High frequency of deletions at the hypoxanthine-guanine phosphoribosyltransferase locus in an ataxia-telangiectasia lymphoblastoid cell line irradiated with gamma-rays.

The molecular nature of gamma-ray-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in an ataxia-telangiectasia (A-T) lymphoblastoid cell line was investigated. Twelve of 15 gamma-ray-induced HPRT-deficient mutants showed deletions. Eight of them had lost the entire HPRT gene, one showed a 1.9-kb deletion, and three had deletions of about 40-150 base pairs. Of the eight mutants that lost the entire gene, five had also lost both DXS79 and DXS86, flanking markers of the HPRT locus. The spectrum of mutations induced by gamma-irradiation in the A-T cells showed a high frequency of deletions in comparison with that in a control cell line, WIL2-NS. Sequence analysis of breakpoint junctions in four mutants revealed that three of them had junctions between short identical sequences at each breakpoint, leaving one copy at the junction. These results suggest that non-homologous end-joining is the major mechanism for deletion formation in A-T cells.

Quantitative and qualitative effect of gamma-ray dose-rate on mutagenesis in human lymphoblastoid cells.

Induction of mutations to 6-thioguanine resistance (TGr) by gamma-rays at three different dose-rates and molecular changes in the HPRT gene were studied in human lymphoblastoid WIL2-NS cells. Mutant induction showed a curvilinear dose-response for acute irradiation (30 Gy/h). The total mutant frequency was lower after irradiation at 0.17 or 0.006 Gy/h compared with acute irradiation. An apparent linear relationship between total dose and mutant frequency was found for the chronic irradiations. Spontaneous mutant frequency increased linearly with the exposure time of protracted irradiation at 0.006 Gy/h. After the spontaneous mutant frequency was subtracted from the total mutant frequency for irradiation at 0.006 Gy/h, no significant difference was found in the mutant frequency as a function of dose between the cultures irradiated at 0.17 Gy/h and those at 0.006 Gy/h. The inverse dose-rate effect, which has been observed in proliferating mouse L5178Y leukemia cells was not evident in WIL2-NS cells at the dose-rates employed. Structural alterations at the HPRT locus in TGr mutants were examined with the multiplex PCR method and compared among cultures irradiated at different dose-rates. Assuming that the mutants isolated were primarily independent, approximately 17% of spontaneous mutants were deletion mutants. When the fraction of spontaneous mutants in the irradiated cultures was subtracted from the total fraction of each type of mutant, it is clear that low dose-rate gamma-rays induced deletion mutations at the HPRT locus just as efficiently (79%) as high dose-rate gamma-rays (74%).

Mutation induction by different dose rates of gamma rays in near-diploid mouse cells in plateau- and log-phase culture.

Induction of mutation to 6-thioguanine resistance was studied in cultured near-diploid mouse cells (m5S) in plateau and log phase after exposure to gamma rays at dose rates of 30 Gy/h, 180 mGy/h, or 13 mGy/h. In plateau-phase culture, lowering the dose rate from 30 Gy/h to 13 mGy/h resulted in an increase in cell survival and a marked decrease in induced mutation frequency. On the other hand, in the log-phase culture, the magnitude of the dose-rate effects was not as marked as in the plateau-phase culture, particularly within a dose range below 5 Gy. These results, together with those indicating the inverse dose-rate effects in growing mouse leukemia cells (Radiat Res. 115, 273-280, 1988), demonstrate the significant influence of cell growth that takes place during protracted irradiation, particularly for the induction of mutation.

Water content in cultured mammalian cells for dosimetry of beta-rays from tritiated water.

One of the critical factors for dosimetry of beta-rays from tritiated water is the water content within the cell. We estimated the cellular water concentration in cultured mammalian cells by measuring accurately the fraction of the extracellular water in the cell sample with [14C]inulin. The net water content (ml.g-1) after correcting for the extra-cellular water fraction was 0.858 for HeLa (human), 0.833 for JTC12P3 (monkey), 0.829 for NRK (rat), 0.843 for C3H10T1/2 (mouse) and 0.846 for L5178Y (mouse) cells. The mean water content (+/- S.E.M.) in these 5 lines of cultured mammalian cells was 0.842 +/- 0.005.

Mutation induction by different dose rates of gamma rays in radiation-sensitive mutants of mouse leukemia cells.

Induction of cell killing and mutation to 6-thioguanine resistance was examined in a radiation-sensitive mutant strain LX830 of mouse leukemia cells following gamma irradiation at dose rates of 30 Gy/h (acute), 20 cGy/h (low dose rate), and 6.2 mGy/h (very low dose rate). LX830 cells were hypersensitive to killing by acute gamma rays. A slight but significant increase was observed in cell survival with decreasing dose rate down to 6.2 mGy/h, where the survival leveled off above certain total doses. The cells were also hypersensitive to mutation induction compared to the wild type. The mutation frequency increased linearly with increasing dose for all dose rates. No significant difference was observed in the frequency of induced mutations versus total dose at the three different dose rates so that the mutation frequency in LX830 cells at 6.2 mGy/h was not significantly different from that for moderate or acute irradiation.

Mutation induction by very low dose rate gamma rays in cultured mouse leukemia cells L5178Y.

Induction of cell killing and mutation to 6-thioguanine resistance was studied in growing mouse leukemia cells in culture following gamma rays at dose rates of 30 Gy/h, 20 cGy/h, and 6.3 mGy/h, i.e., acute, low dose rate, and very low dose rate irradiation. A marked increase was observed in the cell survival with decreasing dose rate; no reduction in the surviving fraction was detected after irradiation at 6.3 mGy/h until a total dose of 4 Gy. Similarly, the induced mutation frequency decreased after low dose rate irradiation compared to acute irradiation. However, the frequency after irradiation at 6.3 mGy/h was unexpectedly high and remained at a level which was intermediate between acute and low dose rate irradiation. No appreciable changes were observed in the responses to acute gamma rays (in terms of cell killing and mutation induction) in the cells which had experienced very low dose rate irradiation.

Cell killing and mutation to 6-thioguanine resistance after exposure to tritiated amino acids and tritiated thymidine in cultured mammalian cells (L5178Y).

Cell killing and mutation to 6-thioguanine resistance were studied in growing mouse leukemia cells in culture after exposure to tritiated amino acids and tritiated thymidine. These effects varied widely among the tritiated compounds tested, being greatest for tritiated thymidine followed by tritiated arginine and tritiated lysine, in that order, for a given concentration of 3H expressed in kBq/ml of 3H in the medium. The differences between each tritiated amino acid disappeared almost totally when the effects were compared on the basis of the absorbed dose to the cells. The effects of tritiated thymidine, however, remained more than twofold greater compared to other tritiated compounds. These results indicate the importance of determining the absorbed dose for assessment of the radiotoxicity of tritiated organic compounds. For an exceptional case (tritiated thymidine), contribution of a mechanism(s) other than beta irradiation should also be taken into account.

Mutation induction by tritiated water and effects of deuterium oxide in cultured mouse leukemia cells.

Induction of mutation to 6-thioguanine resistance was studied in L5178Y mouse leukemia cells after exposure to low-dose-rate gamma rays or tritiated water at dose rates of approximately 0.025 to 0.4 Gy/hr for 20 hr in the presence or absence of 45% (v/v) deuterium oxide. The effect of acute gamma-ray exposure was also examined. A higher frequency of induced mutations was observed after tritium beta rays than after gamma rays, both at equivalent doses and cell survival. Deuterium oxide enhanced the mutation induced by gamma rays and tritium beta rays but did not affect the survival-mutation correlation of the two radiations.