X-ray induced mutants resistant to 8-azaguanine

Synchronous Chinese hamster ovary cells were irradiated in G1 or S phase. Colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 μg/ml 8-azaguanine (AG). An expression period of over three generations (multiplicity of 20) was utilized, with expression times ranging from 58 to 114 h. Both G1 and S phase were practically identical in sensitivity to X-ray-induced mutations, with mutant frequency/viable cell/rad ranging from 1 x 10-7 (75-100 rad) to 8 x 10-7 (1000 rad). The spontaneous mutation rate, shown by Luria-Delbruck fluctuation analysis, was 5 x 10-7 per generation. Thirty-three mutants, isolated at random and grown for over 30 generations in the absence of AG, were analyzed for plating efficiency (PE) in different concentrations of AG or in hypoxanthine-aminopterin-thymidine (HAT) medium. Of these, 64% were resistant (PE > 0.1) to 7.5 μg/ml AG, 85% to 5.0 μg/ml, and 91% to 3.5 μg/ml. Only 42% showed possible hypoxanthine-phosphoribosyltransferase (HPRTase) deficiency as evidenced by HAT sensitivity (PE < 0.1). Wild type controls exhibited PE's in 3.5 μg/ml AG of < 0.001 and in HAT of > 0.5. Of ten mutants studied, all demonstrated survival response to radiation similar to wild type cells (D tilde0 of approx. 120 rad). For radiation protection standards, the radiation dose required to induce mutations at a rate equal to that occurring spontaneously is called the doubling dose. The doubling dose observed for acute irradiation was about 3 rad and was estimated to be 10-60 rad for chronic irradiation, similar to that often reported for in vivo studies