Studies on biodosimetry for estimating radiation dose to the respiratory tract cells from inhaled radon

Exposures to radon and radon progeny lead to α-particle irradiation of cells in the respiratory tract and increase the risk of lung tumor. The absorbed dose in target cells' level, however, remains unclear. Lung model of International Commission of Radiological Protection (ICRP) publication 66 describes that the cells at risk of respiratory tract tissues are secretory and basal cells in bronchial airways, and Clara and Type II cells in alveolar interstitial region. The model requires the calculation of absorbed dose in tissue layers containing the target cells. Recently, biodosimetric approach has been developed and applied to estimate absorbed dose in respiratory tract cells. In the biodosimetry, responses of isolated cells that have been previously exposed to α emitters in vivo are compared to the responses obtained from similar populations of cells that are irradiated in vitro using α sources. The purpose of this study is to investigate the dose-response relationships of radiation-induced cell death and micronucleus formation as a preliminary experiment for biodosimetry in respiratory epithelial cells of rat utilizing immortalized Type II cell line. In addition, a method of isolating primary Type II cells from rat is developed. Survival fractions and micronucleus formation frequencies were studied in rat alveolar epithelial cell line (SV40T2) irradiated in vitro to α-particles (241Am, 0.1 Gy/min, 0.1-1.2 Gy) or X-rays (200 kVp, 1 Gy/min, 0.5-5 Gy). The survival curve for α-particles irradiation was exponential with a D37 of 0.8 Gy, and the relative biological effectiveness (RBE) value was 3.8, as determined at D37. The cytochalasin B blocking technique was employed to measure frequencies of micronucleus induction. A linear relationship between the dose and the micronucleus formation frequencies was observed for doses until 1 Gy of α-particles and 4 Gy of X-rays. The straight line of the dose response curve fitted to Y=a+bD (Y: the micronucleus formation frequency (%), a: the micronucleus formation frequency in control subjects, b: the linear slope, D: the dose in Gy). The frequencies in control (a) were 1.0-3.2 and the slopes for α-particles (b) was 28.5. The RBE value was 4.3, as calculated from the slopes. The slope for α irradiation was dependent on the energy of the α-particles. These results indicate that the micronucleus assay is available for biodosimetry of α-particle irradiation in rat respiratory epithelial cells, although consideration should be given to the range of the linearity and the linear energy transfer (LET) of α-particles. Pneumocytes were isolated from lung of Wister rats by dispase digestion and separated on isotonic Nycodenz gradients. Type II cells rich fraction was recovered from the density range around 1.06 g/ml of the Nycodenz gradient. The cell yield was 5 x 105-2 x 106 cells per rat. Type II cells were identified by immunohistochemistry utilizing epithelial cell- specific rat cytokeratin 17 antibody and by alkaline phosphatase staining. The purity was approximately 40% in the staining. The cells could be cultured on Mylar bottom dishes for α irradiation. It is possible to obtain relatively pure Type II cells from rat that can be used for biodosimetry assay. This approach provides a biologically based method for estimating the α dose to the respiratory tract epithelial cells from inhaled radon and radon progeny. (author)