Inhibition by vitamin C of apoptosis induced by N-nitrosamines in HepG2 and HL-60 cells.

The aim of this study was to evaluate the effect of vitamin C towards N-nitrosopyrrolidine (NPYR)- and N-nitrosodimethylamine (NDMA)-induced apoptosis in human hepatoma (HepG2) and leukemia (HL-60) cell lines using flow cytometry analysis and the terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling assay (TUNEL). None of the vitamin C concentrations tested (1-100 mu M) caused cytotoxicity in HepG2 cells. However, there were significant losses of HL-60 cells viability, measured by MTT assay, 72 h after treatment with 50 and 100 mu M vitamin C (29 and 46%, respectively.). Moreover, an increase of lactate dehydrogenase release was significant with 50 mu M at 72 h (28%) and with 100 mu M of vitamin C at 48 and 72 h (27 and 36%, respectively). Also, the percentage of apoptotic HL-60 cells found in TUNEL assay increased to 21% when they were treated with 100 mu M vitamin C for 72 h. Thus, in subsequent simultaneous treatments with NPYR (30 and 50 mM) or NDMA (27 and 68 mM) and vitamin C, concentrations of 5-50 mu M vitamin C were used. Our results revealed that vitamin C, at all concentrations and times tested, reduced the apoptosis induced by NPYR and NDMA in both cell lines, showing a similar effect in HepG2 and HL-60 cells towards NPYR (50 mM) - 65 and 63% of reduction, respectively - whereas towards NDMA (27 mM) the inhibition was higher in HL-60 than in HepG2 cells - 75 and 57%, respectively. Therefore, our findings suggest that inhibition of apoptosis may be one of the mechanisms by which vitamin C exerts its protective effect. Copyright (C) 2008 John Wiley & Sons, Ltd.