Untargeted effects of ionizing radiation: implications for radiation pathology

The dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation, characteristically associated with the consequences of energy deposition in the cell nucleus, arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that have received damaging signals produced by irradiated cells (radiation-induced bystander effects) or that are the descendants of irradiated cells (radiation-induced genomic instability). Radiation-induced genomic instability is characterized by a number of delayed adverse responses including chromosomal abnormalities, gene mutations and cell death. Similar effects, as well as responses that may be regarded as protective, have been attributed to bystander mechanisms. Whilst the majority of studies to date have used in vitro systems, some adverse non-targeted effects have been demonstrated in vivo. However, at least for haemopoietic tissues, radiation-induced genomic instability in vivo may not necessarily be a reflection of genomically unstable cells. Rather the damage may reflect responses to ongoing production of damaging signals; i.e. bystander responses, but not in the sense used to describe the rapidly induced effects resulting from direct interaction of irradiated and non-irradiated cells. The findings are consistent with a delayed and long-lived tissue reaction to radiation injury characteristic of an inflammatory response with the potential for persisting bystander-mediated damage. An important implication of the findings is that contrary to conventional radiobiological dogma and interpretation of epidemiologically-based risk estimates, ionizing radiation may contribute to malignancy and particularly childhood leukaemia by promoting initiated cells rather than being the initiating agent. Untargeted mechanisms may also contribute to other pathological consequences.     

Dosimetry for quantitative analysis of the effects of low-dose ionizing radiation in radiation therapy patients

We have developed and validated a practical approach to identifying the location on the skin surface that will receive a prespecified biopsy dose (ranging down to 1 cGy) in support of in vivo biological dosimetry in humans. This represents a significant technical challenge since the sites lie on the patient's surface outside the radiation fields. The PEREGRINE Monte Carlo simulation system was used to model radiation dose delivery, and TLDs were used for validation on phantoms and for confirmation during patient treatment. In the developmental studies, the Monte Carlo simulations consistently underestimated the dose at the biopsy site by approximately 15% (of the local dose) for a realistic treatment configuration, most likely due to lack of detail in the simulation of the linear accelerator outside the main beam line. Using a single, thickness-independent correction factor for the clinical calculations, the average of 36 measurements for the predicted 1-cGy point was 0.985 cGy (standard deviation: 0.110 cGy) despite patient breathing motion and other real-world challenges. Since the 10-cGy point is situated in the region of high-dose gradient at the edge of the field, patient motion had a greater effect, and the six measured points averaged 5.90 cGy (standard deviation: 1.01 cGy), a difference that is equivalent to approximately a 6-mm shift on the patient's surface.     

Cell-density dependent effects of low-dose ionizing radiation on E. coli cells

The changes in genome conformational state (GCS) induced by low-dose ionizing radiation in E. coli cells were measured by the method of anomalous viscosity time dependence (AVTD) in cellular lysates. Effects of X-rays at doses 0.1 cGy--1 Gy depended on post-irradiation time. Significant relaxation of DNA loops followed by a decrease in AVTD. The time of maximum relaxation was between 5-80 min depending on the dose of irradiation. U-shaped dose response was observed with increase of AVTD in the range of 0.1-4 Gy and decrease in AVTD at higher doses. No such increase in AVTD was seen upon irradiation of cells at the beginning of cell lysis while the AVTD decrease was the same. Significant differences in the effects of X-rays and gamma-rays at the same doses were observed suggesting a strong dependence of low-dose effects on LET. Effects of 0.01 cGy gamma-rays were studied at different cell densities during irradiation. We show that the radiation-induced changes in GCS lasted longer at higher cell density as compared to lower cell density. Only small amount of cells were hit at this dose and the data suggest cell-to-cell communication in response to low-dose ionizing radiation. This prolonged effect was also observed when cells were irradiated at high cell density and diluted to low cell density immediately after irradiation. These data suggest that cell-to-cell communication occur during irradiation or within 3 min post-irradiation. The cell-density dependent response to low-dose ionizing radiation was compared with previously reported data on exposure of E. coli cells to electromagnetic fields of extremely low frequency and extremely high frequency (millimeter waves). The body of our data show that cells can communicate in response to electromagnetic fields and ionizing radiation, presumably by reemission of secondary photons in infrared-submillimeter frequency range.     

Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells

Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G₁-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G₁/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation.     

The linear no-threshold model does not hold for low-dose ionizing radiation

Almost all of the data on the biological effects of ionizing radiation come from studies of high doses. However, the human population is unlikely to be exposed to such doses. Regulatory limits for radiation exposure are based on the linear no-threshold model, which predicts that the relationship between biological effects and radiation dose is linear, and that any dose has some effect. Chromosomal changes are an important effect of ionizing radiation because of their role in carcinogenesis. Here we exposed pKZ1 mice to single whole-body X-radiation doses as low as 1 microGy. We observed three different phases of response: (1) an induction of inversions at ultra-low doses, (2) a reduction below endogenous inversion frequency at low doses, and (3) an induction of inversions again at higher doses. These results do not fit a linear no-threshold model, and they may have implications for the way in which regulatory standards are presently set and for understanding radiation effects.     

The psychological effects of ionizing radiation

This paper presents a case study of eleven men who were exposed to non-background ionizing radiation as active participants in the United States' atmospheric nuclear tests. Each of the subjects has developed a virtually identical complex of debilitating psychiatric symptoms. The content of these symptoms is almost entirely focused upon the health effects of the radiation to which each of the subjects was exposed. This symptom complex appears to comprise a syndrome. The symptom structure and course of this syndrome suggests three hypotheses: The syndrome appears to be a pathological development of the self diagnostic belief (that one has been physically harmed by radiation) into a set of symptoms that elaborate upon and express this belief. The self diagnostic belief develops as a means of resolving any one of the various medical mysteries that an individual can experience subsequent to exposure to radiation. Development of the syndrome is a consequence of exposure to non-background ionizing radiation. The paper discusses the evidence for these hypotheses and suggests future research directions.     

Biological low-dose radiation effects.

It is theorized that biological responses to ionizing radiation in the low dose range are determined according to a doubly dichotomous pattern. Energy depositions fall into 2 categories: events at thermal energy levels where they may be experienced by cells as rates even at background exposure conditions, and events at energy levels of the order of 10-100 eV where damage to DNA may be caused. Variations in background exposure intensity may or may not lead preemptively to changes in the cell's capacity for response to radiation damage. High-level energy depositions lead post hoc to an initial stabilizing reaction largely leading to the fixation of the initial DNA damage, and to a subsequent restorative or palliative repair process. This model entails reinterpretation of some experimental results. The model has implications for the relationship between scientific analysis of low-dose effects and the regulatory needs for simplicity and homogeneity in risk evaluation. This represents a new challenge for the acceptability of radiation protection norms.     

Population models for the health effects of ionizing radiation

In this paper we review recently-developed extension frailty, quadratic hazard, stochastic process, microsimulation, and linear latent structure models, which have the potential to describe the health effects of human populations exposed to ionizing radiation. We discuss the most common situations for which such models are appropriate. We also provide examples of how to estimate the parameters of these models from datasets of various designs. Carcinogenesis models are reviewed in context of application to epidemiologic data of population exposed to ionizing radiation. We also discuss the ways of how to generalize stochastic process and correlated frailty models for longitudinal and family analyses in radiation epidemiology.     

Membrane effects of ionizing radiation and hyperthermia

Results of numerous studies demonstrate that membranes are important sites of cell damage by both ionizing radiation and hyperthermia. Modification of membrane properties (mainly lipid fluidity) affects the cellular responses to radiation and hyperthermia but former concepts that membrane rigidification sensitizes cells to radiation while membrane fluidization potentiates hyperthermic damage have now been seriously challenged. It seems that the effects of membrane fluidity on cell responses to hyperthermia and radiation are due to an indirect influence on functional membrane proteins. The major role of lipid peroxidation in radiation damage to membranes has also been questioned. The existing evidence makes it unlikely that the interaction between radiation and hyperthermia is determined by the action of both agents on the same membrane components.     

Alteration of cytokine profiles in mice exposed to chronic low-dose ionizing radiation

While a high-dose of ionizing radiation is generally harmful and causes damage to living organisms, a low-dose of radiation has been shown to be beneficial in a variety of animal models. To understand the basis for the effect of low-dose radiation in vivo, we examined the cellular and immunological changes evoked in mice exposed to low-dose radiation at very low (0.7 mGy/h) and low (3.95 mGy/h) dose rate for the total dose of 0.2 and 2 Gy, respectively. Mice exposed to low-dose radiation, either at very low- or low-dose rate, demonstrated normal range of body weight and complete blood counts. Likewise, the number and percentage of peripheral lymphocyte populations, CD4⁺ T, CD8⁺ T, B, or NK cells, stayed unchanged following irradiation. Nonetheless, the sera from these mice exhibited elevated levels of IL-3, IL-4, leptin, MCP-1, MCP-5, MIP-1α, thrombopoietin, and VEGF along with slight reduction of IL-12p70, IL-13, IL-17, and IFN-γ. This pattern of cytokine release suggests the stimulation of innate immunity facilitating myeloid differentiation and activation while suppressing pro-inflammatory responses and promoting differentiation of naïve T cells into T-helper 2, not T-helper 1, types. Collectively, our data highlight the subtle changes of cytokine milieu by chronic low-dose γ-radiation, which may be associated with the functional benefits observed in various experimental models.     

Risk calculations for hereditary effects of ionizing radiation in humans

Summary A prediction of the extent to which an additional dose of ionizing radiation increases the natural germ cell mutation rate, and how much such an increase will affect the health status of future human populations is part of the service that human geneticists are expected to offer to human society. However, more detailed scrutiny of the difficulties involved reveals an extremely complex set of problems. A large number of questions arises before such a prediction can be given with confidence; many such questions cannot be answered at our present state of knowledge. However, such predictions have recently been attempted. The 1988 report of the United Nations Scientific Committee for the Effects of Atomic Radiation and the fifth report of the Committee on Biological Effects of Ionizing Radiation of the US National Research Council have presented a discussion of the human genetics problems involved. Empirical data from studies on children of highly radiation-exposed parents, e.g. parents exposed to the atomic bombs of Hiroshima and Nagasaki, or parents belonging to populations living on soil with high background radiation, have been mentioned in this context. Whereas precise predictions are impossible as yet because of deficiencies in our knowledge of medical genetics at various levels, the bulk of the existing evidence points to only small effects of low or moderate radiation doses, effects that will probably be buried in the background noise of changing patterns of human morbidity and mortality.     

Pulmonary injury after combined exposures to low-dose low-LET radiation and fungal spores

Exposure to infectious microbes is a likely confounder after a nuclear terrorism event. In combination with radiation, morbidity and mortality from an infection may increase significantly. Pulmonary damage after low-dose low-LET irradiation is characterized by an initial diffuse alveolar inflammation. By contrast, inhaled fungal spores produce localized damage around pulmonary bronchioles. In the present study, we assessed lung injury in C57BL/6 mice after combined exposures to whole-body X radiation and inhaled fungal spores. Either animals were exposed to Aspergillus spores and immediately irradiated with 2 Gy, or the inoculation and irradiation were separated by 8 weeks. Pulmonary injury was assessed at 24 and 48 h and 1, 2, 4, 8, and 24 weeks later using standard H&E-stained sections and compared with sham-treated age-matched controls. Immunohistochemistry for invasive inflammatory cells (macrophages, neutrophils and B and T lymphocytes) was performed. A semi-quantitative assessment of pulmonary injury was made using three distinct parameters: local infiltration of inflammatory cells, diffuse inflammation, and thickening and distortion of alveolar architecture. Radiation-induced changes in lung architecture were most evident during the first 2 weeks postexposure. Fungal changes were seen over the first 4 weeks. Simultaneous combined exposures significantly increased the duration of acute pulmonary damage up to 24 weeks (P < 0.01). In contrast, administration of the fungus 8 weeks after irradiation did not produce enhanced levels of acute pulmonary damage. These data imply that the inhalation of fungal spores at the time of a radiation exposure alters the susceptibility of the lungs to radiation-induced injury.     

Non-targeted bystander effects induced by ionizing radiation

Radiation-induced bystander effects refer to those responses occurring in cells that were not subject to energy deposition events following ionizing radiation. These bystander cells may have been neighbors of irradiated cells, or physically separated but subject to soluble secreted signals from irradiated cells. Bystander effects have been observed in vitro and in vivo and for various radiation qualities. In tribute to an old friend and colleague, Anthony V. Carrano, who would have said "well what are the critical questions that should be addressed, and so what?", we review the evidence for non-targeted radiation-induced bystander effects with emphasis on prevailing questions in this rapidly developing research field, and the potential significance of bystander effects in evaluating the detrimental health effects of radiation exposure.     

A microdosimetric understanding of low-dose radiation effects

This paper presents a microdosimetric approach to the problem of radiation response by which effects produced at low doses and dose rates can be understood as the consequences of radiation absorption events in the nucleus of a single relevant cell and in its DNA. Radiation absorption at the cellular level, i.e. in the cell nucleus as a whole, is believed to act through radicals. This kind of action is called 'non-specific' and leads to the definition of an 'elemental dose' and the 'integral response probability' of a cell population. Radiation absorption at the molecular level, i.e. in sensitive parts of the DNA, is thought to act through double-strand breaks. This kind of action is called 'specific' and leads to a 'relative local efficiency'. In general, both mechanisms occur for all types of radiation; however, it is the dose contribution of both specific and non-specific effects that determines the radiation quality of a given radiation. The implications of this approach for the specification of low-dose and low dose-rate regions are discussed.