Advances in Radiation Biology: Effect on Nuclear Medicine Antone L. Brooks, PhD,* and Lawrence T. Dauer, PhD† Over the past 15 years and more, extensive research has been conducted on the responses of biological systems to radiation delivered at a low dose or low dose rate. This research has demonstrated that the molecular-, cellular-, and tissue-level responses are different following low doses than those observed after a single short-term high-dose radiation exposure. Following low-dose exposure, 3 unique responses were observed, these included bystander effects, adaptive protective responses, and genomic instability. Research on the mechanisms of action for each of these observations demonstrates that the molecular and cellular processes activated by low doses of radiation are often related to protective responses, whereas high-dose responses are often associated with extensive damage such as cell killing, tissue disruption, and inflammatory diseases. Thus, the mechanisms of action are unique for low-dose radiation exposure. When the dose is delivered at a low dose rate, the responses typically differ at all levels of biological organization. These data suggest that there must be a dose rate effectiveness factor that is greater than 1 and that the risk following low–dose rate exposure is likely less than that for single short-term exposures. All these observations indicate that using the linear no-threshold model for radiation protection purposes is conservative. Low-dose research therefore supports the current standards and practices. When a nuclear medical procedure is justified, it should be carried out with optimization (lowest radiation dose commensurate with diagnostic or therapeutic outcome). Semin Nucl Med 44:179-186 C 2014 Elsevier Inc. All rights reserved.

Introduction

T

he concern for adverse health effects of ionizing radiation has been an issue since the discovery of radiation. Years of intense research were conducted on the biological responses induced by high radiation doses delivered at a high dose rate. The A-bomb studies1-3 provide the primary link of all the basic research to human data. In contrast to exposure to most other environmental agents, there are extensive data sets on the response of humans to ionizing radiation. These human data made it possible to estimate the risks associated with early lethal effects of high doses of radiation, as well as to estimate the late-occurring effects such as the induction of mutations and cancer. To extrapolate the risk from high doses to that in the low dose (e.g., less than 100 mSv) and dose-rate range, models were developed and used. The most frequently used model is

*Washington State University, Kennewick, WA. †Memorial Sloan Kettering Cancer Center, New York, NY. Address reprint requests to: Antone L. Brooks, PhD, Washington State University, Kennewick, WA 99338. E-mail: [email protected]

http://dx.doi.org/10.1053/j.semnuclmed.2014.03.004 0001-2998/& 2014 Elsevier Inc. All rights reserved.

the linear no-threshold (LNT) model. The basis of this model has been reviewed4 and is based on mutation and DNA damage, which indicated that response for these end points increased linearly with dose and had no threshold. Thus, the assumption is that each and every ionization has the potential to cause a small, but predictable, increase in risk. This model has been deemed to be a prudent approach that may be especially useful for controlling radiation exposure.5-7 Although the LNT model is very useful in setting standards to limit radiation exposure, recent data suggest that it does not appropriately reflect risk in the low-dose region.8-11 A scientific controversy continues to simmer between those who accept the LNT and those who do not. Before research was conducted in the low-dose region, there were several well-accepted paradigms in radiation biology with few data available to challenge them. The “hit theory” was well developed and suggested that the number of cells traversed by radiation with energy deposited in them would determine risk. This information could then be used to predict the hazard and biological response. With this theory, only the cells “hit” with energy deposited in them responded to the exposure. The hit 179

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180 number was carefully calculated and used to predict risk. The second paradigm was that ionizing radiation always produced adverse effects and each ionization increased the risk (ultimately providing the basis for the LNT). Finally, it was assumed that DNA damage and mutations were the primary radiation-related cause of cancer and increased as a linear function of radiation dose. All 3 of these older radiation biology paradigms have not been supported by data in the low-dose region using modern exposure technology combined with newer techniques in cell and molecular biology. This combination of physical advances and biological techniques has made it possible to measure radiation-induced changes at the cellular and molecular levels at low doses and dose rates, which were not possible in the past. Research in the low-dose region resulted in 3 major unique low-dose and low–dose rate observations, which suggested that the field of radiation biology needed to reevaluate and question old paradigms. These observations include the discovery of bystander effects, adaptive responses, and genomic instability. The mechanisms behind these observations are discussed and further research needs are identified. Several effects of these new data and research in the low-dose region are discussed with respect to nuclear medicine and molecular imaging.

Bystander Effects The ability to focus on and irradiate individual cells with known numbers of particles or doses from low–linear energy transfer radiation made it possible to discover the bystander effects. With these tools, individual cells could be exposed, and the biological responses observed in “hit” cells could be compared with those in cells without energy deposited in them. Microbeams were designed to accurately and rapidly expose individual cells to known amounts of energy and to a wide range of different radiation types (alpha particles, x-rays, protons, and electrons).12-15 The development of this equipment paved the way for many studies on bystander effects, demonstrating the importance of both direct cell-to-cell contact and communication through the release of substances from the hit cells that modify the sensitivity and responses in nonhit cells. It was observed that not only the “hit cells” but also the nonhit cells were responding.16 Thus, the target for radiation effects was much larger than an individual cell.17 These effects were observed both in vitro16 and in vivo,18 which suggested that not the single cell, but the whole tissue or organ is responsible for the biological response to radiation, especially low doses.19

Adaptive Responses Adaptive responses were first discovered in human lymphocyte cells. Cells were exposed to low doses of tritium followed by exposure to high radiation doses. The low “priming dose” from tritium decreased the frequency of chromosome aberrations produced by the subsequent high doses of x-rays to below that observed without the priming dose.20 Such studies demonstrated that all radiation exposures do not increase the

amount of biological damage. The low dose caused the cells to change their gene expression profile, to adapt, and increase their radiation resistance. This adaptive response has been reviewed and shown to exist for a wide range of radiation exposures and biological end points.21 The prime argument against a protective adaptive response for radiation has, in the past, involved the way that radiation deposits energy. Radiation deposits its energy in discrete bundles at random sites in cells and molecules. As a result, it was suggested that radiationinduced damage could not be compared with damage from chemicals or stress, where all molecules and cells in a tissue, organ, or organism receive the exposure uniformly and have the potential for being affected. However, extensive research in the low-dose region demonstrated the role of bystander effects following radiation exposure. These effects show that even though the energy is deposited in random, defined sites and the initial DNA damage increases as a linear function of exposure, the biological processing of these changes are not linear and radiation responses are not limited to the individual cells where the energy is deposited. The whole organ or tissue responds to the insult in a way that is similar to that seen for chemicals.22 Radiation delivered before many mutagenic chemicals results in an adaptive response. The mechanisms involved in this nonlinear processing of radiation-induced change are being defined and support the existence of protective adaptation.8 The classic adaptive response has been demonstrated for several different biological end points, including the induction of apoptosis,23 cell killing and radiation hypersensitivity,24 micronuclei,25 gene expression,26 and mutations.27 The second type of adaptive response was the observation that low doses of radiation can decrease the biological response to levels below the spontaneous background level. A number of biological systems were developed to measure the influence of low doses of radiation on the spontaneous frequency of biological alterations and changes related to cancer induction. The most widely cited example of these systems measured the frequency of cell transformation as an end point. Many studies were conducted measuring cell transformation. These showed that low doses of ionizing radiation decreased the spontaneous frequency of cell transformation below that observed in control cells receiving no radiation exposure.28-30 It was noted that the genetic background of the biological materials used in studies on adaptive response plays an important role in the response.31 Many individuals tested for the induction of adaptive responses were “responders” and others were not. Genetic background was shown to play a critical role for each of these observations: bystander effects, adaptive responses, and genomic stability.

Genomic Instability Multiple genetic changes are present in most solid cancers. These reflect the loss of genetic stability of the cells. This loss of genomic stability seems to be one of the hallmarks of the cancer process32 and is critical as the cells take on a cancer

Advances in radiation biology phenotype. However, it is not known if the genomic instability is actually induced by an agent (such as radiation) that “caused” the cancer or is simply a reflection of the cancer process where cells have escaped genetic control present in normal tissues. Radiation exposure has been shown to induce genomic instability. Following short-term high-dose radiation exposure, cells can make multiple, apparently normal cell divisions, then a fraction of the irradiated cells can lose control of their genome. This radiation-induced change was unappreciated in the past. This “genomic instability,” or loss of genetic control, results in multiple genetic changes in the cells. Genomic instability has been defined as the increased rate of acquisition of genetic alterations in the progeny of an irradiated cell.33 These changes are similar to those observed a short time after exposure. One of the earliest reports of genomic instability in vitro was related to the induction of DNA damage and its role on the induction of chromosome instability.34 Radiationinduced late-occurring genomic instability was reported using an in vivo-in vitro method in the bone marrow of mice that were exposed to 239Pu.35 The frequency of chromatid aberrations was increased, showing that the aberrations were not being produced by direct exposure to the α particles from the internal emitter. Similar changes were identified in primary cultures of irradiated human bone marrow cells.36 A wide range of different end points was used as a measure of the induction of genomic instability. Radiation-induced genomic instability was dependent on the genetic background being studied. When 2 different strains of mice that were either sensitive or resistant to radiation-induced breast cancer were studied, it was noted that radiation did not induce genomic instability in the resistant mice, but that it seemed to play an important role in radiation-induced breast cancer in the sensitive strain.37 Studies have been conducted suggesting that genomic instability cannot be induced in stable normal human cells. Genomic instability could not be demonstrated in human cells (RKO cells) in cultures,38 as well as in other normal human and animal cell lines.39 Genomic instability was not detected in severe combined immunodeficiency mice exposed to wholebody low doses of radiation.40 The failure to demonstrate genomic instability in normal cells suggests that it may be part of the process of the cancer development and not induced by the radiation insult. If this is the case, genomic instability would not be detected in A-bomb survivors that received the radiation exposure but had not developed cancer. There has been no genomic instability demonstrated in lymphocytes of the A-bomb survivors.41 Studies have been published to try to resolve the differences in the induction of genomic instability seen in experimental systems and the failure to demonstrate it in normal cell populations and in human populations.42 It was also determined that the frequency of radiation-induced genomic instability could be decreased by previous exposure to a low dose of radiation, a phenomenon indicative of an adaptive response.38 Thus, genomic instability and adaptive response

181 appear to be closely related. Studies were conducted to determine the cellular target for the induction of genomic instability. Induction of chromosome instability using different dose and dose rates suggested that the nucleus was the target for genomic instability.43 As genomic instability can be induced by exposing a single chromosome to ionizing radiation,44 the target for the induction of genomic instability has been demonstrated to be the cell nucleus.

Genetic Susceptibility Research has demonstrated that adaptive response, bystander effects, and genomic instability are all related and seem to be controlled by similar biological processes. The Figure shows the relationship between these processes and the fact that genetic sensitivity plays a key role in all of them. Cancer has long been known to have a genetic component, as many families are cancer prone. The role of genetic background on radiation-induced cancer was carefully reviewed.31 This report demonstrates that genetic differences in many molecular, cellular, and experimental animal systems support the role of genetic background on biological responses to ionizing radiation.

Mechanisms of Action Research in the low-dose region was reviewed by the National Academy of Science in The Biological Effects of Ionizing Radiation (BEIR) VII Phase 2 Report7 and by the French National Academy.10 These reviews were conducted to try to use such data to influence radiation standards. The French academy used cell and molecular data to make the argument that there was a nonlinear dose-response relationship and that linear extrapolation of risk from high doses would overestimate the risk induced by low doses of radiation. However, after a brief discussion of each of these observations (bystander effects, adaptive responses, and genomic instability), the United States National Academy of Science concluded that, without a better understanding of the mechanisms of action, it was not possible to use cell and molecular studies in standard settings.7 Since the publication of the BEIR VII report, extensive research has

Figure Biological responses induced by low doses of radiation.

182 been directed toward increasing the understanding of the mechanisms of action at low doses and dose rates.8 A brief discussion of these mechanisms is included to demonstrate the potential effect of low-dose research on nuclear medicine.

Radiation-Induced DNA Damage Radiation has been shown to produce more complex DNA damage than that which occurs spontaneously.45 This damage is thought to be more difficult to repair. However, data from molecular biology demonstrated that even though DNA damage is induced linearly as a function of radiation dose, the processing of this damage, the cell-cell signaling induced by the damage, and the biological consequences of the damage change as nonlinear functions of dose. Low doses of radiation were thought to be involved in modification and repair of DNA damage. Early studies with gene expression failed to demonstrate that low-dose radiation exposure modified expression of DNA repair genes.46 Thus, in the low-dose region, direct induction and repair of DNA damage may not be as important in the total risk from low doses of ionizing radiation as the signaling processes induced by the damage. At very low doses, there was no DNA repair detected.47 The discovery of repair foci suggested that there was a threshold for DNA damage below which repair was complete.48 When a dose as high as 400 times background was delivered at a low dose rate, it was not possible to detect any DNA damage.49 The same dose delivered at a high dose rate resulted in marked DNA damage. This supports the concept of DNA repair and indicates that the resulting risk from low–dose rate exposure is less that than from the same dose delivered at a high dose rate.

Radiation-Induced Changes in Gene Expression Early research in the field of molecular radiation biology focused on radiation-induced change in gene expression. Techniques were developed that made it possible to measure changes in gene expression in tens of thousands of genes at the same time. Exposures to radiation resulted in up and down regulation of many genes and metabolic pathways. It was determined that the genes modulated by low-dose radiation were not the same genes that high doses of radiation turn on and off.26 This study was one of the first that indicated that cells could indeed detect and respond to low doses of radiation (0.01 Gy). As additional studies were conducted, it became obvious that the number and types of genes with radiationinduced changes in expression were related to stress responses and were dependent on both radiation dose and dose rate.50,51 Many unique genes that are either upregulated or downregulated by low doses of ionizing radiation are not modified by high doses.26 Modification of gene expression by radiation alters many chemical factors and metabolic pathways within the cell that are responsive to low doses of radiation. These pathways have been studied and they influence the responses in cells both with and without energy deposited in them. These effects are important in the subsequent development of biological changes measured in a wide range of systems

A.L. Brooks and L.T. Dauer because they may modify response that either decreases or increases radiation-related risk or effect. For many of these pathways, it is currently not possible to determine which way (beneficial or detrimental) these influences will go. However, there is evidence that supports the observations of protective effects induced by low doses of ionizing radiation.22,28-30

Cell and Tissue Responses to Low-Dose Radiation An important pathway involving radiation-induced changes includes transforming growth factor-β. This pathway was shown to be important in the expression and modification of direct and bystander-induced radiation-induced damage both in vitro16 and in vivo.18 These relationships were extended from the role of transforming growth factor-β at the single-cell level to interactions at the tissue level. This helped demonstrate that it takes a tissue to make a tumor52 and suggests that tissue environments are important to consider as nuclear medicine imaging or therapy techniques are implemented. It has been postulated that radiation-induced genomic instability is important in the generation of cancer. As genomic instability occurs at a very high frequency, it was critical to look for targets larger than traditional radiation-induced gene mutations for the induction of genomic instability. As research on telomeres has advanced, it has been linked to the induction of genomic instability induced both in cell systems and in animal model systems. The telomere provides a larger target and the higher frequency needed to explain radiation-induced genomic instability. Studies with a mouse model (K-ras p53) on the induction of lung cancer indicated that telomere dysfunction promotes genomic instability as well as increasing the metastatic potential for the cancers.53 Review of literature on the interrelationships between genomic instability and telomere dysfunction suggests that telomere dysfunction is one of the major driving forces in radiation-induced genomic instability.54 Past research, at higher radiation doses, suggested that cell killing was a simple function of dose. However, with better techniques to detect cell killing, it was determined that cell killing increased rapidly as a function of low-dose exposures (hyperradiation sensitivity). As the dose increased, the cells became radiation resistant and the cell-killing slope decreased as the dose increased. This was called induced radiation resistance (IRR). Subsequently, numerous studies were conducted to help define the mechanism of action involved in these unique low-dose radiation cell-killing responses. The literature on low-dose hypersensitivity and radiation-induced resistance has been carefully reviewed.55 These phenomena are very important observations relative to the shape of the doseresponse relationships in the low-dose region. If low doses of radiation increase cell killing, this treatment could be eliminating cells from the population that may be at higher risk for the induction of cell transformation. Conversely, low-dose hypersensitivity and radiation-IRR to cell killing could increase cell proliferation in the low-dose region and protect cells that are sensitive to radiation-induced cell transformation in the medium-dose range. It could be postulated that this

Advances in radiation biology combination could result in an increase in cancer risk in the low-dose region. The process of apoptosis, or programmed cell death, has been recognized for a long time and plays a critical role during embryonic development. As cells differentiate and form organs, many of them are programmed to die. For example, in the formation of the hands, the cells between the fingers die on a preprogrammed schedule, thus allowing the fingers to separate. During the early days of radiation biology, it was not widely recognized that radiation produced apoptosis. Cells were thought to be killed by radiation through either the processes of mitotic death or necrosis. Apoptosis has been demonstrated to be a frequent event following exposure to low doses of ionizing radiation and seems to be an important part of the adaptive responses observed following these exposures. Experimental conditions that decreased the frequency of apoptotic cells increased the frequency of mutations in the adenine phosphoribosyltransferase (APRT) gene in mice repeatedly exposed to ionizing radiation.56 Radiation resistance can also be increased by factors that modify cell cycle and reduce apoptosis.57 Such studies suggest the potential for apoptosis to be protective against late-occurring diseases such as cancer. This observation has been related to the induction of protective or adaptive responses in the low-dose region of the dose-response curve. One of the most important observations of apoptosis in radiation biology is the suggestion that low doses of radiation can trigger biochemical and signaling pathways in bystander cells that result in selective apoptosis of cells that are transformed and may be in the process of changing from normal to cancerous cells.23 If low doses of radiation can selectively cause transformed cells to undergo programmed cell death, then it has been postulated that the cancer risk can be directly reduced.58 This would help explain experimental results in the study of cell transformation where low doses of ionizing radiation decrease the frequency of transformed cells to less than the levels seen in controls.59 Similar results on the induction of mutations could be explained by this apoptosisrelated process.60 Such biology made it possible to develop models that show nonlinear low-dose responses with low doses producing less cancer risk than is present in a nonexposed population.61 The reactive oxygen status (ROS) of the cells and oxidative stress play a critical role in the development of radiation-related disease. This is especially true when the dose is given at a low dose rate.62 Following exposure to high doses delivered at a low dose rate, the cancer frequency in Beagles was not increased to more than that seen in the controls, even after doses as high as 20 Gy. As the doses increased, excessive cell killing, tissue disorganization, and chronic inflammatory diseases were induced and the lung cancer increased rapidly. In the low-dose region, it was determined that low doses of radiation activated protective mechanisms. These involved changes in mitochondria, decrease in the ROS status of the cells,63 and the modification of radioprotective chemicals, including the well-known SH-containing radioprotective chemicals.64 Data from such research provided a likely link between low doses of radiation and the observed biological

183 responses such as adaptive response, bystander effects, and genomic instability. Additional research in this area needs to be conducted to evaluate how such mechanisms might affect nuclear medicine approaches to imaging and treatment. Studies on epigenetic changes induced by radiation in utero have been conducted using the Avy strain of mice.65 It has been demonstrated that diet can change the phenotype of these mice. The brown mice are thin, show no increase in diabetes and are resistant to cancer. The yellow mice induced by changes in the diet are obese, diabetic, and have a high frequency of cancer. When female mice were exposed to low doses of radiation at the critical stages of development, which showed changes in offspring, there was a marked shift from yellow to brown offspring. Such data suggest the low doses of radiation reduce the epigenetic effects, increase lifespan, and reduce cancer incidence in the offspring. Radiation in this system was protective. Epigenetic effects of radiation remain an area where additional data are needed.

Summary Mechanisms Implications for Nuclear Medicine This article demonstrates that extensive research has been conducted on the mechanisms involved in the radiationinduced responses in the low-dose region. As the result of this research, additional data are now available to explore the magnitude of the risks from radiation-induced cancer in the low-dose region. Important observations that clarify the mechanisms of action in the low-dose region are summarized here to help those in the medical field understand the potential effect of this research on nuclear medicine.

 Biological systems can detect and respond to very low doses of radiation.

 Direct damage to DNA is an important part of the

 

 



radiation response and increases as a linear function of radiation dose (i.e., the laws of physics hold: increases in energy absorbed increases damage). The biological processing of the damage and the signaling that results from it produce many nonlinear processes. There are multiple genes, chemicals, and metabolic pathways induced by low doses of ionizing radiation that have marked influence on the biological outcome of the exposure. Several of these chemical and metabolic pathways seem to be protective against radiation-induced damage. Low doses of radiation modify the ROS free radical status of the cells. Such modifications are suggestive of protective effects of radiation seen in adaptive and protective responses. Higher doses increase the ROS status of the cells to produce responses that are known to damage cells and increase cancer risks. In the low-dose region, direct radiation effects and the signaling pathways modify cellular responses, including cell transformation, mutations, chromosome aberrations, telomere function, and cell cycle delay, which seem to be

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protective. High doses change all these same end points in a way that would be predicted to be harmful to the organism. Radiation can induce hypersensitivity in the low-dose region. As the dose increases, there is an IRR. Hypersensitivity may be protective by eliminating damaged cells, while induced resistance could be detrimental by protecting damaged cells and allowing them to remain in the population. There is evidence that low doses of radiation produce selective apoptosis in cells that are transformed. This provides a major mechanism of action in the low-dose region and may help explain many of the adaptive responses observed. Extensive research on the role of apoptosis in radiation risk demonstrates a potential protective role. Research has determined that genetic background plays a critical role in all the responses observed in the low-dose region. Early data on the role of radiation-induced epigenetic changes show protective effects in the limited systems investigated.

Research in the low-dose region has several important effects on nuclear medicine. Firstly, the research provides extensive data that suggest that the LNT model used to estimate risks for general radiation protection purposes may overestimate the real risk at low doses and dose rates. Although such observations may not result in significant changes to current radiation protection standards, they do provide extensive data sets that increase our confidence that we have not underestimated the risk in the low-dose region and are, in fact, implementing a prudent approach. Secondly, continuing research on the influence of dose rate and dose distribution suggest that, for many tissues, there are marked dose-rate and dose-distribution effects.62 Therefore, it is likely that nuclear medicine procedures that deliver lower doses and dose rates and focus only on a fraction of the body present risks that are lower than estimated by current radiation protection models. Additional regulatory discussions and research are underway to determine the adequacy of the dose, dose-rate effectiveness factor, and the tissue weighting factors,5 which are currently derived from the A-bomb data where a whole-body dose was delivered at a high dose rate. Many tissues, such as bone,66 lung,62 and liver,6 have been shown to be very radiation resistant when the exposure is confined to that tissue. These data need to be considered in evaluating tissue weighting factors. In nuclear medicine, especially when the dose is delivered at a low dose rate to a specific target tissue, the overall radiation risk is likely reduced below that calculated using current standard methods. Thirdly, new research in the low-dose region has produced data that require changes in basic paradigms in radiation biology. Radiation-effects models must consider adaptive responses, the hit theory must consider bystander effects, and the DNA mutation theory of cancer needs to consider the induction of genomic instability. As such, it is clear that a more complex response model needs to be developed, especially at

low doses and dose rates.8 Priorities should be given to evaluating radiobiological studies with a systems approach at all levels of biological organization (molecular, cellular, tissue, organ, and whole animal.67 This systems approach in nuclear medicine rapidly transitions to increasingly targeted molecular treatment approach.68 Understanding local, regional, and whole-body effects is essential. Finally, as in all applications of radiation in medicine, nuclear medicine imaging or therapy must be justified based on the benefit and risk assessment.69 As most exposures are in the mSv range in nuclear medicine,70 the risk to an individual is very low. It is important to conceptualize the magnitude of the risk. It is critical to note that the frequency of cancer in the US population is about 40% and that approximately 25% of the population currently dies of cancer. The added healthrelated risk, using the possibly conservative LNT model, suggests that radiation risks increase by only approximately 5% per 1000 mSv. As the evidence suggests that this model overestimates the real risk, this exercise should be helpful to the patient that needs the procedure. The bottom line is that the risk from low-dose radiation is very small and the benefits are large.71,72 In fact, a review of recent radiobiological research emphasizes that physicians and patients should not forgo a justified73-75 medical radiation procedure because of the fear of late-occurring radiation effects.76

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