Mutations caused by the main carcinogen found in cigarette

Mutations caused by the main carcinogen found in cigarette



Mutations caused by the main carcinogen found in cigarette

TCR gene mutation test

TCR gene mutation tests were performed as described by Kunugita et al. . Lymphocytes were first isolated from whole blood with Ficoll solution. After exposure of lymphocytes to CSCs for 4 h, cells were resuspended in RPMI 1640 medium containing 10% FCS and recombinant human interleukin-2 (rIL-2, 2 ng/ml). PHA-M (0.2 µg/ml) at 2 × 105 cells per ml were added for mitogenic stimulation, then incubated at 37 °C in a humidified 5% CO2/95% air incubator for 7 days. Culture medium was changed every 2 days. The survival rates of the human peripheral lymphocytes were measured with trypan blue dye exclusion test. The mean values of survival rates of human lymphocytes after the exposure to CSC and after 7 days cultivation period were 92.47 ± 0.81% and 81.69 ± 2.60%, respectively. (D.M. Demarini, R. Gudi, A. Szkudlinska, M. Rao, L. Recio, M. Kehl, P.E. Kirby, G. Polzin and P.A. Richter, 2008, 65-78)

The TCR gene mutation was measured as described by Hongping et al. . Briefly, 1 × 106 cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-CD4 MoAb and phycoerythrin (PE)-labeled anti-CD3 MoAb (Becton Dickinson) for 30 min on ice. After washing the cells, propidium iodide (Sigma) was added to the cell suspension at a final concentration of 10 µg/ml to gate out dead cells and then analyzed by a FACScan flow cytometry (Becton Dickinson, USA). More than 2 × 105 events were acquired in dual parameter (FITC and PE) correlated mode, and the mutant window was set to the region where the surface CD3 level was 1/25 that of normal CD4+ cells. The left and right limits of CD4 FITC fluorescence for the mutant window were set, respectively, at 0.5 and 2 times the mode of FITC fluorescence intensity for normal CD3+4+ T cells. The TCR gene mutation frequency (Mf-TCR) values were calculated as the number of events in the mutant cell window divided by the total number of CD4+ T cells in the flow cytogram. (D.M. Demarini, R. Gudi, A. Szkudlinska, M. Rao, L. Recio, M. Kehl, P.E. Kirby, G. Polzin and P.A. Richter, 2008, 65-78)

Statistical analysis

Each experiment was performed three times (three blood samples from the one donor were measured in the same assay). Paired t-test was used to compare the differences between samples exposed to the same CSC concentration either in the presence or absence of the S9 mixture for each indicator. One-way ANOVA followed by LSD post hoc tests (equal variances) or Dunnett's T3 post hoc tests (unequal variances) were used to analyze differences between samples at different doses. Statistical analyses were performed using the SPSS 11.0 software.

Results

Neutral red uptake (NRU) assay results

The results of the NRU assay are presented in Table 1. Differences in cell viability between the NS and DMSO controls were not significant (P > 0.05; 100% and ...