Evidence that most radiation-induced HPRT mutants are generated directly by the initial radiation exposure

Radiation-induced HPRT mutants are generally assumed to arise directly from DNA damage that is misrepaired within a few hours after X-irradiation. However, there is the possibility that mutations result indirectly from radiation-induced genomic instability that may occur several days after the initial radiation exposure. The protocols that commonly employ a 5-7 day expression period to allow for expression of the mutant phenotype prior to replating for selection of mutants would not be able to discriminate between mutants that occurred initially and those that arose during or after the expression period. To address this question, we performed a fluctuation analysis in which synchronous or asynchronous populations of human bladder carcinoma cells were treated with single doses of X-irradiation. For comparison, radiation was delivered during the expression period, either from an initial dose of 1.0 Gy followed by two 1.0 Gy doses separated by 24 h or from disintegrations resulting from I125dU incorporated into DNA. The mutation frequency observed at the time of replating was used to calculate the average number of mutants in the initial irradiated culture by assuming that the mutants were induced directly at the time of irradiation. Then, this average number was used to calculate the fraction of the irradiated cultures that would be predicted by a Poisson distribution to have zero mutants. There was reasonably good agreement between the predicted poisson distribution and the observed distribution for the cultures that received single doses. Moreover, as expected, when cultures were irradiated during the expression period, the fraction of the cultures having zero mutants was significantly less than that predicted by a Poisson distribution. These results indicate that most radiation-induced HPRT mutations are induced directly by the initial DNA damage, and are not the result of radiation-induced instability during the 5-7 day expression period.