Shigefumi Okada

Radiation-induced DNA damage and cellular lethality in cultured mammalian cells.

X ray-induced DNA scissions and their repair were studied by an alkaline separation method. DNA damage in Chinese hamster V79 cells was assigned to one of three groups based on a repair profile previously used for mouse L5178Y cells: fast-reparable (T1/2 = 5 min), slow-reparable (T1/2 = 70 min), and nonreparable . The three kinds damage were investigated in relation to cellular lethality under conditions where radiosensitivity of cultured cells was modified: (1) different sensitivity in different cell lines, (2) cell cycle fluctuation of radiosensitivity, and (3) recovery after split-dose irradiation. Among the three types of lesions, only nonreparable damage or remaining lesions showed correlation to cell killing. The parallel relationship between nonreparable damage and cell killing implies that this type of damage could play an important role in radiation-induced cell death.

Acute effects of mercuric compounds on cultured mammalian cells.

Abstract The observed acute effects of methyl mercuric chloride on cultured mammalian cells were: (1) retardation of cell multiplication. (2) cell killing, (3) depression of [ 3 H]thymidine and [ 3 H]uridine uptake, and (4) induction of single-strand scissions of DNA. Among the cellular responses studied, [ 3 H]thymidinc and [ 3 H]uridine incorporation were the most sensitive indicators of cellular mercuric poisoning. Toxicities of three mercuric compounds—methyl mercuric chloride, phenyl mercuric acetate and mercuric chloride —were compared by using two indicators, [ 3 H]thymidine incorporation and cell multiplication. Methyl mercuric chloride and phenyl mercuric acetate were equally toxic, while mercuric chloride was the least toxic. Addition of glutathione to cells pretreated with methyl mercuric chloride allowed the cells to recover from the toxic effects of methyl mercuric chloride at a faster rate than those left without glutathione.

Comparative studies of DNA size in various tissues of mice during the aging process

Abstract The sizes of single-strand pieces of DNA were estimated by alkaline sucrose gradient centrifugation in four tissues of C57BL/6 mice at 1–-2 months, 14 months and 22 months of age. When the DNA sizes in 14 and 22 months old mice were compared with those of the 1–2 months old group, the splenic, thymic and cerebellar DNA of old mice were not significantly different from those of the young mice. However, hepatic DNA of 14 months old mice was significantly smaller than that of 2 months old. That of 22 months old was similar to that of 14 months old mice. The increase of DNA scissions from 2 to 14 months old was 3·2×10 4 breaks per cell. If we assume a constant and continuous rate of DNA scission, the rate would be about 88 breaks per cell per day or about 4 breaks per cell per hour. X-iradiation of 500 R administered at 2 months old did not cause any significant difference in DNA size from a non-irradiated group when examined at 14 and 22 months old.

Induction of chromosome aberrations in human lymphocytes by monochromatic X-rays of quantum energy between 4.8 and 14.6 keV

The induction of chromosome aberrations was studied in human peripheral blood lymphocytes irradiated in vitro with synchrotron-produced monochromatic soft X-rays of quantum energy in a range between 4.8 and 14.6 keV. These X-rays were more effective in producing chromosome aberrations (dicentrics and rings) than 60Co gamma-rays. The efficiency increased with increasing LET of the photoelectrons and their associated Auger electrons, reaching a maximum at a track average LET (L delta = 100, T) of around 4 keV/microns, and tended to decrease or become rather refractory with further increase of LET. This unique LET dependency was consistent with the dual nature of chromosome aberration formation, and interpreted as a reflection of a limited range of photoelectrons as compared with the size and intranuclear geometry of the elemental chromatin fibres as vehicles of damage interaction.

Synchrotron system for monochromatic uv irradiation (>140 nm) of biological material

An irradiation system of monochromatic uv radiation down to the wavelength of 140 nm was constructed for biological irradiation experiments in the vacuum-uv range using synchrotron radiation (SR) from the electron storage ring. The system consists of premirror chamber, vacuum-uv monochromator, irradiation chamber, and vacuum systems. Along with the detailed description of all components of the system, the installation at the storage ring and the performance characteristics are presented.

Dose-rate effects of gamma-ray-induced mutations in cultured mammalian cells

The dose-rate dependency of three radiobiological parameters, cell killing and mutations resistant to 6-thioguanine (6-TGr) and to methotrexate (MTXr), were studied in populations of mouse L5178Y cells exposed to gamma-rays. When the dose rate was reduced from 50 rad/min to 0.8 rad/min, the shape of the dose--response curves changed from sigmoidal to exponential for cell killing, from upward concave to linear in 6-TGr mutations and remained linear in MTXr mutations. A linear quadratic model appears capable of explaining the cell killing and 6-TGr mutations but not the MTXr mutations. The declining patterns of induced mutation frequencies of 6-TGr and MTXr with decreasing dose rate seem to be similar. The addition of DMSO resulted in protection of cells from cell killing, 6-TGr and MTXr mutations with acute exposure, but had little effect with chronic exposure. The reduction of mutation frequency of the 6-TGr marker with chronic exposure was eliminated by holding cells in ice-cold condition during irradiation. These results suggest that there may be two components of induced mutation. One results primarily from repairable damage induced by the indirect action of radiation and shows a clear dose-rate dependency. The other is mainly from no-repairable damage by the direct action of radiation and is only slightly dose rate-dependent. Under chronic exposure conditions, the latter may predominate.

Unique increase of superoxide dismutase level in brains of long living mammals.

A possible involvement of superoxide dismutase (SOD) level in determination of mammals lifespan was examined by comparing six different enzyme levels including SOD in brains of eleven mammalian species of different lifespans. When the specific activities of the enzymes were plotted against lifespans on a linear scale, no or weak negative correlations were observed for all enzymes studied except SOD, which revealed positive correlation. The uniqueness of the SOD level suggests that it is one of the enzymes which evolved together with lifespans. It would contribute to long lifespan through protection against oxygen toxicity.

Induction and subsequent repair of DNA damage by fast neutrons in cultured mammalian cells

The induction and repair of DNA damage were studied by a DNA unwinding method in mouse L5178Y cells exposed to fast neutrons. DNA lesions induced by fast neutrons were classified into three types from their repair profiles: fast-reparable breaks (T1/2 = 3-5 min), slow-reparable breaks (T1/2 = 70 min), and nonreparable breaks. The repair rates of both fast-reparable and slow-reparable breaks were almost the same as those of corresponding damage induced by low-LET radiation. Neutrons induced a smaller amount of fast-reparable damage, an almost equal amount of slow-reparable damage, and a larger amount of nonreparable damage than those induced by equal doses of gamma rays or X rays. RBEs for fast- and slow-reparable damage were 0.3 and 0.9, respectively. The RBE for nonreparable damage was dose dependent and was 1.4 at the level of 100 breaks/10(12) Da DNA. Among the three types of lesions, only the nonreparable damage levels correlated with the linear-quadratic shape of the survival curves and with the enhanced killing effectiveness of neutrons (RBE = 1.7 at D0).

Radiation-induced DNA single-strand scission and its rejoining in spermatogonia and spermatozoa of mouse

Gamma-ray-induced DNA single-strand scissions and the ability to repair the scissions in spermatogonia from young mice and in spermatozoa from adult mice were studied quantitatively by an alkaline sucrose density-gradient centrifugation method. The average size of DNAs in non-irradiated spermatogonia was 2.6--3.0 X 10(8) daltons, similar to those of a spermatid-rich population, and the size of DNA in non-irradiated spermatozoa was 1.2 X 10(8) daltons. In spermatogonia, the radiosensitivity of DNA was 0.42 single-strand breaks/10(12) daltons of DNA/rad in oxic conditions and only 0.24 under anoxic conditions. In spermatozoa the break efficiency of DNA was 0.22 single-strand breaks/10(12) daltons of DNA/rad under oxic conditions and altered little under anoxic irradiation. The DNA scissions were efficiently repaired in spermatogonia within 10 min, whereas the breaks in spermatozoa were not rejoined at all even after two days of post-irradiation time. The radiosensitivities of DNA, repair capability and non- and/or slow-reparable DNA scissions were compared in spermatogonium-rich, spermatid-rich and spermatozoan-rich populations.

Isolation of nutrient deficient mutants and quantitative mutation assay by reversion of alanine-requiring L5178Y cells

Nutrient deficient cells for alanine or purine were isolated from L5178Y cells by the BudR-light method of Puck and Kao. The purine requiring cells, named P, showed maximum-plating efficiency in the presence of 10(-3)-10(-4) M inosine. The optimum concentration of L-alpha-alanine for Ala 32, one of alanine requiring mutants, was 10(-3) M. When Ala 32 cells were depleted of alanine, they showed an immediate decrease in incorporation of protein and DNA precursors without much change in that of RNA precursors and they ceased to multiply. Ala 32 cells have been used for experiments and have been phenotypically stable for 4 years. A quantitative mutation assay system for the reversion of L5178Y-Ala 32 cells from auxotrophy to prototrophy was established. The system was applied to some known mutagens, MNNG, 4-NQO, UV and gamma-rays. Some characteristics of the system are discussed and compared to drug-resistant mutation systems.