Biology Research Paper Dr Preston

Abstract

Current understanding of risk associated with low-dose radiation exposure has for many years been embedded in the linear-no-threshold (LNT) approach, based on simple extrapolation from the Japanese atomic bomb survivors. Radiation biology research has supported the LNT approach although much of this has been limited to relatively high-dose studies. Recently, with new advances for studying effects of low-dose exposure in experimental models and advances in molecular and cellular biology, a range of new effects of biological responses to radiation has been observed. These include genomic instability, adaptive responses and bystander effects. Most have one feature in common in that they are observed at low doses and suggest significant non-linear responses. These new observations pose a significant challenge to our understanding of low-dose exposure and require further study to elucidate mechanisms and determine their relevance.

Adaptive response, bystander effect, genomic instability, radiation biology, transgenerational

Introduction

Humans are exposed to multiple physical, chemical and biological agents during their lifetime. Of these, ionizing radiation(s) has long been known to be deleterious after high-dose exposure (>100 mSv) predominantly due to cancer induction although very high dose exposures yield tissue damage and ultimately death (see [1] for a general textbook). Ionizing radiations are widely used in society, play a key role in the treatment of cancer and are an important diagnostic tool. For radiation protection purposes, despite a century of study, the risk estimates for cancer induction in humans are extrapolated from the Japanese atomic bomb survivors, who were exposed to relatively high dose and high dose rates. Several studies of radiation workers have been undertaken as these populations were exposed to protracted low-dose exposures [2,3]. From these epidemiological data, there has been a simple extrapolation of risk to low doses generally found in environmental and most occupational exposures. This has been the linear-no-threshold (LNT) model, which assumes a linear dose-response relationship between dose and risk. Currently, with the exception of radiotherapy, the doses that members of the population can be typically exposed to are lower than the doses typically received by the bomb survivors and are therefore in regions where little epidemiological data are available. Against a typical background dose of ∼3 mSv/year, examples of routine medical exposures include 3 mSv for a breast mammogram and 0.7 mSv for a dental x-ray [4].

The LNT model has been an acceptable compromise with experimental data from radiation biology studies to some extent agreeing with it, although not exclusively. The relevance of the LNT approach has recently been sharply brought into debate with the observation of ‘non-targeted responses’. These are responses which do not follow the standard model of radiation effects. The standard model has been based on direct damage to DNA, leading especially to the production of DNA double-strand breaks and the downstream biological consequences of these [5] (see Figure 1). Non-targeted responses include a range of effects such as the adaptive response, genomic instability and the bystander effect. The aim of this short review is to highlight the key aspects of these new findings.

Figure 1.

Standard model for radiation effects in cells based on direct DNA damage leading to downstream biological responses.

Figure 1.

Standard model for radiation effects in cells based on direct DNA damage leading to downstream biological responses.

Advances in molecular and cell biology

With sequencing of the human genome and technological advances in molecular biology, many studies focus on radiation effects on gene expression using high throughput profiling approaches. The availability of array technology makes screening of individual cells for gene expression changes after irradiation possible, allowing these to be related to cellular responses even at low dose [6]. What has become clear is that the types of genes expressed at low dose (<0.2 Gy) may vary substantially from those expressed at higher doses and that there may be important time- and tissue-dependent differences [7,8]. These approaches open up the possibility of relating whole genome responses to tissue and disease responses under radiation-protection conditions in the future. From the human genome project, it is clear that much of the biological response of cells and tissues is not driven by gene expression changes but by alterations at the protein level. Proteomics aims to study these, using technological approaches such as mass spectrometer–based systems and automated 2D gel array analysis [9]. Coupled with increasing knowledge of gene expression changes and understanding of mechanisms at low doses, these new approaches have the potential to start to contribute to the development of biological-based models of radiation response to allow predictions to be made of tumour induction at low doses below the resolution of the existing epidemiological data [10].

Bystander effect

A major advance in understanding radiation effects has been the observation that cells can respond when their neighbours are irradiated, referred to as a bystander response (see [11,12] for extensive reviews). These responses were first clearly identified in 1992 when Nagasawa and Little [13] observed, under conditions where only 1% of a population of Chinese hamster ovary cells grown in culture had been traversed by a densely ionizing α-particle, that 30% of the population nevertheless experienced the formation of damaged chromosomes. Further studies have shown evidence for these effects in a range of cell types and measuring a range of end-points, including damage to chromosomes [14], mutations [15], cell death [16] and carcinogenesis measured using in vitro transformation assays [17]. Many studies have shown that simply removing the medium from irradiated cells and transferring it to non-irradiated cells is sufficient to observe a bystander response. Another approach is to use sophisticated microbeams which allow individual cells within populations to be selected and irradiated with low doses of charged particles or x-rays [18]. Microbeams have provided defining evidence for bystander responses and the mechanisms underpinning them [19]. In all these approaches, several common features of bystander response have been observed. Firstly, the effect is observed at low dose (<0.2 Gy) and saturates at high dose. Secondly, two main routes of transmittance of the effect have been found: direct cell–cell communication via specific pores between cells called gap junctions [20] and release of factors from irradiated cells into the medium [21]. A range of factors has been observed to play a role. These include reactive oxygen species (ROS), which are highly reactive-free radicals produced during normal cellular oxygen metabolism and after radiation exposure [22], and other molecules including reactive nitrogen species [23], such as nitric oxide and small proteins called cytokines [24]. All of these are also widely reported to be key signalling molecules in cell stress responses.

Some evidence for bystander responses has been observed in vivo. The production of clastogenic factors in the serum from irradiated patients has been studied for many years. These factors lead to cell damage when added to non-exposed lymphocytes and have been postulated to be superoxide products of lipid peroxidation or cytokines, all of which have also been implicated in experimental studies of bystander mechanisms (see [25] for a review). Clinically, abscopal effects, originally defined to describe systemic effects in non-irradiated sites after localized radiotherapy, may be manifestations of bystander responses at the tissue level and have been reported in the literature since the early 1950s [26]. These have been observed in animal models under conditions of relevance to therapy. In a recent study, Camphausen et al. [27] irradiated non-tumour baring legs of mice (five fractions of 10 Gy) which had tumours transplanted at distant sites. They observed reduced tumour growth rates when the leg was irradiated with tumour inhibition decreased when the radiation dose was reduced to 12 fractions of 2 Gy. A similar study in rats under conditions where partial irradiation of lung was undertaken showed damage in the shielded upper part of the lung when only the base was irradiated. The propagation of damage involved ROS and the induction of inflammatory cytokines, such as tumour necrosis factor and interleukin-1 by the irradiation some of which may be due to partial irradiation of the liver [28]. If these responses are proven in humans, they may require the incorporation of directional and geometrical information into calculations of normal tissue complication probabilities for lung, which are currently not considered in conventional dose-volume histograms [29]. Studies with internally deposited radioactive materials have also reported evidence for bystander effect in vivo. When hamsters were injected with the α-particle emitters 239PuO2 or 230Pu citrate, which concentrate in the liver, the induction of chromosome aberrations was independent of large changes in the local dose homogeneity when this was altered by injecting a range of particle sizes, but maintaining a constant total dose to the liver [30]. A similar response was observed when the induction of liver tumours was observed [31]. The authors suggested that the liver was responding to the total energy and to total dose to the liver, not to the numbers of cells traversed by an α-particle or the local dose distribution [32].

Despite advances in understanding of bystander responses, further studies on their role and relevance in vivo are required. An important issue is whether these responses are damaging or protective effects as that will ultimately determine any effect they have on dose-response curves at low dose (see Figure 2). Other studies have shown protective responses such as switching off of cell division via differentiation and the removal of potentially damaged cells by cell-death processes. What will be critical is the relative role of these effects in tissues and individuals in determining overall cancer risk.

Figure 2.

Potential models of non-linearity at low doses for both a damaging (–) or a protective (…..) bystander response versus the standard LNT model (—).

Figure 2.

Potential models of non-linearity at low doses for both a damaging (–) or a protective (…..) bystander response versus the standard LNT model (—).

Genomic instability

For radiation-induced cancer it has been assumed that a multi-step process occurs which starts with an initiation event, such as a radiation-induced mutation, followed by a promotion step where a growth advantage occurs and progression via a series of unstable changes to a tumour phenotype. Radiation biology research has concentrated on short-term assays of biological responses, such as cell killing defined in survival assays as an inability to divide and produce viable colonies. The standard approach for measuring cell killing using in vitro cell models has been the clonogenic assay originally developed by Puck and Marcus [33] in the 1950s. Using this assay, single cells are allowed to divide and colonies consisting of ≥50 cells are classified as ‘viable’. This has underpinned experimental studies of radiation cell killing in cell and tumour models, however it is based on a narrow range of assumptions regarding the definition of clonogenic survival. The accepted criteria assume that a colony derived from a single cell consists of clonal descendents of the initial cell, each with the same proliferative potential as the starting cell. It is clear however that replating of cells after irradiation exposure in many cases does not lead to control survival levels, but delayed cell killing is observed [34,35], also described as lethal mutations and that these appear randomly within the cell progeny [36]. This is a characteristic of the phenomenon of radiation-induced genomic instability. During carcinogenesis, cells and tissues undergo a phase of instability during which multiple mutations are accumulated before a full tumourogenic phenotype is established. This is observed after radiation exposure in cell and tissue models and is defined as an increased rate of acquisition of delayed changes leading to chromosomal aberrations, gene mutations and delayed cell death in the surviving progeny after irradiation [37]. All the various delayed effects are induced at very high frequency and are unlikely to be due to conventional mutational changes. Currently, little is understood of the processes involved in initiation of inducible instabilities and in maintenance and transmission of phenotype over many generations of cell replication. It is becoming evident that expression of inducible instability has a strong dependence on type of radiation exposure [38], cell type irradiated and genetic predisposition of the irradiated cell [39,40]. Instability is also observed in human lymphocytes where only a single radiation track is delivered to a cell population [41]. Elevation of ROS levels may be involved in propagation of the response via cytokine-dependent signalling. At tissue level, it appears that there is a role for an inflammatory type response driven by macrophage activation leading to apoptotic clearance of damaged cells. Several studies have suggested that radiation-induced genomic instability and bystander effect are related, with instability predominantly emanating from bystander cells in some models [42].

Transgenerational effects

The question of whether radiation exposure induces heritable effects in humans has been the subject of debate [43]. Following studies showing genomic instability in the progeny of irradiated cells in vitro and from in vivo studies, it is clear that radiation exposure can induce transgenerational effects (see [44] for a review). Much of this work has been done in mouse models following changes in repeat sequences of DNA found throughout the genome. These sequences, termed minisatellite DNA or extended tandem repeat sequences, are non-coding regions of the genome which can be analysed at the molecular level for mutations. They have a high spontaneous mutation frequency in comparison to single gene loci which means that they can be detected in relatively few cells and at low dose. Dubrova has shown that germline mutations from irradiated parents can be transmitted through sperm to offspring and lead to mutations in both somatic and germline tissues. Similar effects have been reported in humans, specifically from individuals exposed during the Chernobyl accident [45]. The underlying mechanisms of these transgenerational effects are not fully known, however it is thought that they may be a manifestation of radiation-induced genomic instability which can be transmitted.

Adaptive responses

Adaptive responses are another response which has challenged traditional thinking in radiation effects [46]. These are observed when a small dose of (priming) radiation reduces the effect of a larger (challenge) dose, typically given several hours later. The earliest studies were done in human lymphocytes [47], however these responses have also been measured after in vivo exposures in mice for cancer induction [48]. Adaptive responses appear to be highly variable and depending on the cells system and end-point used. Mechanistically, it has been suggested that stimulation of repair processes and antioxidant activity play a role, but the precise molecular mechanisms are still poorly characterized. Adaptive effects have also been related to radiation hormesis which is defined as responses to radiation which are beneficial at low doses. Radiation hormesis predicts that low-dose effects have a threshold dose above which risk increases in contrast to the LNT approach. Most of the evidence for this has been extrapolated from existing epidemiological data and there is a pressing need for further experimental studies [49].

An interesting observation of adaptive responses is that many are observed when very low priming doses are given (<0.2 Gy). These may potentially induce bystander-signalling pathways and there is a need to further study interactions between these two processes [50]. One possibility is that non-targeted responses in general are simply a stress response mechanisms of biological systems to low-dose irradiation.

Concluding remarks

New observations and mechanisms of response to radiation exposure in biological systems are questioning our understanding of the effects of low-dose exposure. These new advances in radiation biology have evolved alongside the tremendous advances in cell and molecular biology that have occurred in the last 10 years and new technological approaches for studying the effects of radiation at low doses. Together, these approaches have the potential of impacting on understanding of the relationship between radiation exposure and human disease.

Conflicts of interest

None declared.

The author acknowledges the support of the Department of Health (RRX92) the European Commission (RISC-RAD), the US Department of Energy (FG02-03ER63633, DE-FG02-02ER63305), Cancer Research UK (C1513/A2676) and the Gray Cancer Institute.

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The goals of our research are to develop and test the efficacy and safety of new treatments for drug abuse and to understand the individual and environmental factors that affect drug taking and relapse. Our primary focus is on evaluating treatments for cocaine and opioid abuse, including both pharmacologic and non-pharmacologic (psychosocial and behavioral) treatments. Related projects are evaluating the effects of specifically targeted counseling programs for reducing high-risk HIV-transmission behaviors, developing new screening and assessment tools (questionnaires, drug-screening methods) for testing potential treatment medications, and assessing the effects of patient characteristics (e.g., co-morbidity, family history, drug metabolism) on compliance, treatment outcome, and other clinical variables. Another major focus of our research is developing field tools to measure the effect of psychosocial stress as it actually occurs in daily life. With these tools, individuals with substance use disorders provide behavioral and physiological data in real time in their usual environments. Behavioral and physiological data are linked with a geographical location that can be codified in terms of objective ratings of neighborhood disarray, enabling us to relate indices of community-level risk to intensive field measurements of individual attempts at behavior change. The field measurements are supplemented with laboratory data from the same pool of participants, enabling assessment of dysregulation of biological responses to stress and its association with time spent in particular environments. The field tools will also enable a more sophisticated, integrative approach to the study of interactions between genes and environment in determining health outcomes.

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