MUTATION-INDUCTION IN GAMMA-IRRADIATED PRIMARY HUMAN BRONCHIAL EPITHELIAL-CELLS AND MOLECULAR ANALYSIS OF THE HPRT(-) MUTANTS

We have examined various radiobiological parameters using commercially -available primary normal human bronchial epithelial (NHBE) cells, whi ch can be subcultured more than 20 population doublings, and have esta blished the mutation system in order to characterize the molecular cha nges in gamma-irradiated primary cells. The survival curve, obtained a fter irradiation of cells with Cs-137 gamma-rays, indicates that the D -0, D-q, and n values are 1.34 Gy, 1.12 Gy, and 2.3, respectively. The induction of HPRT(-) mutation was dose-dependent and the mutant fract ion increased in a non-linear fashion. Since the doubling number of NH BE cells is limited, DNA was extracted directly from the single mutant colonies and alteration in the HPRT gene locus was analyzed using mul tiplex PCR technique. Among spontaneous mutants, the proportion with t otal and partial deletions of the gene was 10.0% (2/20) and 60.0% (12/ 20), respectively, while 30.0% (6/20) did not have any detectable chan ges in the nine exons examined. On the other hand, the fraction of tot al deletion increased by more than 2-fold among mutants induced by gam ma-rays in that 26.3% (10/38) of them showed the total gene deletions. Twenty-five out of 38 gamma-induced mutants (65.8%) had partial delet ions and 3 mutants (7.9%) had no detectable alteration. The present re sults showed that gamma-irradiation efficiently induced HPRT gene muta tion in primary human epithelial cells and that most of the induced mu tants suffered larger deletions compared to that observed in spontaneo us mutants. This system provides an useful tool for determination of m utagenicity and understanding the molecular mechanisms of environmenta l carcinogens in primary human bronchial cells.