Figure 3.

Vitamin E promotes DNA damage and cell transformation. (a) Human fibroblasts (IMR90) were treated with vitamin E (100 µM) and/or neocarzinostatin (NCS, used as positive control), 0.1 µM or 0.5 µM. DMSO was applied in control samples since it was used to dilute vitamin E. After 48 h of pre-incubation with vitamin E the cells were treated with NCS for 2 h and finally with MEM for 24 h (see Table below the histogram). The micronuclei were scored counting at least 1,000 nuclei per sample. A significance difference was observed between control and vitamin E-treated samples (P < 0.05) and between control and NCS-treated samples (P < 0.01 and P < 0.001). Bars represent the mean (±SE). All data were analyzed by the unpaired t-test *P < 0.05; **P < 0.01; ***P < 0.001. (b) An example of DAPI-stained nuclei of cells pre-incubated with DMSO (control, upper) and DMSO plus vitamin E (below). Arrows indicate micronuclei. (c) BALB/c3T3 cells were seeded for cellular transformation assay (CTA), maintained in culture for 24 h and exposed to minimum essential medium (MEM) supplemented with 10% fetal bovine serum (MEMF), 0.1% DMSO or 100 µM vitamin E for 24 h. Then cells were treated with B[a]P (final concentrations 0.01 - 0.1 - 1 µg/ml) for 48 h. Four weeks after seeding, cells were fixed, stained and scored for transformed foci. The type III foci number was determined in each plate, according to the established scoring criteria. Statistical analysis of foci distribution was performed by the Mann–Whitney unpaired t-test. The transformation frequency (TF) was calculated on the basis of the foci number divided by the clonal efficiency (ACE) observed in the cytotoxicity assay performed concurrently. ACE represented the number of cells surviving chemical treatment. Cells treated with vitamin E and then with B[a]P at concentrations leading to cell transformation showed a significant increase in TF (P < 0.01, Exact Poisson test). (Control vs groups of each treatment).