| The effects of ferulic acid, caffeic acid, vanillic acid, aspirin and salicylic acid on oxygen-radical-generated DNA damage in human lymphocytes were studied with single cell gel eletropherosis (comet assay). The tail moment (percent of DNA in the tail x tail length) was used as the cornet parameter. The data associated with structures of the five phenolic acids were analysed and compared. Laser scanning confocal microscope (LSCM) was introduced to investigate the 3-D distribution of DNA within the comet and four fluorescent dyes were compared when the single cell gel eletrophoresis was set up. Main Procedures and methods:1. Comet assay was employed to assess DNA damage of human lymphocytes treated with either various concentrations of H2O2 (0, 10, 20, 40, 80μmol/L) for 20 minutes or 20μ mol/L H2O2 for different time periods (0, 10, 20, 30 minutes). To quantify the DNA damage, three different comet parameters were evaluated: the comet length (Length of the entire comet), the tail length (Difference between the comet length and the diameter of the comet head) and the tail moment. Laser scanning confocal microscope (LSCM) was introduced to investigate the 3-D distribution of DNA within the comet. Four fluorescent dyes including propidium iodide (PI), ethidium bromide (EB), acridine orange (AO) and Hochest 33258 were compared.2. Comet assay was employed to assess DNA damage of human lymphocytes pre-treated with various concentrations (0, 50, 100, 500 u mol/L) of ferulic acid, caffeic acid, vanillic acid, aspirin and salicylic acid at 37℃ for 30min, then challenged with H2O2 at 4℃ for 10min, The fluorescence microscope (200 X) was introduced to observe DNA damage. ANOVA analysis (P<0.05 ) was performed on tail moment (the comet parameter) .3. Comet assay was employed to assess DNA damage of human lymphocytes treatedwith caffeic acid (500μ mol/L), aspirin(500μmol/L) and salicylic acid(500 μ mol/L) at 37℃ for 30min, without H2o2 challenge. The fluorescence microscope (200 X) was used to observe DNA damage. ANOVA analysis (P<0.05) was performed on tail moment. Main Results:1. The tail moments (X±S) were 0.001 ±0.002, 0.31±0.35, 1.23±0.96> 2.82±1.38, 3.52±0.96 (P<0.01), respectively, after treatment with various concentrations of H2O2 (0, 10, 20, 40, 80μmol/L); the tail moments (x±s) were0.05±0.09, 0.22±0.30, 1.62±0.93, 1.89±1.09 (P<0.01), respectively, after treatment for different time(0, 10, 20, 30 minutes ). Tail length and comet length also exhibited significant increases (P<0.01) with the increased dose and extended treating time. The 3-D reconstruction made by LSCM gave a more distinct image of the comet compared with the fluorescence microscope. PI and EB were better dyes in terms of the definition of the image.2. Compared to positive control, the tail moments were all reduced after pretreatment of the cells with different phenolic acids at different concentrations and then challenged with H2O2. The protective effects of ferulic acid and vanillic acid were greater, and the increasing trend were shown with the increased dose. Whereas aspirin, salicylic acid, especially caffeic acid at high dose (500μmol/L) exhibited greater tail moment than low doses(50μmol/L,100μmol/L) groups.3. The tail moment showed markedly increasing compared to control group after treatment of the cells with caffeic acid (500μumol/L) at 37℃ for 30min (without H2O2 challenged), whereas aspirin(500 μumol/L) and salicylic acid(500μ umol/L) showed no effects.Conslusion:1. H2O2 induces dose-dependent and time-dependent cytotoxic effect in terms of DNA damage on human lymphocytes. The LSCM provides substantial and measurable improvement in the definition of the "comet". PI and EB are suitable for comet assay.2. Ferulic acid and vanillic acid offered significant DNA protective effects. Over the concentration range of 50μmol/L to 500μmol/L, the protective effects showed adose-dependent trend. Aspirin and salicylic acid offered significant DNA protective...
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