British Journal of Radiology (2005) Supplement_27, 157-160
© 2005 British Institute of Radiology
doi: 10.1259/bjr/19036098
British Journal of Radiology Supplement_27 (2005),157-160 © 2005 The British Institute of Radiology
New basic science initiatives for improved understanding of radiation-induced multi-organ dysfunction syndrome (MODS)
D A Boothman, PhD1 and
J Reichrath, MD2
1 Departments of Radiation Oncology and Pharmacology, Case Western Reserve University, Cleveland, OH, USA and 2 Dermatology Clinic, The Saarland University Hospital, Homburg/Saar, Germany
Correspondence: David A Boothman, PhD, Professor, Department of Radiation Oncology, Associate Director for Basic Research, Co-Leader, Experimental Therapeutics Program, Ireland Comprehensive Cancer Center, Case School of Medicine, Case Western Reserve University, The University Hospitals, and the Cleveland Clinic Foundation, 10900 Euclid Avenue (BRB-326 East), Cleveland, OH 44106-4942, USA; E-mail: dab30@po.cwru.edu. Priv-Doz Dr Jörg Reichrath, MD, Dermatology Clinic, The Saarland University Hospital, 66421 Homburg/Saar, Germany; E-mail: hajrei@uniklinik-saarland.de
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Abstract
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Multi-organ dysfunction syndrome (MODS), which may lead to multi-organ failure (MOF), is a major clinical problem in patients following accidental exposure to ionising radiation (IR). Although inflammatory "one hit" and "two hit" models have recently been proposed to account for the development of MOF, we are only just beginning to understand the complex pathophysiology of IR-induced MODS and MOF. This paper summarises the main findings of a recent panel discussion at "The Advanced Research Workshop on Radiation-induced Multi-organ Involvement and Failure: a challenge for pathogenetic, diagnostic and therapeutic approaches and research", which was held on 14 November 2003 at the Science Conference Center at Schloss Reisensburg of the University of Ulm. This panel discussion focused on future molecular and clinical research priorities to improve our knowledge regarding possible initiation events following IR exposure that may lead to MODS. Additionally, specific recommendations for improving our understanding of the events that initiate and control MODS were summarised.
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Introduction
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Multi-organ dysfunction syndrome (MODS), which may lead to multi-organ failure (MOF), is a major clinical problem in patients following accidental exposure to ionising radiation (IR), for example after the Chernobyl or Tokai-mura accidents. Unfortunately, we are only just beginning to understand the complex pathophysiology of MODS and MOF induced by IR. Inflammatory "one hit" and "two hit" models have recently been proposed to account for the development of IR-induced MOF. In the "one hit" model, the initial insult is so massive that a systemic inflammatory response syndrome (SIRS) is induced, rapidly leading to MOF. In the "two hit" scenario, initially less severely injured patients eventually develop MOF as a result of a reactivation of their inflammatory response caused by an adverse and often minor intercurrent event. At first sight, these models are attractive because they appear to fit commonly observed clinical patterns in patients exposed to IR. On closer inspection, however, many important questions regarding the clinical outcome and the pathophysiological mechanisms underlying MODS and MOF remain unsolved. A major aim of a recent panel discussion at "The Advanced Research Workshop on Radiation-induced Multi-organ Involvement and Failure: a challenge for pathogenetic, diagnostic and therapeutic approaches and research", which was held on 14 November 2003 at the Science Conference Center at Schloss Reisensburg of the University of Ulm, was to discuss the known basic research on the possible initiating events in MODS. Future basic research direction was also discussed. Speakers within these sessions included: Drs A Brooks, N Daniak, D A Boothman, C Mothersill, M H Barcellos-Hoff, J E Trosko, L Roy and N Cordes. Significant discussion followed at the conclusion of these seminars regarding possible initiation events following IR exposure that may lead to MODS, as well as specific recommendations for improving our understanding of the events that initiate and control MODS. These recommendations for further molecular and clinical research priorities into the causes of MODS are summarised below.
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Initiation events
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Based on the research of a number of basic and clinical scientists presenting at the Workshop, two overriding and probably interconnected mechanisms for initiating MODS were discussed. These interrelated initiation events included: (a) the creation of extreme levels of reactive oxygen species (ROS) following IR exposure; and (b) the creation of cell debris (specifically the formation of lipid peroxides), possibly originating from IR-induced necrotic cells, as well as sloughing off of groups of apoptotic cells. Clearly, significant levels of ROS are formed after doses of IR above 4 Gy. The effects of ROS scavengers, however, have not been investigated in detail in animal models under conditions that trigger MODS. Furthermore, differential effects of ROS formation in various cell types (fibroblasts, epithelial cells or endothelial cells), before and after IR exposure, in whole animals will be required to elucidate the role(s) of ROS formation in MODS. Furthermore, there is a paucity of data on the effects of chemical and specific enzyme scavenging enzymes in the initiation of MODS. Given the short half-life of ROS, it seems clear that ROS formation alone cannot explain the distant "bystander effects" of high dose acute exposure to IR observed in patients following certain radiation accidents.
ROS-induced cell death responses
It was theorized by various investigators that ROS formation would trigger apoptotic or necrotic cell death responses in specific cell types, for example endothelial cells, which would initiate microvascular bleeding and initial tissue leakage events. It is also possible that groups of cells simultaneously undergoing apoptotic responses in endothelial and epithelial tissue could be initiated from the extreme IR exposures. Moreover, IR exposure could adversely affect normal cell phagocytic responses that would otherwise clear apoptotic cells in normal tissue cell turnover responses. Such cell death responses would lead to ROS-induced cell debris. It was also suggested that ROS exposure could initiate calcium (Ca2+) release responses from the endoplasmic reticulum (ER), and this could be a further molecular initiation effector of necrosis and apoptosis, eventually leading to MODS.
Cell debris
Cell debris arising from ROS-induced cell death responses in normal tissues and in endothelial microvasculature may then rapidly distribute throughout the irradiated tissue, with enhanced spreading resulting from microvascular destruction. This cell debris could: (a) overwhelm normal cell processes involved in the turnover of cell debris within the immediate organ and within other organs where cell debris would distribute; and (b) induce specific responses not present in normal tissues owing to the ROS-induced formation of unique cell debris species, including lipid peroxidation. Some specific factors that were proposed to be involved in cell debris clearing included: (a) the production of clusterin (also known as the testosterone-repressed prostate message-2 (TRPM-2) and apolipoprotein J (apoJ) protein), which acts as a molecular chaperone to bind and clear debris, and the megalin receptor, whose ligand includes the secretory form of clusterin (sCLU) and whose binding to the GP330 megalin receptor is thought to initiate lysosome-mediated clearance of cell debris [1, 2].
Altered gene expression
Several presentations suggested that IR damage could lead to new gene expression in certain cells, which would subsequently enhance or prevent the cell death responses discussed above. However, a systematic analysis of global gene expression responses after high dose acute IR has not been performed.
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Unresolved and relatively unexplored basic research questions
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The abovementioned discussions highlighted several deficiencies in our current knowledge of the basic molecular and cellular responses that act to initiate MODS, and these are discussed below.
More basic research funding is needed to delineate the initiation of MODS
(a) A clear role for specific ROS that induce MODS needs to be delineated. For example, is superoxide the key initiation species of free radical oxygen? Another important question is whether intervention is possible. Can free radical scavenging chemicals or overexpressing scavenging enzymes (e.g. superoxide dismutase (SOD)) be used to suppress, moderate or block MODS? (b) A role for Ca2+ release and induced ER stress needs to be examined. (c) A role for cell debris, and particularly specific types of cell debris (e.g. cell debris containing various levels of lipid peroxidation), needs to be determined. (d) Roles for necrosis and apoptosis need to be delineated. How does IR exposure affect normal cell phagocytosis? Are mice with altered apoptotic processes (pro- or anti-apoptotic responses) more or less susceptible to IR-induced MODS [3]? (e) There is an urgent need to characterise the pathophysiological mechanisms leading to reversible vs irreversible responses of various organ stem cells [4]. A better understanding of these mechanisms will enable development of improved protocols for the treatment of MODS and MOF. Recent studies have suggested that ex vivo expansion of autologous haematopoietic cells could be a therapy of choice for the treatment of bone marrow failure. The potential of a combined infusion of autologous ex vivo expanded haematopoietic cells with mesenchymal stem cells (MSCs) for the treatment of multi-organ failure syndrome following irradiation has been investigated in a non-human primate model [5]. In this study, homing of expanded MSCs was shown in numerous tissues following a severe multi-organ injury in primates. Localisation of transduced MSCs correlated with the severity and geometry of irradiation. A repair process was observed in various tissues. The plasticity potential of MSCs and their contribution to the repair process in vivo remain to be studied [5].
More basic research funding is required for the generation of animal models that are altered in MODS responses
(a) Small animal models (e.g. mouse) are desperately needed. Examples of informative models could include: (i) the wasted mouse model; (ii) tumour necrosis factor-
(TNF-), transforming growth factor-
1 (TGF-1), interferon- (IFN-) and clusterin knockout mice; (iii) known IR-sensitive mouse models, such as Ku70, Ku80, SCID (DNA-Pkcs) knockout mice; and (iv) investigation of the influence of genetic backgrounds, and elucidation of response modifiers. (b) Development of a large animal model is needed. It was clear from the discussion that a primate animal model was desperately required. However, prior investigations of dogs and monkeys could be useful and prior research at the National Institutes for Health (NIH) and Department of Energy (DOE) on large animal models should be made available in the appropriate databases for subsequent analyses. (c) Development of a systemic inflammatory response syndrome (SIRS) animal model is needed. Additional basic research on the differential effects of IR on various organ stem cells is needed.
The role of molecular effectors of acute IR-induced bystander effects need to be elucidated
(a) The role of gap junctions should be elucidated. (b) The role of exfoliation in the induction of MODS needs to be clarified; and (c) the role of secreted factors, such as TGF-1, in MODS responses needs to be clarified [6].
IR exposure of the extracellular matrix can modify both the pattern of gene expression and the phenotype of cells, which may result in cell transformation without direct mutation. IR exposure results in a range of dose–response relationships for changes in the number, types and patterns of gene expression. Low dose research using microbeams demonstrated that cells do not require a direct "hit" to result in significant biological alterations. These "bystander effects" demonstrate that "non-hit" cells respond with changes in gene expression, DNA repair, chromosome aberrations, mutations and cell killing [7].
The role of the immune system to both the "first hit" and "second hit" of IR needs to be characterised
(a) Extensive IR exposure inevitably produces a large amount of devitalised tissue. Accumulated data have shown that devitalised tissue may activate macrophages, lymphocytes and neutrophils, and may liberate oxygen free radicals, lysozymes, lipoprotein complexes, cytokines and eicosanoids, all of which are detrimental to the integrity of cells. (b) The role of infection for the "second hit" in the development of IR-induced MODS has to be analysed. Bacterial cell products such as endotoxin and exotoxin are the most potent immunoinflammatory factors identified. They induce the generation of pro-inflammatory mediators, and at the same time activate the pre-primed inflammatory cells, leading to a state of uncontrolled systemic inflammatory response that may finally culminate in MODS. However, it is still unclear whether infection is a major triggering factor for the development of IR-induced MODS.
Biomarkers of exposure to radiation need to be analysed and defined
(a) The role(s) of mRNA biomarkers need(s) to be elucidated [8]. (b) Dose-dependent effects of IR need to be characterised for various biomarkers, including cytokine and adhesion molecule pattern [7–10]. IR exposure of the extracellular matrix can modify both the pattern of gene expression and the phenotype of the cells, which results in cell transformation without direct mutation. (c) Research to link genomic instability with biological effects of IR also needs to be completed. Detection of radiosensitivity genes as markers of genetic susceptibility in individuals and populations can be used in epidemiological studies to determine how molecular changes may impact risk following IR exposure.
Specific cell and tissue repositories need to be established
(a) Existing animal tissues from prior DOE and NIH studies, as well as other sources, need to be catalogued and organised for availability, and the information organised into a MODS website [7]. (b) Existing human tissues from prior accident victims need to be catalogued and organised for availability, and the information organised into a MODS website.
Development of a complete human database, linked to the tissue repository (see above)
Do MODS or MOF induced by IR differ from MODS and MOF induced by other agents (e.g. trauma, sepsis)?
The question of whether MODS or MOF induced by IR differs from MODS and MOF induced by other agents (e.g. trauma, sepsis) must be answered. In this context, the particular role of IR-induced DNA damage and the role of the DNA repair system has to be addressed. Animal models, e.g. DNA repair-deficient knockout mice, represent a valuable tool to analyse the importance of IR-induced DNA damage for the development of MODS and MOF. Many of these mouse models have already been established and they should enable us to analyse systematically the importance of different kinds of DNA damage (e.g. double-strand breaks), and of specific components of the DNA repair system for the development of MODS and MOF.
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Conclusions
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The above recommendations developed by this panel discussion will undoubtedly require significant resources that should be established from the world community. Clearly, there is a significant void in our understanding of the effects of acute IR exposure on human and animal tissues. The development of animal models will be necessary for subsequent delineation of molecular effectors mediating MODS. It can be speculated that these basic and clinical research initiatives will eventually greatly improve our knowledge regarding IR-induced MODS. These studies will finally answer the question of whether IR-induced MODS is characterised by IR-specific features, or whether it can be compared with MODS induced by other agents. The US Department of Energy's Low Dose Radiation Research Program is an example of ongoing activities that will lead to a better understanding of the biological effects of IR, including MODS. This programme is a 10-year activity currently funded at $21 million per year. It focuses on biological responses to low doses (<0.1 Gy) of low-LET (linear energy transfer) IR [7]. A similar initiative for understanding high dose IR exposure responses at the cellular and molecular levels in vivo should be developed for better understanding and intervening in high dose IR exposures, especially those that would unfortunately accompany terrorist attacks. In the USA, it is recommended that the Office of Homeland Security undertake such an initiative. The overall goal of these programmes is to provide sound scientific bases for radiation protection standards and potential future intervention strategies. These programmes (would) support basic research that combines modern genomic, molecular and cellular techniques with recent advances in scientific instrumentation. These combinations make it possible to detect responses and to test paradigms associated with the mechanisms of low dose radiation action not previously measurable or testable [7]. At present, it is not possible to determine how future research initiatives will influence current radiation standards. However, the goal of these research programmes will ensure that radiation standards are set using the best scientific data available, and that they are adequate and appropriate for the protection of workers and the public. [7].
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References
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Abstract
Introduction
