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 deoxynucleotidyl transferase-mediated dUTP nick end labelling assay (TUNEL). None of the vitamin C concentrations tested (1-100 microM) 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 microM vitamin C (29 and 46%, respectively). Moreover, an increase of lactate dehydrogenase release was significant with 50 microM at 72 h (28%) and with 100 microM 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 microM 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 microM 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.
Arranz
Nuria
N
Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Haza
Ana I
AI
García
Almudena
A
Delgado
Ma Eugenia
ME
Rafter
Joseph
J
Morales
Paloma
P
D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001205:Ascorbic Acid / Q000494:pharmacology*; D002273:Carcinogens / Q000633:toxicity; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity; D018922:HL-60 Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008113:Liver Neoplasms / Q000473:pathology; D016202:N-Methylaspartate / Q000037:antagonists & inhibitors / Q000633:toxicity; D009242:N-Nitrosopyrrolidine / Q000633:toxicity; D009602:Nitrosamines / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D013778:Tetrazolium Salts; D013844:Thiazoles
D000975:Antioxidants; D002273:Carcinogens; D018690:Excitatory Amino Acid Agonists; D009602:Nitrosamines; D013778:Tetrazolium Salts; D013844:Thiazoles; D016202:N-Methylaspartate; D003577:Cytochrome P-450 Enzyme System; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue; D001205:Ascorbic Acid; D009242:N-Nitrosopyrrolidine