Trp53 activity is repressed in radio-adapted cultured murine limb bud cells.

Understanding the effects of of ionizing radiation (IR) at low dose in fetal models is of great importance, because the fetus is considered to be at the most radiosensitive stage of the development and prenatal radiation might influence subsequent development. We previously demonstrated the existence of an adaptive response (AR) in murine fetuses after pre-exposure to low doses of X-rays. Trp53-dependent apoptosis was suggested to be responsible for the teratogenic effects of IR; decreased apoptosis was observed in adapted animals. In this study, in order to investigate the role of Trp53 in AR, we developed a new model of irradiated micromass culture of fetal limb bud cells, which replicated proliferation, differentiation and response to IR in murine embryos. Murine fetuses were exposed to whole-body priming irradiation of 0.3 Gy or 0.5 Gy at embryonic day 11 (E11). Limb bud cells (collected from digital ray areas exhibiting radiation-induced apoptosis) were cultured and exposed to a challenging dose of 4 Gy at E12 equivalent. The levels of Trp53 protein and its phosphorylated form at Ser18 were investigated. Our results suggested that the induction of AR in mouse embryos was correlated with a repression of Trp53 activity.

Human exposure to high natural background radiation: what can it teach us about radiation risks?

Natural radiation is the major source of human exposure to ionising radiation, and its largest contributing component to effective dose arises from inhalation of (222)Rn and its radioactive progeny. However, despite extensive knowledge of radiation risks gained through epidemiologic investigations and mechanistic considerations, the health effects of chronic low-level radiation exposure are still poorly understood. The present paper reviews the possible contribution of studies of populations living in high natural background radiation (HNBR) areas (Guarapari, Brazil; Kerala, India; Ramsar, Iran; Yangjiang, China), including radon-prone areas, to low dose risk estimation. Much of the direct information about risk related to HNBR comes from case-control studies of radon and lung cancer, which provide convincing evidence of an association between long-term protracted radiation exposures in the general population and disease incidence. The success of these studies is mainly due to the careful organ dose reconstruction (with relatively high doses to the lung), and to the fact that large-scale collaborative studies have been conducted to maximise the statistical power and to ensure the systematic collection of information on potential confounding factors. In contrast, studies in other (non-radon) HNBR areas have provided little information, relying mainly on ecological designs and very rough effective dose categorisations. Recent steps taken in China and India to establish cohorts for follow-up and to conduct nested case-control studies may provide useful information about risks in the future, provided that careful organ dose reconstruction is possible and information is collected on potential confounding factors.

Dose limits below which the effect of radiation on health becomes undetectable due to background variation.

To clarify the low-dose limit at which the effect of radiation on health becomes undetectable is important in the regulation of radiation. As one of a series of cytogenetical studies on the effect of radiation on health, we present low-dose limits determined by analyzing the background frequencies of translocations in the lymphocytes of people living in normal circumstances. The frequencies of translocations in the lymphocytes were analyzed in 20 non-smokers (61.2-year-old on the average) in a large city, and 16 non-smokers (64.4-year-old on the average) and 8 children (12.3-year-old on the average) in a remote village. The radiation dose was calculated based on the background frequencies of translocations assuming that all the translocations had been induced by radiation. The calculated doses were 384+/-200, 336+/-124 and 128+/-80 mSv in the case of chronic exposure, and 248+/-153, 225+/-104 and 107+/-72 mSv in acute exposure. Standard deviation of the calculated doses is considered to be the dose level below which the effect of radiation becomes undetectable due to the background variation in the effects of all kind of mutagenic factors, i.e., the dose level below which an epidemiological study will not be able to show any significant increase in malignant diseases. The results obtained from epidemiological studies are in fairly good agreement with our results.

International study of factors affecting human chromosome translocations.

Chromosome translocations in peripheral blood lymphocytes of normal, healthy humans increase with age, but the effects of gender, race, and cigarette smoking on background translocation yields have not been examined systematically. Further, the shape of the relationship between age and translocation frequency (TF) has not been definitively determined. We collected existing data from 16 laboratories in North America, Europe, and Asia on TFs measured in peripheral blood lymphocytes by fluorescence in situ hybridization whole chromosome painting among 1933 individuals. In Poisson regression models, age, ranging from newborns (cord blood) to 85 years, was strongly associated with TF and this relationship showed significant upward curvature at older ages versus a linear relationship (p<0.001). Ever smokers had significantly higher TFs than non-smokers (rate ratio (RR)=1.19, 95% confidence interval (CI), 1.09-1.30) and smoking modified the effect of age on TFs with a steeper age-related increase among ever smokers compared to non-smokers (p<0.001). TFs did not differ by gender. Interpreting an independent effect of race was difficult owing to laboratory variation. Our study is three times larger than any pooled effort to date, confirming a suspected curvilinear relationship of TF with age. The significant effect of cigarette smoking has not been observed with previous pooled studies of TF in humans. Our data provide stable estimates of background TF by age, gender, race, and smoking status and suggest an acceleration of chromosome damage above age 60 and among those with a history of smoking cigarettes.

Combined genotoxic effects of radiation and a tobacco-specific nitrosamine in the lung of gpt delta transgenic mice.

It is important to evaluate the health effects of low-dose-rate or low-dose radiation in combination with chemicals as humans are exposed to a variety of chemical agents. Here, we examined combined genotoxic effects of low-dose-rate radiation and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. In this mouse model, base substitutions and deletions can be separately analyzed by gpt and Spi- selections, respectively. Female gpt delta mice were either treated with gamma-irradiation alone at a dose rate of 0.5, 1.0 or 1.5 mGy/h for 22 h/day for 31 days or combined with NNK treatments at a dose of 2 mg/mouse/day, i.p. for four consecutive days in the middle course of irradiation. In the gpt selection, the NNK treatments enhanced the mutation frequencies (MFs) significantly, but no obvious combined effects of gamma-irradiation were observable at any given radiation dose. In contrast, NNK treatments appeared to suppress the Spi- large deletions. In the Spi- selection, the MFs of deletions more than 1 kb in size increased in a dose-dependent manner. When NNK treatments were combined, the dose-response curve became bell-shaped where the MF at the highest radiation dose decreased substantially. These results suggest that NNK treatments may elicit an adaptive response that eliminates cells bearing radiation-induced double-strand breaks in DNA. Possible mechanisms underlying the combined genotoxicity of radiation and NNK are discussed, and the importance of evaluation of combined genotoxicity of more than one agent is emphasized.

Regulation of radiation-induced protein kinase Cdelta activation in radiation-induced apoptosis differs between radiosensitive and radioresistant mouse thymic lymphoma cell lines.

Protein kinase Cdelta (PKCdelta) has an important role in radiation-induced apoptosis. The expression and function of PKCdelta in radiation-induced apoptosis were assessed in a radiation-sensitive mouse thymic lymphoma cell line, 3SBH5, and its radioresistant variant, XR223. Rottlerin, a PKCdelta-specific inhibitor, completely abolished radiation-induced apoptosis in 3SBH5. Radiation-induced PKCdelta activation correlated with the degradation of PKCdelta, indicating that PKCdelta activation through degradation is involved in radiation-induced apoptosis in radiosensitive 3SBH5. In radioresistant XR223, radiation-induced PKCdelta activation was lower than that in radiosensitive 3SBH5. Cytosol PKCdelta levels in 3SBH5 decreased markedly after irradiation, while those in XR223 did not. There was no apparent change after irradiation in the membrane fractions of either cell type. In addition, basal cytosol PKCdelta levels in XR223 were higher than those in 3SBH5. These results suggest that the radioresistance in XR223 to radiation-induced apoptosis is due to a difference in the regulation of radiation-induced PKCdelta activation compared to that of 3SBH5. On the other hand, Atm(-/-) mouse thymic lymphoma cells were more radioresistant to radiation-induced apoptosis than wild-type mouse thymic lymphoma cells. Irradiated wild-type cells, but not Atm(-/-) cells, had decreased PKCdelta levels, indicating that the Atm protein is involved in radiation-induced apoptosis through the induction of PKCdelta degradation. The decreased Atm protein levels induced by treatment with Atm small interfering RNA had no effect on radiation-induced apoptosis in 3SBH5 cells. These results suggest that the regulation of radiation-induced PKCdelta activation, which is distinct from the Atm-mediated cascade, determines radiation sensitivity in radiosensitive 3SBH5 cells.

Effect of smoking on chromosomes compared with that of radiation in the residents of a high-background radiation area in China.

Cytogenetic investigation of stable-type aberrations (translocations) was carried out with our improved methods on 28 elderly individuals in a high-background radiation area (HBRA) in China, and on 24 elderly individuals in a control area (CA). The level of radiation in HBRA is 3 to 5 times higher than in CA. The mean frequencies of translocations per 1,000 cells in HBRA and CA were 12.4 +/- 5.3 and 10.0 +/- 3.8, respectively. No significant difference was found in the frequencies between HBRA and CA (P>0.05, Mann-Whitney U test). When elderly individuals in HBRA and CA were classified into four subgroups of HBRA nonsmokers, HBRA smokers, CA nonsmokers, and CA smokers, a significant difference was found in the frequencies between CA smokers and CA nonsmokers (P<0.05, Mann-Whitney U test). Furthermore a tendency of difference (a near T-value of 0.05 level) was found in a comparison of HBRA smokers vs. CA nonsmokers. The present results indicate that the elevated level of natural radiation in HBRA plays a less significant part than smoking in bringing about the induction rate of stable-type aberrations (translocations) in those areas.

Characterization of ionizing radiation-induced ring chromosomes by atomic force microscopy.

We applied atomic force microscopy (AFM) to the structural analysis of radiation-induced ring chromosomes. Constrictions observed on the metaphase ring chromosome were found to correspond to the centromere regions of the ring chromosome in comparison with the AFM image of the centromere of rod chromosomes and with the fluorescence in situ hybridization (FISH) technique. Section analysis by AFM revealed that some ring-like chromosome fragments and ring-like chromatid fragments were thicker than standard chromosomes or chromatids, suggesting that they were ring chromosomes viewed edge on. Topographic analysis by AFM makes it possible to distinguish a ring viewed edge on that is difficult to recognize as a ring by light microscopy and to discriminate between a centric ring chromosome and an acentric ring chromosome using the same slides prepared for light microscopy.

Involvement of protein kinase C-related anti-apoptosis signaling in radiation-induced apoptosis in murine thymic lymphoma(3SBH5) cells.

Protein kinase C (PKC; also known as PRKC) is known to be an important participant in radiation-induced apoptosis. However, its role is not fully clarified. Using 3SBH5 cells, which are radiation-sensitive thymic lymphoma cells, the involvement and functions of PKC were assessed in radiation- induced apoptosis. PMA (phorbol 12-myristate 13-acetate), a PKC activator, inhibited the radiation-induced apoptosis in 3SBH5 cells. On the other hand, chelerythrine, a PKC inhibitor, potentiated apoptosis. In addition, Gö6976, a classical PKC (cPKC) inhibitor, which specifically inhibits PKC (alpha and betaI), also promoted apoptosis. Interestingly, post-treatment (20 min after irradiation) with Gö6976 had no effect on the radiation-induced apoptosis. These results suggest that cPKC is activated early after irradiation for anti-apoptosis signaling and contributes to the balance between cell survival and death. Indeed, an increase of cPKC activity involving PKC (alpha, betaI and betaII) was observed in the cytosolic fraction 3 min after irradiation with 0.5 Gy. However, no translocation of cPKC was observed in the cells after irradiation. Our findings indicate that activation of cPKC (alpha or beta) soon after irradiation is critical to the understanding of the regulation of radiation-induced apoptosis in radiation-sensitive cells.

Adaptive response in embryogenesis: IV. Protective and detrimental bystander effects induced by X radiation in cultured limb bud cells of fetal mice.

The radioadaptive response and the bystander effect represent important phenomena in radiobiology that have an impact on novel biological response mechanisms and risk estimates. Micromass cultures of limb bud cells provide an in vitro cellular maturation system in which the progression of cell proliferation and differentiation parallels that in vivo. This paper presents for the first time evidence for the correlation and interaction in a micromass culture system between the radioadaptive response and the bystander effect. A radioadaptive response was induced in limb bud cells of embryonic day 11 ICR mice. Conditioning irradiation of the embryonic day 11 cells with 0.3 Gy resulted in a significant protective effect against the occurrence of apoptosis, inhibition of cell proliferation, and differentiation induced by a challenging dose of 5 Gy given the next day. Both protective and detrimental bystander effects were observed; namely, irradiating 50% of the embryonic day 11 cells with 0.3 Gy led to a successful induction of the protective effect, and irradiating 70% of the embryonic day 12 cells with 5 Gy produced a detrimental effect comparable to that seen when all the cells were irradiated. Further, the bystander effect was markedly decreased by pretreatment of the cells with an inhibitor to block the gap junction-mediated intercellular communication. These results indicate that the bystander effect plays an important role in both the induction of a protective effect by the conditioning dose and the detrimental effect of the challenge irradiation. Gap junction-mediated intercellular communication was suggested to be involved in the induction of the bystander effect.

An improved culture system of mouse peripheral blood lymphocytes for analysis of radiation-induced chromosome aberrations.

There is an incentive to develop a culture system of mouse peripheral blood lymphocytes (PBLs) to serve as models for studying genotoxic effects in humans exposed to mutagens, including ionizing radiation. However, many past approaches have been laborious, complex and only partly reproducible. In the present study, we established an improved culture system of mouse PBLs by removing blood and/or plasma, which was found to inhibit in vitro mitotic stimulation or proceeding cell cycles of lymphocytes. We compared the reactions of isolated PBLs to mitogens between the classical method and the present improved one. Then, we applied this method to the cytogenetic analysis using chemically induced premature chromosome condensation (PCC) as well as the conventional analysis, and demonstrated that the frequency of excess fragments observed in PCC cells might be useful to quantify the radiation-induced damages on chromosomes.

An accidental exposure by a medical linear accelerator under construction.

A radiation accident occurred at a medical linear accelerator facility under construction in Japan. The radiation source was a 3- and 6-MV potential drop accelerator designed to produce X-rays for radiation therapy. This accelerator was also capable of producing a 5 to14-MV swept electron beam. During setting up, an operator turned on the accelerator to test the beam not knowing that a man was working on the ceiling above the accelerator. Thus, an X-ray beam was emitted against the ceiling and the man was exposed to 10-MV of X-ray irradiation. However, no obvious physical symptoms were noted. Dose estimation was made from reconstruction of the accident and clinical examinations including chromosome analysis. Mean dose of the whole body ranged from 70 to 180 mSv. Estimated dose from his right foot to hand was between180 to 900 mSv.

Preferential reduction of dicentrics in reciprocal exchanges due to the combination of the size of broken chromosome segments by radiation.

Induction rates of the dicentrics and translocations involving chromosomes 2 and 4 in peripheral lymphocytes irradiated with X-rays at a dose of 3 Gy were examined using a conventional Giemsa staining method and a chromosome painting method. In total, 228 reciprocal exchanges detected in 982 metaphases were classified into three groups according to the break points of the original chromosomes. The incidence of both acentric fragments being larger than half of the original chromosome (combination 1) was only seven (3%) and did not contribute significantly to induction rates. When the broken acentric fragments of two affected chromosomes were smaller than half of the original chromosomes (combination 2), which was found in 175 (77%) rearrangements, the induction rates of dicentrics and translocations were about the same (86:89). But if the sizes of the broken segments were unequal in both chromosomes (combination 3: one with a larger acentric part and the other with a smaller acentric part), the yield of dicentrics was significantly lower than that of translocations (16:30). It was suggested that there was a special mechanism causing preferential reduction of dicentrics in reciprocal exchanges originated from the heteromorphic size of broken chromosomes in the last combination.

Imperceptible effect of radiation based on stable type chromosome aberrations accumulated in the lymphocytes of residents in the high background radiation area in China.

Cytogenetic investigation of stable type aberrations (translocations) was performed with our improved methods in 6 children and 15 elderly persons in a high background radiation area (HBRA) in China, and in 8 children and 11 elderly persons in a control area. The total numbers of cells analyzed in elderly persons were 68,297 in HBRA and 35,378 in controls and in children were 45,535 in HBRA and 56,198 in controls. On average 5138 cells per subject were analyzed. The variation in the frequencies of translocations per 1000 cells was small in children while it was large in elderly persons. No significant difference was found in the frequencies between HBRA and control (P > 0.05, Mann-Whitney U test). On the other hand, correlation between age and translocation frequencies was significant at the 1% level (rs = 0.658 with 37DF, Spearman rank correlation test). The contribution of an elevated level of natural radiation in HBRA in China to the induction of stable type chromosome aberrations does not have a significant effect compared with the contribution of chemical mutagens and/or metabolic factors. The present study suggests that the probability of the risk of causing malignant and/or congenital diseases by the increased amount of radiation is imperceptible in HBRA where the level of natural radiation is 3 to 5 times higher than that in the control area.