Handbook of Vitamin C Research

100Ana I. Haza, Almudena García and Paloma Moralesa humidified atmosphere. After the cells were washed with PBS, the label incorporated intothe damaged sites of DNA was detected using a FACS Calibur flow cytometer (Becton andDickinson) and the Cell Quest software. For each experiment 10 4 cells were analyzed. Theresults are expressed as percentage of apoptotic cells over the total cells, and data are mean ±standard deviation (S.D.) of three independent experiments.Measurement of ROSROS production was determined using 2´,7´-dichlorodihydrofluorescein diacetate(H 2 DCFDA) from Molecules Probes (Eugene, Oregon, USA). H 2 DCFDA diffuses throughthe cell membrane and is hydrolyzed by esterases to non fluorescent dichlorofluorescein(DCFH). In the presence of ROS, this compound is oxidized to highly fluorescentdichlorofluorescein (DCF).In our previous works we have showed that both NPIP and NDBA increase the ROSproduction in HL-60 cells (5-20 mM and 0.5-2.5 mM, respectively) after 0.5 h incubationtime [44], whereas it was only observed a significant increase of ROS levels in HepG2 cellsone hour after treatment with NPIP (10-45 mM) but not with NDBA (1-3.5 mM) [45]. Forthese experiments, HepG2 and HL-60 cells were cultured in Dulbecco´s Modified Eagle´sMedium and RPMI 1640 Medium, respectively, without phenol red and without fetal calfserum and subsequently were pre-incubated with vitamin C (5, 10 and 50 M) for 1 h. Afterpre-treatment with vitamin C, 25 mM NPIP was added for 1 h in HepG2 cells and 20 mMNPIP or 2 mM NDBA was added for 0.5 h in HL-60 cells. Then, 2.5 10 5 cells were washedwith PBS loaded for 30 min with H 2 DCFDA (10 M) and incubated in a water bath (37ºC).The cells were kept on ice and fluorescence intensity was read immediately with FACSCalibur flow cytometer (Becton & Dickinson) and the CellQuest software. For eachexperiment 10 4 cells were analyzed. DCF fluorescence is expressed as percentage of control,and data are mean ± standard deviation (S.D.) of three independent experiments.Statistical Analyses of DataImages of 50 randomly selected cells per concentration were evaluated and the test wascarried out three times. The reported OTM is the mean ± standard deviation (S.D.) of thethree independent experiments. Cultures without N-Nitrosamines and vitamin C wereconsidered as negative control and cultures with N-Nitrosamines as positive controls (C 1 ).Induction of oxidative DNA damage by N-Nitrosamines was defined as 100% ofgenotoxicity. On the other hand, in the analysis of DNA damage induced by a simultaneoustreatment of H 2 O 2 and vitamin C, cultures without H 2 O 2 and vitamin C were considered asnegative controls (C 0 ) and cultures with H 2 O 2 as positive controls (C 1 ). Induction of DNAdamage by H 2 O 2 was defined as 100% of genotoxicity.Student‘s t-test was used for statistical comparison and differences were consideredsignificant at P≤0.05 or P≤0.01. Descriptive and graphical methods were used to characterizethe data. All tests were performed with the software package Statgraphics Plus 5.0.