| Objective: Immortalized human bronchial epithelial cells(BEAS-2B) were exposed to cigarette smoke and radon either alone or in combination, to measure changes in ROS, 8-OHdG, cell cycle, apoptosis, malignant transformation and the expression of several genes, in order to investigate the mechanisms through which the two factors, either alone or in combination to induce lung cancer .Methods: 1.5×105 BEAS-2B cells in exponential growth phase were seeded on transwell membrane,with the density of 1×105 cells. When the cells were attached, they were exposed to tobacco smoke or radon. The concentration of oxygen was 21%, flow rate was 10ml/min. The suitable time and concentration would be determined by cell inhibition rate.Then the cells were divided into negative control group, tobacco smoke exposed group, radon exposed group, smoke treatment followed by radon exposed group, radon treatment followed by smoke exposed group(named as C group, Sm group, Rn group, Sm+Rn group, Rn+Sm group respectively).The cells were exposed to cigarette smoke for 10 minutes once at a smoke concentration of 20% in Sm group; and the cells were exposed to radon for 20 minutes with a concentration of radon of 20000Bq/m3 in Rn group; Sm+Rn group and Rn+Sm group were the same concentration and time with Sm group and Rn group. The cells were subcultured twice per week after exposing to either tobacco smoke or radon. The cells were exposed to smoke or radon again after freshly reseeded in exponential phase. The normal control group was exposed to filtrated air, and the other conditions were the same with the exposed group. Then the following endpoints were examined:(1)To detect the cell inhibition rate by CCK-8 assay.(2) The mean content of ROS in cells were detected by the laser scanning confocal microscope with fluorescent probe technique.(3)The mean content of 8-OHdG were detected by enzyme-linked immunosorbent assay in the once and five-time passage treated cells .(4)The cell cycle and apoptosis were detected by flow cytometer in the once and five-time treated cells.(5)After treating for five consecutive times, stopped treating and subcultured, the results of neopalstic changes were detected by cell doubling time, serum antibody experiment, colony formation experiment in soft agar, and expression of the FHIT, mutant p53 and p16 genes in the fifth generation.Results: (1)The higher the concentration of tobacco smoke and radon gas, the higher the inhibition rate of the exposed cells was.(2) The mean content of ROS and 8-OHdG in the combined treatment groups were higher than single exposure groups. The mean content of ROS and 8-OHdG in all the fifth generation of exposed groups were higher than that in the corresponding groups of the first generation.(3)Compared with the normal control group, the percentage of G2/M phase in the first generation of exposed cells was less and the apoptosis rate in the exposed groups was higher; The apoptosis rate in the first generation of combined treatment cells was higher than single exposed cells. The percentage of G2/M phase in the fifth generation of exposed cells was higher and the apoptosis rate was significantly lower than the corresponding first generation of exposed cells.(4) Compared with the normal control group,the anchorage independent growth rate in the combined treatment groups was higher and the doubling time was shorter. (5) The expression of p16 and FHIT genes of the cells in Rn+Sm group was lower and the mutant p53 gene was higher than normal control group.Conclution: (1) A cell model was successfully established to study the neoplastic changes in response to tobacco smoke and radon in vitro. (2) The results showed that tobacco smoke and radon caused DNA oxidative damage, and cell apoptosis. Tobacco smoke in combination with radon caused a synergistic induction in oxidative damage and malignant transformation. (3) Exposure to radon and followed by smoke decreased the mRNA expression of p16 and FHIT gene, and increased the mRNA expression of mutant p53 gene.
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