DNA migration was detected at lower concentrations of acrolein (12.5- 25μM); but at the higher tested concentrations (50-100μM), a reduction in the migration compared to the maximum migration at 25μM was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of acrolein (50-100μM). These results indicated that acrolein caused DNA strand breaks at lower concentrations of acrolein and DNA-protein crosslinks (DPC) formation at higher concentrations. The present study showed that acrolein induced the increased levels of ROS and depletion of GSH in HepG2 cells, the doses being 50-100μM and 25-100μM, respectively. Moreover, acrolein significantly caused 8-hydroxydeoxyguanosine (8-OHdG) formation in HepG2 cells at concentrations from 25 to 100μM. N-acetylcysteine (NAC), a precursor of GSH and intracellular ROS scavenger, prevented the formation of DNA strand breaks caused by acrolein.Conclusion: we conclude that the DNA damage of acrolein is mediated by the formation of ROS and depletion of GSH, which cause oxidative DNA damage, formation of DNA strand breaks and DPC.