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246 National Food Safety Toxicology Center, Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI 48824, USA
Correspondence: J E Trosko. E-mail: james.trosko@ht.msu.edu
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Abstract |
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Introduction |
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0.01 mSv day–1) and below high dose radiation (150 mSv day–1) [1]), a re-examination of this fundamental paradigm must be performed. If any health effect following low level acute or chronic radiation exposure is measured, there must be underlying biological effects that can be measured. However, even if one can measure some biological effects after low level radiation exposure, it does not necessarily mean that there will be negative health effects.
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Biological consequences of ionising radiation |
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A second concept that is frequently ignored is the fact that in a multicelled organism, there exist three basic cell types: the pluripotent stem cells; their progenitor cells; and the terminally differentiated cells. Assuming their equal sensitivities to ionising radiation could lead to erroneous risk assessments. A third concept that has been ignored has been the fundamental concept of homeostatic control of cellular functions, namely cell communication mechanisms. Lastly, the concept of the hierarchical nature of a multicellular organism must be taken into account in order to translate any molecular and cellular consequences in an intact organism into a potential health effect [4].
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Health consequences of low level radiation exposure |
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It becomes difficult to draw hard conclusions from any of these studies for a number of reasons. Assuming that the low level exposure was given acutely, it then becomes necessary to bring in the concept of the multistage, multimechanism basis of carcinogensis. The reason is simple: "No one thing ‘causes' cancer". The "initiation/promotion/progression" concept of carcinogenesis clearly illustrated the fact that multiple distinct molecular, cellular and physiological events must take place within the biological architecture/milieu of different cell types in tissues [5].
Therefore, the first experimental question that must be answered is, "Is low level ionising radiation an effective ‘initiator’?" Next, "What might be the underlying molecular mechanisms that leads to the initiation event?" and "Can ionising radiation bring about this event?" With experimental molecular oncological, in vitro, whole animal and human epidemiological data suggesting multiple genetic and epigenetic events having to be altered during the carcinogenic process (activating mutator genes, activating oncogenes, inactivating tumour suppressor genes, etc. [6]), it becomes impossible, based on current knowledge of the speculated roles of ionising radiation, that a single acute exposure to low levels of ionising radiation could bring about all the mechanisms and steps required to convert a normal cell to a malignant, invasive and metastatic cancer cell.
Operationally, "initiation" occurs after an animal is exposed to an agent that induces an "irreversible" change in a single cell [7]. This initiated cell appears to be blocked in its ability to terminally differentiate and to apoptose normally, as well as to be able to proliferate indefinitely upon stimulation by mitogens or by compensatory hyperplasia due to death of surrounding cells [8]. At the whole animal level, agents that can induce DNA damage and mutations appear to be effective "initiators", for example as ultraviolet (UV) light, a known effective point mutagen and skin cancer inducer both in rodents and human beings [9] at doses that are associated with significant cell killing. At low doses in animals, where little or no cell killing is induced, the acute UV light radiation must be followed by sustained exposure to a promoting condition or agent.
The promotion phase of carcinogenesis is defined as that process which allows a single initiated cell to clonally expand both by stimulation of cell proliferation and inhibition of apoptosis [10]. If, at low doses, the initiated cell is restrained from cell proliferation by "contact inhibition" [11], that cell will remain in this early "pre-malignant state" during the organism's normal life span. Agents that can inhibit both contact inhibition and apoptosis are called promoters and can contribute to the clonal expansion of this initiated cell. Tumour promoters (e.g. phorbol esters, ochratoxin), both synthetic (DDT, Phenobarbital) and endogenous (growth factors, hormones), which do not damage genomic DNA or induce mutations, can trigger signal transduction-altered gene expression at non-cytotoxic levels [12]. Promoting conditions, such as sustained hyperplasia due to irritants, compensatory hyperplasia due to surgery, or tissue damage due to cell killing by either mutagenic agents at cytotoxic doses or non-mutagenic chemicals (e.g. carbon tetrachloride, chloroform) could cause a surviving initiated cell to repopulate the dead or removed cells. In the previous example of UV radiation-induced skin cancer, compensatory hyperplasia, caused by the killing doses of UV light, caused the surviving mutated or initiated cell to multiply.
The progression phase of carcinogenesis is that phase whereby the initiated cell, having been multiplied by some external promoting agent or condition, has accrued enough genetic or epigenetic changes so that the phenotype of one of the cells in the clone has become independent of external mitogenic stimuli and can endogenously escape contact inhibition of the surrounding normal cells. During this phase, the additional altered phenotypic changes can occur during continued proliferation that confers invasive, metastatic and angiogenetic abilities to these cells.
Can ionising radiation at low levels induce DNA damage, induce mutations and "initiate" carcinogenesis [13]? Little or no evidence has been generated testing the hypothesis that low level, acute ionising radiation followed by some tumour promoter or promoting condition can be an initiator, although a few studies showed that at higher dose exposures it appears to be a weak initiator [14–16]. After an acute low dose exposure, it has been shown that the final incidence of solid neoplasms is indistinguishable from the spontaneous incidence [17]. In terms of the molecular and cellular evidence of DNA damage and mutations, ionising radiation has long been assumed to be a rather weak point mutagen compared with its ability to induce single- and double-strand breaks [18]. Recent reports have, however, suggested that double-strand breaks can occur in human cells exposed to very low X-ray doses [13]. With the known observation that ionising radiation is efficient at neither "immortalising" human fibroblast in vitro [19] nor of inducing in vitro neoplastic transformation (consistent with the multistage, multimechanism processes of carcinogenesis), these authors speculate that these unrepaired lesions are contained in cells that die. This then sets the stage for newer concepts in radiation and chemical toxicology and carcinogenesis, namely the "bystander", "adaptive response" or "hormesis" effects, and genomic instability (see later).
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Hallmarks of carcinogenesis: where might ionising radiation fit in? |
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To answer that question, several concepts seem to be absent, not only in the characterisation of the cancer hallmarks, but with many analyses of the origin of cancers. The two main concepts are: (a) the stem cell theory of cancer; and (b) the role of gap junctional intercellular communication (GJIC) [21]. Whilst cancer has been described as a "disease of differentiation" [22], a stem cell disease [23], or as "oncogeny as partially blocked ontogeny" [24], most newer ideas have ignored these more holistic and biologically-based hypotheses. Potter stated that "The cancer problem is not merely a cell problem, it is a problem of cell interaction, not only within tissues, but also with distal cells in other tissues" [24].
How might low dose ionising radiation play a role in the conversion of a single normal cell to accrue all these hallmarks of cancer? Clearly, a single low dose exposure at the start of the multistage, multimechanism process cannot induce all the genetic and epigenetic changes needed to bring about all these hallmarks, even with the rather new hypothesis of radiation-induced "genomic instability".
Again, assuming that the low level radiation exposure is acute, above background levels and below the 0.15 mSv day–1 high dose level, to extrapolate back from a population of atomic bomb survivors there are several other factors that have been ignored. First, it is assumed that age itself prior to exposure to a low level dose must account for the number of "spontaneous" initiated cells. Thus, the spontaneous initiated cell number would be expected to increase with age, as well as with exposure to other initiating agents. Second, one of the most powerful risk reducers to chronic diseases, including cancers in experimental animals, is caloric restriction. Could the reduced caloric intake of the atomic bomb survivors, prior to and after the exposure, have lowered the frequencies of tumours so far accrued? Third, at the time of exposure, the acute ionising radiation at the different doses would have been expected to affect different stages of either normal cells (see later discussion of stem cells) or of other spontaneously initiated cells that either were or were not clonally expanded (this would alter the probability of the radiation effects).
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Role of GJIC in radiation carcinogenesis |
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GJIC is the integrating link in the metazoan. It is mediated by the formation of a membrane-associated hemi-channel structure, the connexon, which consist of six self-organised proteins, coded by at least 20 connexin genes [26]. Following synthesis, transport, and assembly into functional gap junctions after multiple connexons align with the juxtaposed connexons of the neighbouring cells, regulatory ions and small molecules (below 1000 Da) can freely equilibrate [27]. Upon coupling by these gap junctions, depending on the types of connexins that make up the connexons, synchronisation of either electrotonic or metabolic functions within a tissue between homologous or heterologous cell types can take place [28]. Most normal cells (endothelial, epithelial, fibroblastic) express connexin genes and can adaptively have functional GJIC. Adaptive upregulation or downregulation of GJIC is necessary for tissue growth, wound healing, differentiation, synchronised contraction of electrotonic-dependent cells, apoptosis, and adaptive responses of differentiated cells [29]. A few normal cells do not express connexin genes or have functional gap junctions, e.g. free-existing cell types, such as blood cells, neutrophils or many tested pluripotent stem cells [30].
What is relevant to the issue of carcinogenesis is the observation that an ignored hallmark of cancer is either the absence of the expression of any connexin gene or the dysfunction of GJIC in cancer cells [21]. Since cancers do not have growth control, do not terminally differentiate, are immortal and have the ability to escape apoptosis, at the same time that they do not have functional GJIC, suggests a possible "connection" between GJIC and cancer. This link was first hypothesised by Loewenstein [31] and later modified by others [5]. The evidence supporting this linkage has been reviewed [8, 29].
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Role of stem cells in radiation carcinogenesis |
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The fact that cancers appear to be clonally derived, are either very embryonic-like or are not terminally differentiated [23], and are immortal suggests that they must have been derived from a single cell that was capable of unlimited cell proliferation, either because it was naturally immortal or it could be induced to be immortal very easily. The prevailing paradigm has it that carcinogenesis starts when a normal, "mortal" cell is "immortalised" [34]. After which, this immortal cell can be neoplastically transformed. An alternative hypothesis is that, starting from a naturally immortal cell, namely the pluripotent stem cell, the first step of carcinogenesis is to block "mortalisation" or terminal differentiation of the immortal stem cell [31]. Only then can this cell be neoplastically transformed by accruing all the hallmarks needed to become a cancer cell.
Stem cells have been described as cells capable of unlimited cell proliferation, and capable of either symmetrical or asymmetrical cell division. In the case of symmetrical division, a stem cell could increase its numbers. By asymmetrical cell division, the mother stem cell could divide to form one daughter, which losses its telomerase and can differentiate and or senesce. The other daughter cell must maintain "stemness" in order to provide cells needed for growth and replacement of dead or lost cells.
Therefore, if a normal, immortal stem cell, which lacks GJIC, is exposed to an initiator that can block asymmetrical cell division (i.e. it now cannot differentiate, is kept in the immortal mode and is resistant to apoptosis), this cell could proliferate in a symmetrical manner when stimulated by a promoter (see Figure 1
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Critical to these explanations is how does the pluripotent stem cell (GJIC-deficient) and early partially differentiated progenitor cell control their growth. Obviously, normal and pre-malignant or initiated stem cells and early pre-malignant or initiated progenitor cells are growth inhibited. Stem cells, deficient in expressed connexins and GJIC, must be growth inhibited by either cell–matrix microenvironmental or secreted negative growth factors [21]. Tumour promoters for this class of initiated stem cells must either chronically reduce the production of negative growth regulators or prevent the initiated stem cell from responding to the secreted negative growth regulators. When the initiated stem cell stably becomes resistant to these negative growth regulators, they become tumour promoter-independent. Without transcription of connexins and functional GJIC, these initiated stem cells will never differentiate and ultimately become invasive and metastatic tumour cells like HeLa and MCF-7 cells.
Another possibility, as supported by the recent demonstration of a human breast cancer stem cell [35], is that, under the right microenvironmental conditions, the initiated stem cell could be induced to express their connexins and start to partially differentiate. In other words, the tumour consists of both the initiated stem cell with no expressed connexins or functional GJIC and abnormally differentiated connexin-expressing initiated cells, which exhibit genomic instability, and a variety of phenotypes due to altered genotypes and altered epigenetic expressions.
On the other hand, early stem cell-derived progenitor cells still have telomerase activity and are growth inhibited and partially differentiated because they do express connexins and have functional GJIC. When these cells are exposed to initiators, they cannot continue their differentiation to the terminal state, either because they cannot lose their telomerase activity or cannot respond to GJIC-transmitted intercellular signals. Upon exposure to promoters that transiently downregulate GJIC, they can only proliferate via symmetrical cell division. When during the long, sustained promotion process, an initiated progenitor cell becomes tumour promoter-independent via some stable activation of an oncogene [10], the cell has been converted to the "progression" phase of carcinogenesis where, via chromosome instability, abnormal gene/chromosome modifications and altered gene expression, cells can acquire the other hallmarks of cancer.
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GJIC and the "adaptive response", "bystander effects" and "genomic instability" phenomena |
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The bystander effect is defined as the transmitted effect of a targeted cell's response to a genotoxic agent to surrounding non-hit cells. The consequence of this transmitted signal could be either negative or positive, depending on circumstances. The transmission of the signal(s) appears to be via secreted processes [39, 40] or via gap junctions [41, 42], depending on the biological system being studied. The biological consequence of this (these) transmitted signal(s) can, in principle, be cell death, mutations or epigenetic changes (such as induction of p53 enzymes). Whilst introduction of the term bystander effect has been in the context of recent radiation biology studies, it had its origin in the observation of "metabolic cooperation" [43]. This phenomenon would have major consequences on either increasing or decreasing the biological risk of low level radiation of a tissue composed of both gap junction-coupled or non-gap junction-coupled cells.
The concept of genomic instability demonstrated in many experimental studies could provide a mechanism by which an initial radiation or chemical-induced genetic or epigenetic change in a single cell could alter the internal homeostatic control of either or both the quality or the integrity of the nuclear DNA or the transcriptional regulation of the genome [44]. Although, at present, there is no consensus as to how this phenomenon comes about or even whether it is the cause or consequence of radiation-induced carcinogenesis, it has the potential of acting as an amplifier of a single hit that could assist in generating additional hits needed to bring about all the known hallmarks of carcinogenesis.
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Radiation-induced cell death by necrosis or apoptosis: a paradox |
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Would the quantity and quality of the surviving stem cells of each organ be the deciding factor or whole body radiation be the ultimate determinate of multi-organ involvement and failure? |
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One of the key concepts of tissue/organ functioning is the ability of the stem cells and the finite lifespan progenitor cells to replace injured, dead and lost cells. The quantity of stem cells remaining after acute high dose in any organ will determine whether that organ can survive and function to maintain physiological homeostasis affecting the functioning of other organs. Replacing blood-forming cells in the haematopoietic organ system after total destruction of this system after whole body radiation will not "cure" the regeneration of other organs (i.e. skin, gastrointestinal tract or central nervous system) if these organ systems lose all their stem cells.
On the other hand, if the radiation is not whole body or does not eliminate all the stem cells of any organ system, potential acute recovery is possible with appropriate acute medical intervention if any one organ system is not totally dysfunctional and if multi-organ involvement/failure is not affected by even a transient low threshold level function of that organ on other organ systems. However, the quality of any surviving stem cell after acute sublethal total or partial body exposure could affect the health status of any system because dysfunctional genes in those surviving stem cells will give rise to progenitor cells that will not function as perfect differentiated cells. The amount of damaged stem cells and the genomic defects they carry would determine the function of that organ system. An example of a stem cell-derived chronic disease caused by sublethal exposure of radiation would be cancer.
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Paradoxes as the "grists" for new scientific solutions |
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However, in the case of carcinogenesis, the acquisition of all those hallmarks of cancer in order that a normal cell become an invasive, metastatic tumour challenges the prevailing paradigm with new observations being made in and out of the field of radiation carcinogenesis. The induction of gene expression by low dose exposure (an epigenetic mechanism, not a mutagenic event), the multiple distinct gene-dependent phenotypic changes that occur during the multistage, multimechanism process of carcinogenesis, the roles of intercellular communication in modifying radiation effects (both stromal–epithelial or extracellular communication and GJIC in the adaptive response, hormesis, genomic instability, bystander effects), and the differential response of stem, progenitor and terminal differentiated cells to ionising radiation contributes to a new paradigm.
After the recent discovery of normal adult stem cells and their potential role in radiation carcinogenesis [30, 49], the observation that human breast and brain cancer stem cells has been noted [35, 50, 51]. The observation that metastatic cancer cells have gene expression patterns similar to the cells of the primary tumour mass from which they were derived implies that the dominant cell population in the primary tumour mass is phenotypically and possibly genotypically (almost) identical to cells in the metastases [52]. This suggests that the metastatic cell may be derived from a normal stem cell that already expresses many of those genes needed to be immortal, not to terminally differentiate, not to apoptose, and to invade tissues and metastasise. In other words, multiple radiation-induced mutations might not be needed, but only a few to prevent a normal stem cell from terminally differentiating or "mortalising".
In the field of chemical carcinogenesis, the role of chemicals as the agents causing oncomutations via oxidative stress has been challenged [53–55]. Rather, these chemicals are seen as potential promoters of pre-existing initiated cells. The cause of these pre-existing initiated cells is unknown (possibly due to errors in replication, errors in methylation/acetylation of genes/histone proteins, spontaneous oxidative damage, low level background radiation).
Moreover, most of the radiation-induced cell killing, and gene and chromosomal mutagenesis data have been derived from in vitro studies done under very irrelevant conditions (high oxygen tension, isolated from co-culture epithelial–stromal interactions, use of non-stem cells as target cells). Even all the DNA repair studies have been done either on bacterial models or non-stem cells. As of now, there are no data existing that demonstrate whether normal stem cells have DNA repair kinetics/qualities that are the same as or different from the non-stem cells that have been used for our current knowledge of DNA repair.
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Summary |
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However, in the context of the charge of this Workshop, it seems that a potential conceptual way to view either high or low level radiation exposure on acute, short-term or long-term chronic health effects might be related to the quantity and quality of the stem cells that remain in each organ system. Whether whole body or partial body exposure is added to the list of factors that could contribute to either short-term survival or multisystem involvement and failure has to be considered. Lastly, the issue of whether ionising radiation's effects on multisystem involvement and failure would be different than trauma induced by burns, surgery, massive physical injury or infection can only be surmised at present as being due to the unique character of ionising radiation being able to penetrate all tissues of all organs of the body, all areas within a cell and all cell types of cells within the tissues.
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Abstract
Introduction
1=rate of terminal differentiation and death of stem cell daughter; 
) but not of the proliferation of the initiated cell. This initiated cell can no longer divide asymmetrically but can divide symmetrically; 
1=rate of stem cell renewal after stem cell divides asymmetrically;