Handbook of Vitamin C Research

110Ana I. Haza, Almudena García and Paloma MoralesHepG2 and HL-60 cells were incubated with various concentrations of vitamin C (1-100μM) for 72 h. Then, the percentage of apoptotic cells was measured by flow cytometry. Theresults are expressed as percentage of apoptotic cells over the total cells, and are the mean ±standard deviation (SD) of three independent experiments. No significant changes wereobserved when HepG2 and HL-60 cells were treated with 5-50 μM vitamin C for 72 h(Figure 14). However, substantial increase of apoptotic cells was observed when HL-60 cellswere treated with 100 μM vitamin C, about 21%. Based on the results, 5-50 μM vitamin Cwas used in our apoptosis studies.Figure 14.Figure 14. Percentage of apoptotic cells obtained by TUNEL assay and flow cytometry after treatmentwith vitamin C. C 0 , ( ) untreated HepG2 and ( ) HL-60 cells. ( ) HepG2 and ( ) HL-60 cells treatedwith vitamin C for 72 h. Asterisks indicate significant difference from control * p 0.05.Effect of Vitamin C on N-Nitrosamines-Induced Apoptosis by the TUNELAssayIn subsequent simultaneous treatments with N-Nitrosamines and vitamin C, the HepG2and HL-60 cells were incubated with the selected concentrations of 5-50 μM vitamin C. Thepercentage of apoptosis is the mean ± SD of three independent experiments. The effect ofvitamin C on N-Nitrosamines-induced apoptosis is shown in Figure 15.Figure 15 A shows the results obtained with a simultaneous treatment of NDMA (68mM) and vitamin C (5–50 M) in HepG2 and HL-60 cells. The protective effect of vitamin Cwas dose-dependent and significant reductions in the percentage of apoptosis in NDMAHepG2 treated cells (68 mM) were observed at exposure concentrations of 5, 10 and 50 µM(37, 43 and 49% of reduction, respectively). The inhibitory effect of vitamin C towardsNDMA-induced apoptosis in HL-60 cells was significantly higher than in HepG2 cells.Vitamin C at 5, 10 and 50 μM reduced the percentage of apoptotic HL-60 cells induced by 68mM NDMA, by about 56, 65 and 66%, respectively.