Akiko M. Ueno

Iodine contrast medium sensitizes cultured mammalian cells to x rays but not to. gamma. rays

The influence of an iodine contrast medium on several responses to radiation was examined in mammalian cells in culture (L5178Y). The presence of the medium at the time of irradiation enhanced cell killing, frequency of micronuclei, and yield of DNA single-strand breaks induced by x rays, depending on the concentration used, whereas no such effect was found with ..gamma.. rays. It was concluded that the contrast medium sensitizes mammalian cells in culture primarily by means of the photoelectric effect, thereby increasing the absorbed dose of x rays in the cells.

A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1− mutants in human-hamster hybrid AL cells

We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

Induction of cell killing, micronuclei, and mutation to 6-thioguanine resistance after exposure to low-dose-rate gamma rays and tritiated water in cultured mammalian cells (L5178Y).

Cell killing and induction of micronuclei and mutation to 6-thioguanine resistance were studied in growing cultured mammalian cells (L5178Y) after exposure for a fixed period to low-dose-rate ..gamma.. rays and tritiated water (HTO) at comparable dose rates, from about 10 to 40 rad/hr. From the analysis of dose-response relationships, RBE values of HTO ..beta.. rays relative to ..gamma.. rays were calculated. These were 1.5 for cell killing, 2.0 for micronuclei, and 1.8 for mutation induction. Reported RBE values for HTO were reviewed.

Heavy ion mutagenesis: linear energy transfer effects and genetic linkage.

We have characterized a series of 69 independent mutants at the endogenoushprt locus of human TK6 lymphoblasts and over 200 independent S 1-deficient mutants of the humanxhamster hybrid cell line AL arising spontaneously or following low-fluence exposures to densely ionizing Fe ions (600 MeV/amu, linear energy transfer = 190 keV/µm). We find that large deletions are common. The entirehprt gene (>44 kb) was missing in 19/39 Fe-induced mutants, while only 2/30 spontaneous mutants lost the entirehprt coding sequence. When the gene of interest (S1 locus = M1C1 gene) is located on a nonessential human chromosome 11, multilocus deletions of several million base pairs are observed frequently. The S1 mutation frequency is more than 50-fold greater than the frequency ofhprt mutants in the same cells. Taken together, these results suggest that low-fluence exposures to Fe ions are often cytotoxic due to their ability to create multilocus deletions that may often include the loss of essential genes. In addition, the tumorigenic potential of these HZE heavy ions may be due to the high potential for loss of tumor suppressor genes. The relative insensitivity of thehprt locus to mutation is likely due to tight linkage to a gene that is required for viability.

Inhibition of gamma-ray dose-rate effects by D2O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells.

Effects of deuterium oxide (D2O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of gamma rays. Growth of irradiated and unirradiated cells was inhibited by 45% D2O but not by 3-aminobenzamide at 10 mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. The D2O did not inhibit the radiation-induced increase in poly(ADP-ribose) synthesis as measured by the incorporation of [14C]NAD into the acid insoluble fraction, contrary to 3-aminobenzamide. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of gamma rays. Possible mechanisms underlying the inhibition are discussed.

Deficient repair and degradation of DNA in X-irradiated L5178Y S/S cells: cell-cycle and temperature dependence

The rejoining of DNA strand breaks induced by X rays in the radiosensitive S/S variant of the L5178Y murine leukemic lymphoblast has been studied by alkaline-EDTA-sucrose sedimentation using swinging-bucket and zonal rotors. After irradiation, incubation resulted in an increase in DNA size, but the DNA structures were not restored in all cells, even when the x-ray dose was only 50 rad. Subsequently, 10 to 20 h after irradiation, heavily degraded DNA began to appear. When cells were irradiated at different parts of the cycle, the extent of DNA degradation varied in a fashion similar to survival: Least DNA degradation was found after irradiation at the most radioresistant stage (G/sub 1/ + 8 h), and most DNA degradation occurred after irradiation at the radiosensitive stage (G/sub 1/). Changes in cell survival caused by postirradiation hypothermia were also reflected in the extent of DNA degradation. Populations of G/sub 1/ cells, which show marked increases in survival after postirradiation hypothermic exposure, exhibited a lower level of DNA degradation, whereas populations of G/sub 1/ + 8 h cells, whose survival is affected little by postirradiation hypothermia, showed limited changes in DNA degradation. The onset of degradation was delayed by hypothermia in all cases.

Measuring the Spectrum of Mutation Induced by Nitrogen Ions and Protons in the Human–Hamster Hybrid Cell Line ALC

Abstract Kraemer, S. M., Kronenberg, A., Ueno, A. and Waldren, C. A. Measuring the Spectrum of Mutation Induced by Nitrogen Ions and Protons in the Human–Hamster Hybrid Cell Line ALC. Astronauts can be exposed to charged particles, including protons, α particles and heavier ions, during space flights. Therefore, studying the biological effectiveness of these sparsely and densely ionizing radiations is important to understanding the potential health effects for astronauts. We evaluated the mutagenic effectiveness of sparsely ionizing 55 MeV protons and densely ionizing 32 MeV/nucleon nitrogen ions using cells of two human–hamster cell lines, AL and ALC. We have previously characterized a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in the human–hamster hybrid cell lines ALC and AL. CD59– mutants have lost expression of a human cell surface antigen encoded by the CD59 gene located at 11p13. Deletion of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the AL hybrid, so that CD59 mutants that lose the entire chromosome 11 die and escape detection. In contrast, deletion of the 11p15.5 region is not lethal in the hybrid ALC, allowing for the detection of chromosome loss or other chromosomal mutations involving 11p15.5. The 55 MeV protons and 32 MeV/nucleon nitrogen ions were each about 10 times more mutagenic per unit dose at the CD59 locus in ALC cells than in AL cells. In the case of nitrogen ions, the mutations observed in ALC cells were predominantly due to chromosome loss events or 11p deletions, often containing a breakpoint in the pericentromeric region. The increase in the CD59– mutant fraction for ALC cells exposed to protons was associated with either translocation of portions of 11q onto a hamster chromosome, or discontinuous or “skipping” mutations. We demonstrate here that ALC cells are a powerful tool that will aid in the understanding of the mutagenic effects of different types of ionizing radiation.

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.

Incorporation of tritium from tritiated water into nucleic acids of Oryzias latipes eggs.

Eggs of Oryzias latipes were kept at 25°C in tritiated water (HTO) from a few hours after fertilization to the day before hatching (9 days). When eggs were kept at 25°C in 10 μCi/ml of HTO, a radio...

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.