ABSTRACT
OBJECTIVETo assess the DNA damage of copper 8-quinolinolate (CuQ) and to elucidate the plausible mechanisms.METHODSHepG2 cells were treated with CuQ0-4 μmol·L-1 for different time,DNA damage was measured by Comet assay.Catalase (CAT) activity,glutathione(GSH) level and thiobarbituric acid reactive substances (TBARS) were measured.NF-κB was examined using Western blotting.8-Hydroxydeoxyguanosine (8-OHdG) was measured by immunoperoxidase staining analysis.RESULTSCuQ 0.5 -4 μmol·L-1 caused significant increase of DNA migration in HepG2 cells.CuQ significantly decreased levels of GSH and activity of CAT in HepG2 cells (P ＜0.05).Moreover,CuQ significantly increased accumulation of the p65 subunit of NF-κB into nucleus,levels of lipid peroxidation product TBARS and the formation of 8-OHdG (P ＜0.05).The intracellular GSH level was modulated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO),depletion of GSH in HepG2 cells pre-treated with BSO dramatically increased susceptibility of HepG2 cells to CuQ-induced DNA damage.CONCLUSIONCuQ exerts DNA damage by oxidative stress and increases accumulation of p65 subunit of NF-κB into nucleus in HepG2 cells.
ABSTRACT
Objective:To investigate whether hydroxytyrosol (HT) may ameliorate oxidative stress and nuclear factor kappa B (NF-?B) activation in the lipopolysaccharide (LPS)-stimulated THP-1 cell line.Methods:The intracellular formation of reactive oxygen species (ROS) was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe.Intracellular glutathione (GSH) level was estimated by fluorometric methods;Nitric oxide (NO) production was measured as nitrite (a stable metabolite of NO) concentrations.Western blot analysis was used to detect the expressin level of NF-?B.Results:The results showed that treatment of THP-1 cells with HT significantly reduced LPS-stimulated NO production and ROS formation in a concentration-dependent manner.HT at 50 and 100 ?mol/L concentrations increased the GSH level.The specific DNA-binding activities of NF-?B in nuclear extracts from 50 and 100 ?mol/L HT treatments were significantly suppressed.The antioxidant N-acetylcysteine (NAC) also showed the same effects as HT on LPS-induced ROS and NO production,change of GSH level,and NF-?B activation.Conclusion:These findings suggest that HT has antioxidant activity by suppressing intracellular oxidative stress and NF-?B activation in THP-1 cells.