Chromosome Damage Caused by Accidental Chronic Whole-Body Gamma Radiation Exposure in Thailand.

In February 2000, a radiation incident involving a medical (60)Co source occurred in a metal scrapyard in Thailand. Several individuals were suspected to have received chronic or fractionated exposures ranging from a few mGy to a several Gy. Using fluorescence in situ hybridization to paint chromosomes, we determined the frequencies of chromosome aberrations in peripheral blood lymphocytes of 13 people who entered the scrapyard, 3 people who involved in recovering the source, and 9 nearby residents. Aberration frequencies greater than controls were observed in 13 of the donors at 3 months postexposure. The predominant form of aberration observed was simple, complete, symmetrical translocations. An approximate 50% decrease in these aberrations and in total color junctions was observed in 7 donors resampled at 16 months postexposure. Although high, acute exposures are known to have detrimental effects, the biological consequences of chronic, low dose-rate radiation exposures are unclear. Thirteen of the donors had elevated aberration frequencies, and 6 also had symptoms of acute radiation syndrome. If there are any long-term health consequences of this incident, it will most likely occur among this group of individuals. The consequences for the remaining donors, who presumably received lower total doses delivered at lower dose rates, are less clear.

A critical role of toll-like receptor 4 (TLR4) and its' in vivo ligands in basal radio-resistance.

Toll-like receptor-4 (TLR4) plays a critical role in innate and acquired immunity, but its role in radio-resistance is unknown. We used TLR4 knockout (KO,(-/-)) mice and gut commensal depletion methods, to test the influence of TLR4 and its' in vivo agonist on basal radio-resistance. We found that mice deficient in TLR4 were more susceptible to IR-induced mortality and morbidity. Mortality of TLR4-deficient mice after IR was associated with a severe and persistent bone marrow cell loss. Injection of lipopolysaccharide into normal mice, which is known to activate TLR4 in vivo, induced radio-resistance. Moreover, TLR4 in vivo ligands are required for basal radio-resistance. We found that exposure to radiation leads to significant endotoxemia that also confers endogenous protection from irradiation. The circulating endotoxins appear to originate from the gut, as sterilization of the gut with antibiotics lead to increased mortality from radiation. Further data indicated that Myd88, but not TRIF, may be the critical adaptor in TLR4-induced radio-resistance. Taken together, these data strongly suggest that TLR4 plays a critical role in basal radio-resistance. Our data suggest, it is important not to give antibiotics that may sterilize the gut before the whole body irradiation. Further, these data also suggest that management of gut flora through antibiotic or possibly probiotic therapy may alter the innate response to the total body irradiation.

Low-dose radiation exposure and protection against atherosclerosis in ApoE(-/-) mice: the influence of P53 heterozygosity.

We recently described the effects of low-dose γ-radiation exposures on atherosclerosis in genetically susceptible (ApoE(-/-)) mice with normal p53 function. Doses as low as 25 mGy, given at either early or late stage disease, generally protected against atherosclerosis in a manner distinctly nonlinear with dose. We now report the influence of low doses (25-500 mGy) on atherosclerosis in ApoE(-/-) mice with reduced p53 function (Trp53(+/-)). Single exposures were given at either low or high dose rate (1 or 150 mGy/min) to female C57BL/6J ApoE(-/-) Trp53(+/-) mice. Mice were exposed at either early stage disease (2 months of age) and examined 3 or 6 months later, or at late stage disease (7 months of age) and examined 2 or 4 months later. In unirradiated mice, reduced p53 functionality elevated serum cholesterol and accelerated both aortic root lesion growth and severity in young mice. Radiation exposure to doses as low as 25 mGy at early stage disease, at either the high or the low dose rate, inhibited lesion growth, decreased lesion frequency and slowed the progression of lesion severity in the aortic root. In contrast, exposure at late stage disease produced generally detrimental effects. Both low-and high-dose-rate exposures accelerated lesion growth and high dose rate exposures also increased serum cholesterol levels. These results show that at early stage disease, reduced p53 function does not influence the protective effects against atherosclerosis of low doses given at low dose rate. In contrast, when exposed to the same doses at late stage disease, reduced p53 function produced detrimental effects, rather than the protective effects seen in Trp53 normal mice. As in the Trp53 normal mice, all effects were highly nonlinear with dose. These results indicate that variations in p53 functionality can dramatically alter the outcome of a low-dose exposure, and that the assumption of a linear response with dose for human populations is probably unwarranted.

miR-200c enhances radiosensitivity of human breast cancer cells.

Due to the intrinsic resistance of many tumors to radiotherapy, current methods to improve the survival of cancer patients largely depend on increasing tumor radiosensitivity. It is well-known that miR-200c inhibits epithelial-mesenchymal transition (EMT), and enhances cancer cell chemosensitivity. We sought to clarify the effects of miR-200c on the radiosensitization of human breast cancer cells. In this study, we found that low levels of miR-200c expression correlated with radiotolerance in breast cancer cells. miR-200c overexpression could increase radiosensitivity in breast cancer cells by inhibiting cell proliferation, and by increasing apoptosis and DNA double-strand breaks. Additionally, we found that miR-200c directly targeted TANK-binding kinase 1 (TBK1). However, overexpression of TBK1 partially rescued miR-200c mediated apoptosis induced by ionizing radiation. In summary, miR-200c can be a potential target for enhancing the effect of radiation treatment on breast cancer cells.

MiR-21 plays an important role in radiation induced carcinogenesis in BALB/c mice by directly targeting the tumor suppressor gene Big-h3.

Dysregulation of certain microRNAs (miRNAs) in cancer can promote tumorigenesis, metastasis and invasion. However, the functions and targets of only a few mammalian miRNAs are known. In particular, the miRNAs that participates in radiation induced carcinogenesis and the miRNAs that target the tumor suppressor gene Big-h3 remain undefined. Here in this study, using a radiation induced thymic lymphoma model in BALB/c mice, we found that the tumor suppressor gene Big-h3 is down-regulated and miR-21 is up-regulated in radiation induced thymic lymphoma tissue samples. We also found inverse correlations between Big-h3 protein and miR-21 expression level among different tissue samples. Furthermore, our data indicated that miR-21 could directly target Big-h3 in a 3'UTR dependent manner. Finally, we found that miR-21 could be induced by TGFß, and miR-21 has both positive and negative effects in regulating TGFß signaling. We conclude that miR-21 participates in radiation induced carcinogenesis and it regulates TGFß signaling.

Low-dose radiation exposure and atherosclerosis in ApoE⁻/⁻ mice.

The hypothesis that single low-dose exposures (0.025-0.5 Gy) to low-LET radiation given at either high (about 150 mGy/min) or low (1 mGy/min) dose rate would promote aortic atherosclerosis was tested in female C57BL/6J mice genetically predisposed to this disease (ApoE⁻/⁻). Mice were exposed either at an early stage of disease (2 months of age) and examined 3 or 6 months later or at a late stage of disease (8 months of age) and examined 2 or 4 months later. Changes in aortic lesion frequency, size and severity as well as total serum cholesterol levels and the uptake of lesion lipids by lesion-associated macrophages were assessed. Statistically significant changes in each of these measures were observed, depending on dose, dose rate and disease stage. In all cases, the results were distinctly non-linear with dose, with maximum effects tending to occur at 25 or 50 mGy. In general, low doses given at low dose rate during either early- or late-stage disease were protective, slowing the progression of the disease by one or more of these measures. Most effects appeared and persisted for months after the single exposures, but some were ultimately transitory. In contrast to exposure at low dose rate, high-dose-rate exposure during early-stage disease produced both protective and detrimental effects, suggesting that low doses may influence this disease by more than one mechanism and that dose rate is an important parameter. These results contrast with the known, generally detrimental effects of high doses on the progression of this disease in the same mice and in humans, suggesting that a linear extrapolation of the known increased risk from high doses to low doses is not appropriate.

Cancer and non-cancer risks in normal and cancer-prone Trp53 heterozygous mice exposed to high-dose radiation.

This report tests the hypotheses that cancer proneness elevates risk from a high radiation exposure and that the risk response to high doses is qualitatively similar to that from low doses. Groups of about 170 female mice heterozygous for Trp53 (Trp53(+/-)) and their normal female littermates (Trp53(+/+)) were exposed at 7-8 weeks of age to (60)Co gamma-radiation doses of 0, 1, 2, 3 or 4 Gy at a high dose rate (0.5 Gy/min) or 4 Gy at a low dose rate (0.5 mGy/min). In the absence of radiation exposure, Trp53 heterozygosity reduced life span approximately equally for death from either cancer or non-cancer disease. Heterozygosity alone produced a 1.5-fold greater shortening of life span than a 4-Gy acute exposure. Per unit dose, life shortening from cancer or non-cancer disease was the same for normal mice and Trp53 heterozygous animals, indicating that, contrary to previous reports, Trp53 heterozygosity did not confer radiation sensitivity to high doses of gamma rays. In Trp53(+/-) mice with cancer, life shortening from acute doses up to 4 Gy was related to both increased tumor formation and decreased tumor latency. A similar tumor response was observed in normal mice, but only up to 2 Gy, indicating that above 2 Gy, normal Trp53 function protected against tumor initiation, and further life shortening reflected only decreased latency for cancer and non-cancer disease. Dose-rate reduction factors were 1.7-3.0 for both genotypes and all end points. We conclude that Trp53 gene function influences both cancer and non-cancer mortality in unexposed female mice and that Trp53-associated cancer proneness in vivo is not correlated with elevated radiation risk. Increased risk from high acute radiation doses contrasts with the decreased risk seen previously after low doses of radiation in both Trp53 normal and heterozygous female mice.

A lower dose threshold for the in vivo protective adaptive response to radiation. Tumorigenesis in chronically exposed normal and Trp53 heterozygous C57BL/6 mice.

Low doses of ionizing radiation to cells and animals may induce adaptive responses that reduce the risk of cancer. However, there are upper dose thresholds above which these protective adaptive responses do not occur. We have now tested the hypothesis that there are similar lower dose thresholds that must be exceeded to induce protective effects in vivo. We examined the effects of low-dose/low-dose-rate fractionated exposures on cancer formation in Trp53 normal or cancer-prone Trp53 heterozygous female C57BL/6 mice. Beginning at 6 weeks of age, mice were exposed 5 days/week to single daily doses (0.33 mGy, 0.7 mGy/h) totaling 48, 97 or 146 mGy over 30, 60 or 90 weeks. The exposures for shorter times (up to 60 weeks) appeared to be below the level necessary to induce overall protective adaptive responses in Trp53 normal mice, and detrimental effects (shortened life span, increased frequency) evident for only specific tumor types (B- and T-cell lymphomas) were produced. Only when the exposures were continued for 90 weeks did the dose become sufficient to induce protective adaptive responses, balancing the detrimental effects for these specific cancers and reducing the risk level back to that of the unexposed animals. Detrimental effects were not seen for other tumor types, and a protective effect was seen for sarcomas after 60 weeks of exposure, which was then lost when the exposure continued for 90 weeks. As shown previously for the upper dose threshold for protection by low doses, the lower dose boundary between protection and harm was influenced by Trp53 functionality. Neither protection nor harm was observed in exposed Trp53 heterozygous mice, indicating that reduced Trp53 function raises the lower dose/ dose-rate threshold for both detrimental and protective tumorigenic effects.

Fractionated, low-dose-rate ionizing radiation exposure and chronic ulcerative dermatitis in normal and Trp53 heterozygous C57BL/6 mice.

The influence of low-dose-rate chronic radiation exposure and adaptive responses on non-cancer diseases is largely unknown. We examined the effect of low-dose/low-dose-rate fractionated or single exposures on spontaneous chronic ulcerative dermatitis in Trp53 normal or heterozygous female C57BL/6 mice. From 6 weeks of age, mice were exposed 5 days/week to single daily doses (0.33 mGy, 0.7 mGy/h) totaling 48, 97 or 146 mGy over 30, 60 or 90 weeks, and other Trp53+/- mice were exposed to a single dose of 10 mGy (0.5 mGy/min) at 20 weeks of age. The 90-week exposure produced an adaptive response, decreasing both disease frequency and severity in Trp53+/+ mice and extending the life span of older animals euthanized due to severe disease. The 30- or 60-week exposures had no significant protective or detrimental effect. In contrast, the chronic, fractionated exposure for 30 or 60 weeks significantly increased the frequency and severity of the disease in older Trp53+/- mice, significantly decreasing the life span of the animals required to be euthanized for disease. Similarly, the single 10-mGy exposure also increased disease frequency in older animals. However, the chronic, fractionated exposure for 90 weeks prevented these detrimental effects, with disease frequency and severity not different from unexposed controls. We conclude that very low-dose fractionated exposures can induce a protective adaptive response in both Trp53 normal and heterozygous mice, but that a lower threshold level of exposure, similar in both cases, must first be passed. In mice with reduced Trp53 functionality, doses below the threshold can produce detrimental effects.

Detrimental and protective bystander effects: a model approach.

This work integrates two important cellular responses to low doses, detrimental bystander effects and apoptosis-mediated protective bystander effects, into a multistage model for chromosome aberrations and in vitro neoplastic transformation: the State-Vector Model. The new models were tested on representative data sets that show supralinear or U-shaped dose responses. The original model without the new low-dose features was also tested for consistency with LNT-shaped dose responses. Reductions of in vitro neoplastic transformation frequencies below the spontaneous level have been reported after exposure of cells to low doses of low-LET radiation. In the current study, this protective effect is explained with bystander-induced apoptosis. An important data set that shows a low-dose detrimental bystander effect for chromosome aberrations was successfully fitted by additional terms within the cell initiation stage. It was found that this approach is equivalent to bystander-induced clonal expansion of initiated cells. This study is an important step toward a comprehensive model that contains all essential biological mechanisms that can influence dose-response curves at low doses.

Cancer and low dose responses in vivo: implications for radiation protection.

The Linear No Threshold (LNT) hypothesis states that ionizing radiation risk is directly proportional to dose, without a threshold. This hypothesis, along with a number of additional derived or auxiliary concepts such as radiation and tissue type weighting factors, and dose rate reduction factors, are used to calculate radiation risk estimates for humans, and are therefore fundamental for radiation protection practices. This system is based mainly on epidemiological data of cancer risk in human populations exposed to relatively high doses (above 100 mSv), with the results linearly extrapolated back to the low doses typical of current exposures. The system therefore uses dose as a surrogate for risk. There is now a large body of information indicating that, at low doses, the LNT hypothesis, along with most of the derived and auxiliary concepts, is incorrect. The use of dose as a predictor of risk needs to be re-examined and the use of dose limits, as a means of limiting risk needs to be re-evaluated. This re-evaluation could lead to large changes in radiation protection practices.

A model for the induction of chromosome aberrations through direct and bystander mechanisms.

A state vector model (SVM) for chromosome aberrations and neoplastic transformation has been adapted to describe detrimental bystander effects. The model describes initiation (formation of translocations) and promotion (clonal expansion and loss of contact inhibition of initiated cells). Additional terms either in the initiation model or in the rate of clonal expansion of initiated cells, describe detrimental bystander effects for chromosome aberrations as reported in the scientific literature. In the present study, the SVM with bystander effects is tested on a suitable dataset. In addition to the simulation of non-linear effects, a classical dataset for neoplastic transformation in C3H 10T1/2 cells after alpha particle irradiation is used to show that the model without bystander features can also describe LNT-like dose responses. A published model for bystander induced neoplastic transformation was adapted for chromosome aberration induction and used to compare the results obtained with the different models.

Low doses of radiation reduce risk in vivo.

The "Linear No Threshold" hypothesis, used in all radiation protection practices, assumes that all doses, no matter how low, increase the risk of cancer, birth defects and heritable mutations. In vitro cell based experiments show adaptive processes in response to low doses and dose rates of low LET radiation, and do not support the hypothesis. This talk will present cellular data and data from animal experiments that test the hypothesis in vivo for cancer risk. The data show that a single, low, whole body dose (less than about 100 mGy) of low LET radiation, given at low dose rate, increased cancer latency and consequently reduced both spontaneous and radiation-induced cancer risk in both genetically normal and cancer-prone mice. This adaptive response lasted for the entire lifespan of all the animals that developed these tumors, and effectively restored a portion of the life that would have been lost due to the cancer in the absence of the low dose. Overall, the results demonstrate that the assumption of a linear increase in risk with increasing dose in vivo is not warranted, and that low doses actually reduce risk.

The adaptive response and protection against heritable mutations and fetal malformation.

There are a number of studies that show radiation can cause heritable mutations in the offspring of irradiated organisms. These "germ-line mutations" have been shown to occur in unique sequences of DNA called "minisatellite loci". The high frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be measured at low doses of exposure in a small population, making minisatellite mutation a powerful tool to investigate radiation-induced heritable mutations. However, the biological significance of these mutations is uncertain, and their relationship to health risk or population fitness is unknown. We have adopted this mutation assay to study the role of adaptive response in protecting mice against radiation-induced heritable defects. We have shown that male mice, adapted to radiation with a low dose priming exposure, do not pass on mutations to their offspring caused by a subsequent large radiation exposure to the adapted males. This presentation and paper provide a general overview of radiation-induced mutations in offspring and explain the effect of low dose exposures and the adaptive response on these mutations.It is also known that exposure of pregnant females to high doses of radiation can cause death or malformation (teratogenesis) in developing fetuses. Malformation can only occur during a specialized stage of organ formation known as organogenesis. Studies in rodents show that radiation-induced fetal death and malformation can be significantly reduced when a pregnant female is exposed to a prior low dose of ionizing radiation. The mechanism of this protective effect, through an adaptive response, depends on the stage of organogenesis when the low dose exposures are delivered. To better understand this process, we have investigated the role of an important gene known as p53. Therefore, this report will also discuss fetal effects of ionizing radiation and explain the critical stages of development when fetuses are at risk. Research will be explained that investigates the biological and genetic systems (p53) that protect the developing fetus and discuss the role of low dose radiation adaptive response in these processes.

Radiation risk prediction and genetics: the influence of the TP53 gene in vivo.

Risk prediction and dose limits for human radiation exposure are based on the assumption that risk is proportional to total dose. However, there is concern about the appropriateness of those limits for people who may be genetically cancer prone. The TP53 gene product functions in regulatory pathways for DNA repair, cell cycle checkpoints and apoptosis, processes critical in determining ionizing radiation risk for both carcinogenesis and teratogenesis. Mice that are deficient in TP53 function are cancer prone. This review examines the influence of variations in TP53 gene activity on cancer and teratogenic risk in mice exposed to radiation in vivo, and compares those observations to the assumptions and predictions of radiation risk inherent in the existing system of radiation protection. Current assumptions concerning a linear response with dose, dose additivity, lack of thresholds and dose rate reduction factors all appear incorrect at low doses. TP53 functional variations can further modify radiation risk from either high or low doses, or risk from radiation exposures combined with other stresses, and those modifications can result in both quantitative and qualitative changes in risk.

Low-LET-induced radioprotective mechanisms within a stochastic two-stage cancer model.

A stochastic two-stage cancer model with clonal expansion was used to investigate the potential impact on human lung cancer incidence of some aspects of the hormesis mechanisms suggested by Feinendegen (Health Phys. 52 663-669, 1987). The model was applied to low doses of low-LET radiation delivered at low dose rates. Non-linear responses arise in the model because radiologically induced adaptations in radical scavenging and DNA repair may reduce the biological consequences of DNA damage formed by endogenous processes and ionizing radiation. Sensitivity studies were conducted to identify critical model inputs and to help define the changes in cellular defense mechanisms necessary to produce a lifetime probability for lung cancer that deviates from a linear no-threshold (LNT) type of response. Our studies suggest that lung cancer risk predictions may be very sensitive to the induction of DNA damage by endogenous processes. For doses comparable to background radiation levels, endogenous DNA damage may account for as much as 50 to 80% of the predicted lung cancers. For an additional lifetime dose of 1 Gy from low-LET radiation, endogenous processes may still account for as much as 20% of the predicted cancers (Fig. 2). When both repair and scavengers are considered as inducible, radiation must enhance DNA repair and radical scavenging in excess of 30 to 40% of the baseline values to produce lifetime probabilities for lung cancer outside the range expected for endogenous processes and background radiation.

Low doses of radiation are protective in vitro and in vivo: evolutionary origins.

Research reports using cells from bacteria, yeast, alga, nematodes, fish, plants, insects, amphibians, birds and mammals, including wild deer, rodents or humans show non-linear radio-adaptive processes in response to low doses of low LET radiation. Low doses increased cellular DNA double-strand break repair capacity, reduced the risk of cell death, reduced radiation or chemically-induced chromosomal aberrations and mutations, and reduced spontaneous or radiation-induced malignant transformation in vitro. In animals, a single low, whole body dose of low LET radiation, increased cancer latency and restored a portion of the life that would have been lost due to either spontaneous or radiation-induced cancer in the absence of the low dose. In genetically normal fetal mice, a prior low dose protected against radiation-induced birth defects. In genetically normal adult-male mice, a low dose prior to a high dose protected the offspring of the mice from heritable mutations produced by the large dose. The results show that low doses of low-LET radiation induce protective effects and that these induced responses have been tightly conserved throughout evolution, suggesting that they are basic responses critical to life. The results also argue strongly that the assumption of a linear increase in risk with increasing dose in humans is unlikely to be correct, and that low doses actually reduce risk.