Responses to ionizing radiation mediated by inflammatory mechanisms.

Since the early years of the twentieth century, the biological consequences of exposure to ionizing radiation have been attributed solely to mutational DNA damage or cell death induced in irradiated cells at the time of exposure. However, numerous observations have been at variance with this dogma. In the 1950s, attention was drawn to abscopal effects in areas of the body not directly irradiated. In the 1960s reports began appearing that plasma factors induced by irradiation could affect unirradiated cells, and since 1990 a growing literature has documented an increased rate of DNA damage in the progeny of irradiated cells many cell generations after the initial exposure (radiation-induced genomic instability) and responses in non-irradiated cells neighbouring irradiated cells (radiation-induced bystander effects). All these studies have in common the induction of effects not in directly irradiated cells but in unirradiated cells as a consequence of intercellular signalling. Recently, it has become clear that all the various effects demonstrated in vivo may reflect an ongoing inflammatory response to the initial radiation-induced injury that, in a genotype-dependent manner, has the potential to contribute primary and/or ongoing damage displaced in time and/or space from the initial insult. Importantly, there is direct evidence that non-steroidal anti-inflammatory drug treatment reduces such damage in vivo. These new findings highlight the importance of tissue responses and indicate additional mechanisms of radiation action, including the likelihood that radiation effects are not restricted to the initiation stage of neoplastic diseases, but may also contribute to tumour promotion and progression. The various developments in understanding the responses to radiation exposures have implications not only for radiation pathology but also for therapeutic interventions.

Radiation and the microenvironment - tumorigenesis and therapy.

Radiation rapidly and persistently alters the soluble and insoluble components of the tissue microenvironment. This affects the cell phenotype, tissue composition and the physical interactions and signalling between cells. These alterations in the microenvironment can contribute to carcinogenesis and alter the tissue response to anticancer therapy. Examples of these responses and their implications are discussed with a view to therapeutic intervention.

Manifestations and mechanisms of non-targeted effects of ionizing radiation.

A well-established radiobiological paradigm is that the biological effects of ionizing radiation occur in irradiated cells as a consequence of the DNA damage they incur. However, many observations of, so-called, non-targeted effects indicate that genetic alterations are not restricted to directly irradiated cells. Non-targeted effects are responses exhibited by non-irradiated cells that are the descendants of irradiated cells (radiation-induced genomic instability) or by cells that have communicated with irradiated cells (radiation-induced bystander effects). Radiation-induced genomic instability is characterized by 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. The majority of studies to date have used in vitro systems but some non-targeted effects have been demonstrated in vivo and there is also evidence for radiation-induced instability in the mammalian germ line. However, there may be situations where radiation-induced genomic instability in vivo may not necessarily identify genomically unstable somatic cells but the manifestation of responses to ongoing production of damaging signals generated by genotype-dependent mechanisms having properties in common with inflammatory processes. Non-targeted mechanisms have significant implications for understanding mechanisms of radiation action but the current state of knowledge does not permit definitive statements about whether these phenomena have implications for assessing radiation risk.

Microenvironmental and genetic factors in haemopoietic radiation responses.

PURPOSE: To review studies of radiation responses in the haemopoietic system in the context of radiation-induced chromosomal instability, bystander effects, the influence of the microenvironment and genetic factors. CONCLUSIONS: Blood cells are continuously produced by the proliferation and differentiation of lineage-specific precursor cells that, in turn, are all derived from a small population of multipotential stem cells. The homeostatic regulation of this hemopoietic hierarchy involves multiple regulatory factors and interactions with the tissue microenvironment and responses of the hemopoietic system are major determinants of outcome after exposure to ionizing radiation. A sub-optimal or aberrant response to radiation-induced damage may divert the system away from effective restoration of tissue homeostasis into responses that ultimately result in pathological changes. DNA damage in irradiated cells that has not been correctly restored by metabolic repair processes is conventionally regarded as the reason for the adverse consequences of radiation exposures. However, reports of radiation-induced genomic instability and radiation-induced bystander effects challenge this conventional paradigm. In the context of the haemopoietic system, these, so called, non-targeted effects can be inter-related and an instability phenotype need not necessarily be a reflection of genomically unstable cells but a reflection of responses to ongoing production of damaging bystander signals in the tissue microenvironment. Both the production of and the response to such signals are influenced by genetic factors and the cell interactions have properties in common with inflammatory mechanisms.

Genotype-dependent induction of transmissible chromosomal instability by gamma-radiation and the benzene metabolite hydroquinone.

Although it is well established that ionizing radiation and benzene are epidemiologically linked to acute myeloid leukemia (AML), the underlying mechanisms are not understood. We have shown that gamma-radiation can induce a persisting genomic instability in the clonal descendants of hemopoietic stem cells manifested as a high frequency of nonclonal chromosome and chromatid aberrations. A strikingly similar instability is shown after exposure to the benzene metabolite hydroquinone. The CBA/Ca but not the C57BL/6 genotype is susceptible to the induction of instability by both ionizing radiation and hydroquinone and exposure of CBA/Ca, but not C57BL/6, mice to either agent is known to be associated with the development of AML. The results are consistent with the proposal that chromosomal instability induced by either agent may contribute to AML development by increasing the number of genetic lesions in hemopoietic cells. Genotype-dependent chromosomal instability can be induced by hydroquinone doses that are not acutely stem cell toxic and this may have important implications for current assessment of safe levels of exposure to benzene as well as for mechanistic understanding of the hemotoxic and leukemogenic effects.

Chromosomal instability in unirradiated hemopoietic cells resulting from a delayed in vivo bystander effect of gamma radiation.

Untargeted effects of ionizing radiation (de novo effects in the unirradiated descendants or neighbors of irradiated cells) challenge widely held views about the mechanisms of radiation-induced DNA damage with implications for the health consequences of radiation exposures particularly in the context of the induction of malignancy. To investigate in vivo untargeted effects of sparsely ionizing (low linear energy transfer) radiation, a congenic sex-mismatch bone marrow transplantation protocol has been used to repopulate the hemopoietic system from a mixture of gamma-irradiated and nonirradiated hemopoietic stem cells such that host-, irradiated donor- and unirradiated donor-derived cells can be distinguished. Chromosomal instability in the progeny of irradiated hemopoietic stem cells accompanied by a reduction in their contribution to the repopulated hemopoietic system is consistent with a delayed genomic instability phenotype being expressed in vivo. However, chromosomal instability was also shown in the progeny of the nonirradiated hemopoietic stem cells implicating a bystander mechanism. Studies of the influence of irradiated recipient stromal microenvironment and experiments replacing irradiated cells with irradiated cell-conditioned medium reveal the source of the in vivo bystander effect to be the descendants of irradiated cells, rather than irradiated cell themselves. Thus, it is possible that a radiation-induced genomic instability phenotype in vivo need not necessarily be a reflection of intrinsically unstable cells but the responses to ongoing production of inflammatory-type damaging signals as a long-term unexpected consequence of the initial single radiation exposure.

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.

Radiation-induced genomic instability and bystander effects: inter-related nontargeted effects of exposure to ionizing radiation.

The paradigm of genetic alterations being restricted to direct DNA damage after exposure to ionizing radiation has been challenged by observations in which cells that are not exposed to ionizing radiation exhibit responses typically associated with direct radiation exposure. These effects are demonstrated in cells that are the descendants of irradiated cells (radiation-induced genomic instability) or in cells that are in contact with irradiated cells or receive certain signals from irradiated cells (radiation-induced bystander effects). There is accumulating evidence that radiation-induced genomic instability may be a consequence of, and in some cell systems may also produce, bystander interactions involving intercellular signalling, production of cytokines and free-radical generation. These processes are also features of inflammatory responses that are known to have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. Thus, radiation-induced genomic instability and untargeted bystander effects may reflect inter-related aspects of inflammatory-type responses to radiation-induced stress and injury and contribute to the variety of pathological consequences of radiation exposures.

Damaging and protective cell signalling in the untargeted effects of ionizing radiation.

The major adverse consequences of radiation exposures are attributed to DNA damage in irradiated cells that has not been correctly restored by metabolic repair processes. However, the dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that are the descendants of irradiated cells either directly or via media transfer (radiation-induced genomic instability) or in cells that have communicated with irradiated cells (radiation-induced bystander effects). Radiation-induced genomic instability is characterized by a number of delayed responses including chromosomal abnormalities, gene mutations and cell death. Bystander effects include increases or decreases in damage-inducible and stress-related proteins, increases or decreases in reactive oxygen and nitrogen species, cell death or cell proliferation, cell differentiation, radioadaptation, induction of mutations and chromosome aberrations and chromosomal instability. The phenotypic expression of untargeted effects and the potential consequences of these effects in tissues reflect a balance between the type of bystander signals produced and the responses of cell populations to such signals, both of which may be significantly influenced by cell type and genotype. Thus, in addition to targeted effects of damage induced directly in cells by irradiation, a variety of untargeted effects may also make important short-term and long-term contributions to determining overall outcome after radiation exposures.

Commentary on radiation-induced bystander effects.

The paradigm of genetic alterations being restricted to direct DNA damage after exposure to ionizing radiation has been challenged by observations in which effects of ionizing radiation arise in cells that in themselves receive no radiation exposure. These effects are demonstrated in cells that are the descendants of irradiated cells (radiation-induced genomic instability) or in cells that are in contact with irradiated cells or receive certain signals from irradiated cells (radiation-induced bystander effects). Bystander signals may be transmitted either by direct intercellular communication through gap junctions, or by diffusible factors, such as cytokines released from irradiated cells. In both phenomena, the untargeted effects of ionizing radiation appear to be associated with free radical-mediated processes. There is evidence that radiation-induced genomic instability may be a consequence of, and in some cell systems may also produce, bystander interactions involving intercellular signalling, production of cytokines and free radical generation. These processes are also features of inflammatory responses that are known to have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. Thus, radiation-induced genomic instability and untargeted bystander effects may reflect interrelated aspects of inflammatory type responses to radiation-induced stress and injury and contribute to the variety of the pathological consequences of radiation exposures.

Interactions of apoptotic cells with macrophages in radiation-induced bystander signaling.

Nontargeted effects that result in ongoing cellular and tissue damage show genotype-dependency in murine models with CBA/Ca, but not C57BL/6, exhibiting sensitivity to induced genomic instability. In vivo, radiation exposure is associated with genotype-dependent macrophage activation, and these cells are a source of bystander signaling involving cytokines and reactive oxygen and nitrogen species. The mechanisms responsible for macrophage activation and production of damaging bystander signals after irradiation are unclear. Macrophages from CBA/Ca exhibit an M1 (proinflammatory) phenotype compared to the M2 (anti-inflammatory) phenotype of C57BL/6 macrophages. Using the murine RAW264.7 macrophage-like cell line, we show that the ability of macrophages to interact with apoptotic cells and their responses to interaction varies significantly according to macrophage phenotype. Nonstimulated and M2 macrophages induce anti-inflammatory markers arginase and TGFß after engulfment of apoptotic cells. In contrast, M1 macrophages do not induce anti-inflammatory responses, but express the proinflammatory markers NOS2, IL-6, TNFα, superoxide and NO, able to contribute to a damaging microenvironment. Macrophages stimulated with both inflammatory and anti-inflammatory agents prior to exposure to apoptotic cells induce a mixed response. The results indicate a complex cross-talk between macrophages and apoptotic cells and demonstrate that phagocytic clearance of apoptotic cells induced by genotoxic stress can produce microenvironmental responses consistent with the induction of a chromosomal instability phenotype in sensitive CBA/Ca mice with M1 macrophage activation, but not in resistant C57BL/6 mice with M2 macrophage activation. Modulation of macrophage phenotypes may represent a novel approach for reducing the nontargeted effects of radiation.

Radiation-induced bone marrow apoptosis, inflammatory bystander-type signaling and tissue cytotoxicity.

PURPOSE: A study of irradiated (0.25-2 Gy) murine bone marrow has investigated the relationships between apoptotic responses of cells exposed in vivo and in vitro and between in vivo apoptosis and tissue cytotoxicity. MATERIALS AND METHODS: The time course of reduction in bone marrow cellularity in vivo was determined by femoral cell counts and apoptosis measurements obtained using three commonly used assays. Inflammatory pro-apoptotic cytokine production at 24 h post-exposure in vivo was investigated using a bystander protocol. RESULTS: In vivo, there is a dose- and time-dependent non-linear reduction in bone marrow cellularity up to 24 h post- irradiation not directly represented by apoptosis measurements. The majority of cells are killed within 6 h but there is on-going cell loss in vivo up to 24 h post-irradiation in the absence of elevated levels of apoptosis and associated with the induction of cytokines produced in response to the initial tumor protein 53 (p53)-dependent apoptosis. CONCLUSION: The results demonstrate that small increases in measured apoptosis can reflect significant intramedullary cell death and with apoptotic processes being responsible for pro-inflammatory mechanisms that can contribute to additional on-going cell death. The findings demonstrate the importance of studying tissue responses when considering the mechanisms underlying the consequences of radiation exposures.

The influence of p53 functions on radiation-induced inflammatory bystander-type signaling in murine bone marrow.

Radiation-induced bystander and abscopal effects, in which DNA damage is produced by inter-cellular communication, indicate mechanisms of generating damage in addition to those observed in directly irradiated cells. In this article, we show that the bone marrow of irradiated p53(+/+) mice, but not p53(-/-) mice, produces the inflammatory pro-apoptotic cytokines FasL and TNF-α able to induce p53-independent apoptosis in vitro in nonirradiated p53(-/-) bone marrow cells. Using a congenic sex-mismatch bone marrow transplantation protocol to generate chimeric mice, p53(-/-) hemopoietic cells functioning in a p53(+/+) bone marrow stromal microenvironment exhibited greater cell killing after irradiation than p53(-/-) hemopoietic cells in a p53(-/-) microenvironment. Cytogenetic analysis demonstrated fewer damaged p53(-/-) cells in a p53(+/+) microenvironment than p53(-/-) cells in a p53(-/-) microenvironment. Using the two different model systems, the findings implicate inflammatory tissue processes induced as a consequence of p53-dependent cellular responses to the initial radiation damage, producing cytokines that subsequently induce ongoing p53-independent apoptosis. As inactivation of the p53 tumor suppressor pathway is a common event in malignant cells developing in a stromal microenvironment that has normal p53 function, the signaling processes identified in the current investigations have potential implications for disease pathogenesis and therapy.

Bystander-type effects mediated by long-lived inflammatory signaling in irradiated bone marrow.

Radiation-induced bystander and abscopal effects, in which DNA damage is produced in nonirradiated cells as a consequence of communication with irradiated cells, indicate mechanisms of inducing damage and cell death additional to the conventional model of deposition of energy in the cell nucleus at the time of irradiation. In this study we show that signals generated in vivo in the bone marrow of mice irradiated with 4 Gy γ rays 18 h to 15 months previously are able to induce DNA damage and apoptosis in nonirradiated bone marrow cells but that comparable signals are not detected at earlier times postirradiation or at doses below 100 mGy. Bone marrow cells of both CBA/Ca and C57BL/6 genotypes exhibit responses to signals produced by either irradiated CBA/Ca or C57BL/6 mice, and the responses are mediated by the cytokines FasL and TNF-α converging on a COX-2-dependent pathway. The findings are consistent with indirect inflammatory signaling induced as a response to the initial radiation damage rather than to direct signaling between irradiated and nonirradiated cells. The findings also demonstrate the importance of studying tissue responses when considering the mechanisms underlying the consequences of radiation exposures.

The in vivo expression of radiation-induced chromosomal instability has an inflammatory mechanism.

Ionizing radiation is unequivocally leukemogenic and carcinogenic, and this is generally attributed to DNA damage arising as a consequence of deposition of energy in the cell nucleus at the time of exposure. However, nontargeted effects, in which DNA damage is produced in nonirradiated cells as a consequence of cell signaling processes, indicate additional mechanisms. Radiation-induced chromosomal instability, a nontargeted effect with the potential to produce pathological consequences, is characterized by an increased rate of chromosome aberrations many generations after the initial insult. In this study, using a mouse model that has been well characterized with respect to its susceptibility to both radiation-induced chromosomal instability and acute myeloid leukemia, we investigated whether the underlying signaling mechanism was an inflammatory process by studying the effects of a nonsteroidal anti-inflammatory drug. Treated mice showed significant reduction in expression of the chromosomal instability phenotype 100 days postirradiation associated with reduced expression of inflammatory markers. The data support the hypothesis that the radiation-induced chromosomal instability phenotype is not an intrinsic property of the cells but a consequence of inflammatory processes having the potential to contribute secondary damage expressed as nontargeted and delayed radiation effects.

Lack of nontargeted effects in murine bone marrow after low-dose in vivo X irradiation.

Exposure to high doses of ionizing radiation unequivocally produces adverse health effects including malignancy. At low doses the situation is much less clear, because effects are generally too small to be estimated directly by epidemiology, and extrapolation of risk and establishment of international rules and standards rely on the linear no-threshold (LNT) concept. Claims that low doses are more damaging than would be expected from LNT have been made on the basis of in vitro studies of nontargeted bystander effects and genomic instability, but relevant investigations of primary cells and tissues are limited. Here we show that after low-dose low-LET in vivo radiation exposures in the 0-100-mGy range of murine bone marrow there is no evidence of a bystander effect, assessed by p53 pathway signaling, nor is there any evidence for longer-term chromosomal instability in the bone marrow at doses below 1000 mGy. The data are not consistent with speculations based on in vitro nontargeted effects that low-dose X radiation is more damaging than would be expected from linear extrapolation.

Long-lived inflammatory signaling in irradiated bone marrow is genome dependent.

Ionizing radiation is carcinogenic, but genotype is a key determinant of susceptibility. Mutational DNA damage is generally attributed to cause disease, but irradiation also affects multicellular interactions as a result of poorly understood bystander effects that may influence carcinogenic susceptibility. In this study, we show that the bone marrow of irradiated mice will retain the ability to kill hemopoietic clonogenic stem cells and to induce chromosomal instability for up to 3 months after irradiation. Chromosomal instability was induced in bone marrow cells derived from CBA/Ca mice, a strain that is susceptible to radiation-induced acute myeloid leukemia (r-AML), but not in C57BL6 mice that are resistant to r-AML. Similarly, clonogenic cell lethality was exhibited in C57BL/6 mice but not CBA/Ca mice. Mechanistic investigations revealed that these genotype-dependent effects involved cytokine-mediated signaling and were mediated by a cyclooxygenase-2-dependent mechanism. Thus, our results suggested that inflammatory processes were responsible for mediating and sustaining the durable effects of ionizing radiation observed on bone marrow cells. Because most exposures to ionizing radiation are directed to only part of the body, our findings imply that genotype-directed tissue responses may be important determinants of understanding the specific consequence of radiation exposure in different individuals.

Radiation-induced delayed bystander-type effects mediated by hemopoietic cells.

Genetic lesions and cell death associated with exposure to ionizing radiation have generally been attributed to DNA damage arising as a consequence of deposition of energy in the cell nucleus. However, reports of radiation-induced bystander effects, in which DNA damage is produced in nonirradiated cells as a consequence of communication with irradiated cells, indicate additional mechanisms. At present, most information has been obtained using in vitro systems, and the in vivo significance of bystander factors is not clear. In this study we show that signals generated in vivo in the bone marrow of CBA/Ca mice irradiated with 4 Gy gamma rays 24 h previously, but not immediately postirradiation, are able to induce DNA damage and apoptosis in nonirradiated bone marrow cells. The signaling mechanism involves FasL, TNF-alpha, nitric oxide and superoxide and macrophages are implicated as a source of damaging signals. Such delayed bystander-type damage demonstrates the importance of studying tissue responses subsequent to the radiation exposure as well as effects at the time of irradiation when considering the mechanisms underlying the consequences of radiation exposures.

Differential contextual responses of normal human breast epithelium to ionizing radiation in a mouse xenograft model.

Radiotherapy is a key treatment option for breast cancer, yet the molecular responses of normal human breast epithelial cells to ionizing radiation are unclear. A murine subcutaneous xenograft model was developed in which nonneoplastic human breast tissue was maintained with the preservation of normal tissue architecture, allowing us to study for the first time the radiation response of normal human breast tissue in situ. Ionizing radiation induced dose-dependent p53 stabilization and p53 phosphorylation, together with the induction of p21(CDKN1A) and apoptosis of normal breast epithelium. Although p53 was stabilized in both luminal and basal cells, induction of Ser392-phosphorylated p53 and p21 was higher in basal cells and varied along the length of the ductal system. Basal breast epithelial cells expressed ΔNp63, which was unchanged on irradiation. Although stromal responses themselves were minimal, the response of normal breast epithelium to ionizing radiation differed according to the stromal setting. We also demonstrated a dose-dependent induction of γ-H2AX foci in epithelial cells that was similarly dependent on the stromal environment and differed between basal and luminal epithelial cells. The intrinsic differences between human mammary cell types in response to in vivo irradiation are consistent with clinical observation that therapeutic ionizing radiation is associated with the development of basal-type breast carcinomas. Furthermore, there may be clinically important stromal-epithelial interactions that influence DNA damage responses in the normal breast. These findings demonstrate highly complex responses of normal human breast epithelium following ionizing radiation exposure and emphasize the importance of studying whole-tissue effects rather than single-cell systems.