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.

A shuttle vector system for studying ionizing radiation-induced mutagenesis in mammalian cells.

A shuttle vector system was developed to quantitate and analyze ionizing radiation-induced mutation in mammalian host cells, COS-1 and CV-1. The shuttle vector pSV2-lacY, which was constructed to detect both point mutations and deletions, was irradiated in vitro with 60Co gamma rays before introduction into unirradiated host cells. The plasmid was then isolated and reintroduced into HB101 (lacY-) bacterial host cells for identification of mutated lacY marker genes. Gamma-irradiation produced a decrease of the survival (recovery) and an increase of mutation of the shuttle vector. The mutated shuttle vector molecules were examined for structural changes by means of restriction endonuclease digestion and agarose gel electrophoresis. A dose dependent increase was observed in the percentages of gross alteration events of total mutations in mammalian host. This system will be useful for studies of ionizing radiation-induced mutagenesis.

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.

Induction of malignant transformation in mouse 10T1/2 cells by low-dose-rate exposure to tritiated water and gamma-rays at two different temperatures, 4 degrees C and 37 degrees C.

Cultured mouse (C3H 10T1/2) cells in contact-inhibited state were subjected to protracted exposure at 4 degrees C or 37 degrees C to either beta-rays from HTO or 60Co gamma-rays. The duration of exposure was 20 h and the total dose was varied by changing the dose-rate. The dose-survival and dose-transformation curves for gamma-irradiation at 4 degrees C were close to those obtained after a single acute X-ray dose (0.5 Gy/min). When gamma-irradiation was administered at 37 degrees C, both the lethality and transformation induction were lower than those after the corresponding doses at 4 degrees C, presumably owing to repair of lethal and transformational damage during gamma-irradiation at 37 degrees C. The same effect of temperature was observed in the case of HTO exposure, indicating again the existence of repair of damage during beta-irradiation at 37 degrees C but not at 4 degrees C. These facts strongly suggest that when irradiated at a low temperature such as 4 degrees C, both the dose-lethality and dose-transformation induction relationships were independent of the dose-rate for either gamma- or beta-exposure, at least the dose-rates used in this experiment. Thus, the comparison of radiation effectiveness between beta- and gamma-exposures at 4 degrees C gave reliable values of relative biological effectiveness (RBE) of tritium beta-rays which were ca. 1.4 at the D0 for lethality and 1.6 for cell transformation within the dose range examined (1 to 6 Gy of beta-rays). The comparison between exposures at 37 degrees C resulted in RBE values (1.4 and 1.7, respectively) very close to those at 4 degrees C.

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.

Method of formation of artificial organ components using a plastic dispenser system manipulated by a robot.

In order to produce an artificial organ component having a complicated structure, a plastic-forming method was proposed in which small amounts of material were accumulated successively by using a dispenser system manipulated by a robot; a preliminary experiment with silicone rubber was conducted. Components of different shapes were produced, including straight tubes, tapered tubes, bifurcating tubes, tubes having leaflets on the inside, cones, spiral tubes, and tubes with a sack. This method provides the possibility of producing complicated artificial organ components, which are difficult to produce by conventional plastic-processing methods.

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.

Higher susceptibility to N-methyl-N'-nitro-N-nitrosoguanidine-induced tumorigenesis in an interstrain hybrid of the fish, Oryzias latipes (medaka).

Adult fish of an interstrain hybrid (F1) of inbred medaka, obtained from crosses between HO4C and HB32C, were exposed for 2 hr to an aqueous solution of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine at concentrations of 15, 20, 25, 30, and 35 ppm. Survival and neoplastic changes were examined over a 6-month period. A large variety of neoplasms were induced, including melanoma, papilloma, ovarian tumors, olfactory epithelioma, branchioblastoma and fibroma. More than 60% of the tumors were classified as melanoma on the basis of histological examinations. A markedly higher cumulative incidence of the melanoma with a dose-related response was demonstrated in the F1 hybrid fish compared to the parental strains. The latent period for melanoma development, however, remained unchanged in F1 compared to the parents. The variety of tumors induced in the F1 fish was greater than in the parental strains. The results indicate the usefulness of F1 hybrid fish in testing the carcinogenicity of certain water-soluble chemicals, due to their high sensitivity.

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.

Induction of transplantable melanoma by treatment with N-methyl-N'-nitro-N-nitrosoguanidine in an inbred strain of the teleost Oryzias latipes.

Adult fish of two different inbred strains (HO4C and HB32C) of Oryzias latipes that were established in this laboratory were exposed for 2 hours to an aqueous solution of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine [(MNNG) CAS: 70-25-7; 1-methyl-3-nitro-1-nitrosoguanidine] at concentrations from 20 to 100 ppm. The acute toxicity and carcinogenicity of MNNG differed between two inbred strains. Almost no tumors were observed in strain HO4C fish, although these fish were sensitive to the acute toxicity of MNNG, with the median lethal dose being 28 ppm at 48 hours after the treatment. In contrast, in the HB32C strain, a dose-related tumorigenic response was observed, with the median lethal dose being 38 ppm at 48 hours after treatment. Most MNNG-induced tumors were considered amelanotic melanomas on the basis of histologic findings and a positive dopa reaction in selected samples. By the technique of transplantation of tumor tissue into the eye chamber and intraperitoneal cavity of fish of the syngeneic strain, serial transplantation of the tumor was successful. Tumor transplantation into one of the allogeneic strains (HO5) was also successful. The tumors have now been serially grown in the eye chambers in syngeneic fish for more than 14 generations.

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.

Factors influencing methylazoxymethanol acetate initiation of liver tumors in Oryzias latipes: carcinogen dosage and time of exposure.

Hepatic tumors were produced in the medaka (Oryzias latipes) kept at 25 degrees C in aquaria after exposure to methyl azoxymethanol (MAM) acetate. The tumors, including trabecular carcinomas and cholangiomas, were observable after 2-3 months. A similar incidence, i.e., nearly 100%, was found after a long-time exposure to a low level (0.1 ppm for 120 days) and after a short exposure to a high level (10 ppm for 1 hr) of MAM acetate. The relationship between the level of MAM acetate (0-2.0 ppm) and tumor incidence was studied after exposure for 1 day. The incidence increased as the level of drug increased, although an apparent threshold was seen at low levels. The incorporation of [3H]thymidine into the liver greatly increased over the period of 6-20 days after treatment with 0.5 ppm for 3 days. However, only a slight increase in incorporation was found when fish were treated continuously with a low level (0.1 ppm) from days 30 through 90. No reduction of tumor incidence was found when the carcinogen was given in 2 divided doses separated by various intervals, compared with a single exposure.

Incidence of chromatoblastomas in aging goldfish (Carassius auratus).

The incidence of chromatoblastomas in aging goldfish, Carassius auratus, was observed for 2 successive years. Tumors first appeared in fish at 5 years of age, and the tumor frequency increased with increasing age of the fish. The cumulative incidence rate of chromatoblastomas and the mean number of tumors per fish were estimated for each age. The mean number of tumors per fish fitted a straight line with a slope of approximately 3 on a double logarithmic graph against age. Thus tumor incidence seemed proportional to about the third power of age, and tumor appearance seemed dependent on some mutational events.

Induction and repair of DNA strand breaks in cultured mammalian cells following fast neutron irradiation.

Induction and repair of DNA breaks following irradiation with NIRS cyclotron neutrons were studied in cultured mammalian cells (L5178Y) in comparison to those following gamma-rays. The yield of the total single-strand breaks, 3'OH terminals and sites susceptible to S1 endonuclease following fast neutrons was found to be approximately 50 per cent of that following gamma-irradiation. On the other hand, the yield of double-strand breaks was slightly higher after fast neutrons than after gamma-rays. The percentage of the total single-strand breaks remaining unrejoined at 3 hours after post-irradiation incubation was found to be distinctly higher after the fast neutrons than after gamma-rays. The neutron-induced damage appears to carry a higher proportion of alkali-labile lesions compared to gamma-rays. It was concluded that the increase in the yield of double-strand breaks and of unrejoinable breaks is responsible for a high r.b.e. of the cyclotron neutrons.