Radiation Protection Dosimetry Advance Access published April 20, 2015 Radiation Protection Dosimetry (2015), pp. 1–7
doi:10.1093/rpd/ncv167
NON-TARGETED EFFECTS OF RADIATION EXPOSURE: RECENT ADVANCES AND IMPLICATIONS M. A. Kadhim1,* and M. A. Hill2 1 Genomic Instability Group, Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, UK 2 CRUK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, ORCRB Roosevelt Drive, Oxford OX3 7DQ, UK *Corresponding author: [email protected] The target theory of radiation-induced effects has been challenged by numerous studies, which indicate that in addition to biological effects resulting from direct DNA damage within the cell, a variety of non-DNA targeted effects (NTE) may make important contributions to the overall outcome. Ionising radiation induces complex, global cellular responses, such as genomic instability (GI) in both irradiated and never-irradiated ‘bystander’ cells that receive molecular signals produced by irradiated cells. GI is a well-known feature of many cancers, increasing the probability of cells to acquire the ‘hallmarks of cancer’ during the development of tumours. Although epidemiological data include contributions of both direct and NTE, they lack (i) statistical power at low dose where differences in dose response for NTE and direct effects are likely to be more important and (ii) heterogeneity of non-targeted responses due to genetic variability between individuals. In this article, NTE focussing on GI and bystander effects were critically examined, the specific principles of NTE were discussed and the potential influence on human health risk assessment from low-dose radiation was considered.
INTRODUCTION The interpretation of radiation-induced damage and associated consequences has followed a classical target theory for many years, implying that biological effects produced after radiation such as cell death, chromosomal aberrations, mutation and ultimately cancer induction are a direct result of energy deposition from ionising radiation (IR) in the effected cell. Specifically, in assessing radiation effects, it is assumed that DNA is the primary target with biological consequences relating to radiation interactions in the DNA and its immediate environment leading to DNA damage. However, evidence of non-targeted effects (NTE) emerged in the early 1990s to challenge conventional target theory and substantial work in this area has been extensively discussed in recent reviews(1, 2). NTE of radiation exposure are characterised by cellular responses that occur in (i) progeny of irradiated somatic/germ cells and (ii) cells influenced by energy deposition in neighbouring irradiated cells as a result of intercellular signalling. Distinct classes of NTE include genomic instability (GI) and bystander effects (BEs) (Figure 1), the latter includes abscopal effects and bystander-mediated adaptive response (AR). Here, the discussion is limited to effects of low dose on the induction of GI and associated BE responses in normal tissue encountered from occupational, environmental and medical diagnostic exposures. GENOMIC INSTABILITY GI is defined by its novel biological effects, i.e. delayed gene mutations, gene amplifications, chromosomal
damage, micronucleus formation and de novo chromosomal aberrations that occur in the progeny of cells many generations after the original radiation event(3) and is a well-known characteristic of many tumours assisting the cells transition from normal to oncogenic(4). GI does not support the conventional target theory of IR as it occurs at a higher frequency than can be explained by mutation of specific genes (discussed later). It is also heterogeneous arising non-clonally within the clonal descendants multiple generations from the irradiated population and germ cells resulting in transgenerational GI(3). The induction of GI follows a non-conventional dose response compared with that seen for biological effects initiated by radiation-induced DNA damage and influenced by several factors such as genetic predisposition, cell type and radiation quality. Although the multiple phenotypes produced are well characterised, the initiating molecular, biochemical and cellular events are still not fully understood(3, 5).
Mechanisms of genomic instability Evidence suggests that radiation-induced DNA damage is not the initiating factor of GI(6) but are likely to be associated with epigenetic mechanisms, where small adjustments are made to individual DNA bases modulating gene expression and irregularities in these processes have also been associated with cancer(7). The unique properties and mechanisms of the GI phenotype have been recently reviewed by Kadhim et al. (3). Mechanistic studies implicate elevated intracellular oxidative stress, as the potential
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
M. A. KADHIM AND M. A. HILL
Figure 1. Paradigms of radiation exposure effects.
dominant candidate for initiating GI. This could involve direct interaction of reactive oxygen species (ROS)(8) with DNA or effects of ROS on intracellular compartments and organelles(9). BYSTANDER EFFECTS Radiation-induced BEs are generally defined as biological effects observed in un-irradiated cells as a consequence of cellular communication with irradiated cells; however, similar intercellular signalling across dose gradients has also been observed to modify the response of irradiated cells(10). BEs have been observed in a range of cell types for several biological end points, for external beam as well as radionuclide exposures(11 – 13). The in vitro and in vivo evidence, and proposed mechanisms, has been extensively reviewed(1, 5, 14). Possibly related to these in vitro observations of BE are abscopal, out-of-field effects observed in vivo and cohort effects between irradiated cells within an irradiated volume(1, 15). The interrelationship between these ‘long-range’ BE and shortrange cellular effects has not been clearly defined but are probably manifestations of an integrated stress response to localised irradiation.
Mechanisms of bystander effects There are two main mechanisms by which radiationinduced BE signalling molecules can be transmitted between cells (1) via gap junction intercellular communication (GJIC) and (2) via soluble factors within cells/tissue’s microenvironment and/or medium(1, 12, 16). Gap junction channels permit the transfer of small-molecular-weight molecules and ROS, and their importance in mediating the bystander and cell population responses as an integrated unit was demonstrated by Azzam et al.(17), who showed that bystander-induced DNA damage was eliminated when using GJIC-deficient cultures. The role of media-borne signals has been demonstrated using a range of experimental techniques including media transfer from irradiated cells, coculture insert systems, partial shielding or microbeams. Some types of transmissible signals produced from irradiated cells have been shown to play a role in BE (reviewed by Hei et al.(18)); these include ROS, nitric oxide species (NOS)(19), cytokines(13), calcium fluxes(20), plasma membrane-bound lipid rafts(21), signals associated with the inflammatory response(22) and also microvesicles such as exosomes(23, 24). Albanese and Dainiak(23) showed that irradiated cells shed vesicles such as exosomes that affect the
Page 2 of 7
NON-TARGETED EFFECTS OF RADIATION
recipient cells(25). Exosomes are small (,150 nm)(26) membrane-bound vesicles containing cargo material from endosomes, cytosol, plasma membrane and reportedly microRNA molecules and released by a number of non-cancer and cancer cells into both the extracellular microenvironment (27) and body fluids, such as blood plasma. Exosomes can be taken up by recipient cells, resulting in the delivery of their protein and RNA cargo(28). They have been shown to play roles in a variety of biological responses including cell –cell communication, and their function is determined by cell type and conditions of release; in particular, increased blood exosome levels have been reported in cancer patients and may mediate signalling of the cancer ‘field effect’(29). Recent work(24) has demonstrated that exosomes released from irradiated cells are responsible in part for BE in MCF7 breast cancer cells. MCF7 cells were irradiated with 2 Gy of X rays, and the extracellular media was harvested. RNase treatment abrogated the ability of media to induce early and late chromosomal damages in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the BE, and GI, are at least in part mediated by exosomes and implicate a role for RNA(27, 30, 31). The transportation of exosomes through body fluids and their relative stability means that they have the potential to induce biological effects over greater distances than normally associated with BE and therefore has the potential to play a role in abscopal effects.
models of radiation effects. These principals include the following: (1) DNA is NOT the sole direct target. (2) NTE do not follow conventional dose dependency. (3) NTE are not universally expressed due to influencing factors (e.g. genetic predisposition, cell/ tissue type, radiation dose and quality). (4) NTE response results in non-clonal aberrations and heterogeneity within the progeny of the exposed cell population. (5) NTE are induced at higher frequency than expected for mutation in a single gene: epigenetic mechanism. (6) Transmission of information is NOT one way, and biological functionality is multilevel. DNA is NOT the sole direct target NTE represent a paradigm shift from the ‘DNA centric’ view that IR only elicits biological effects and subsequent health consequences as a result of energy deposition in the cell nucleus and more specifically the DNA. Cytoplasmic irradiation using microbeams is not only capable of inducing genotoxic effects in the irradiated cells, i.e. mutation and transformation(35), but also in neighbouring un-irradiated bystander cells(36). NTE responses (radiation-induced GI) are exhibited by the descendants of irradiated cells or by cells that have communicated with irradiated cells (BE). NTE do not follow conventional dose dependency
LINK BETWEEN BYSTANDER EFFECT AND GENOMIC INSTABILITY (32 – 34)
and share important BE and GI are interlinked phenotypes (see Figure 1), e.g. micronuclei formation, increased mutation and chromosomal rearrangements and up-regulation of oxidative stress; these are expressed at high frequency, and there is generally an absence of conventional dose response. This link has been demonstrated experimentally using various different approaches such as the grid technique(32) and co-culture system(33) in the haemopoietic cell system. Results from these experiments show that most of the progeny demonstrating GI were from the un-irradiated cell population resulting in the conclusion that the induction of this damage was caused by intercellular communication, i.e. a bystander mechanism. Furthermore, in other studies(34), the up-regulation of oxidative stress in bystander cells is similar to that observed in radiation-induced GI. PRINCIPLES OF NTE Many of the principles of NTE are unique and do not conform to the expectations of the conventional
Another notable feature is the non-conventional dose dependence of induction of GI and BE. In general, there is a tendency for the instability response to reach a plateau at low doses(37) and in some cases even decrease at high doses, contrasting to the steadily increasing responses conventionally observed. For low linear energy transfer (LET) X rays, the GI response in the progeny of irradiated cells was high for a relatively low dose of 0.1 Gy. However, in the case of CBA/H haemopoietic stem cells, the response was observed to reduce at higher doses and at 3 Gy was not significantly different to the control level(38). While for low to medium doses of high-LET particles, the induction of GI appears to be relatively dose independent (39). Therefore, radiation-induced GI could profoundly impact the interpretation of dose– response relationships in two ways: firstly, an increased level of response at low doses in comparison with that expected by a conventional linear or linear-quadratic extrapolation from higher doses, and secondly, the uncertainties associated with consequences of delayed non-clonal expression of damage and therefore the potential to play a major role in radiation-induced carcinogenesis. Also, as with GI, BE has been observed
Page 3 of 7
M. A. KADHIM AND M. A. HILL
at very low doses for low- and high-LET radiations, e.g. Portess et al. (40) showed significant BE for 2 mGy g-rays and 0.3 mGy a-particles with response saturation at low dose (above 25 mGy) with no additional increase with increasing dose, similar dose responses have been observed for a range of end points and radiation types. Work from Mothersill’s lab using media transfer methods also showed that bystander cells have decreased survival relative to un-irradiated controls; the magnitude of BE depended on the number of cells irradiated but was dose independent with 0.02–5 Gy(41). In situations where single cells are traversed following microbeam exposure, there is evidence that the event that triggers the emission of the bystander signal may be a binary, all-or-nothing response, with the probability of effect increasing with radiation dose to the single cell irradiated(42). NTE are not universally expressed due to influencing factors As expected from any biological process, there are likely to be multiple pathways to a particular end point associated with NTE and as such there are some inconsistencies in NTE studies, as reviewed by Morgan and Sowa(2). Some studies showed that following irradiation, GI is not universally expressed in mammalian cells in vivo or in vitro (43 – 45). In other studies, whether animal or human, its expression has been reported to depend on the genotype of the irradiated cell(46), with considerable inter-individual variation even in those genotypes that may express high levels of instability(47). Conflicting issues and observations may be attributed to cell type, genotype or other, poorly understood factors. Similarly, as with GI, BE is not universally expressed(48), these conflicts confirm that biological/experimental systems, radiation type and dose, dose rate, genotype, biological end points/time points should all be considered in comparing results of different experiments. All together, these findings clearly indicate a need for caution in drawing generalised conclusions from limited data or attempting to extract a simple coherent picture from the literature in general. NTE response results in non-clonal aberrations and heterogeneity within the progeny of the exposed populations As a result of radiation-induced GI, responses may be observed in the progeny of both irradiated and unirradiated cells, multiple generations after the initial insult (Figure 1). Multiple end points have been observed including chromosomal alterations, micronucleus formation, gene mutations and amplifications, microsatellite instabilities and/or decreased plating efficiency. These effects are heterogeneous and expressed non-clonally(5).
NTE induced at higher frequency than expected for mutation in a single gene: epigenetic mechanism Expression of GI is observed in a larger proportion of cells irradiated with low to medium doses (about 10– 20 %) than can be explained by specific/single gene mutations from conventional targeted effects (typically ,1024 per cell per Gy)(49). Rather than a genetic mutation, instability might arise through epigenetic mechanisms, for example, induced expression of a mutator gene(50). The observed high frequency of instability responses, as well as the lack of evidence of involvement of DNA double-strand breaks per se in instability initiation, has led Baverstock(37) to speculate that alterations in gene expression that disrupt cellular homeostasis may underlie induced instability. Rugo et al.(51) demonstrated the role of methylation in GI transmission in embryonic stem cells, showing that elimination of DNA cytosine methylation genes, Dmmt1 and Dmmt3a, completely eliminated transmission of GI. Like GI, BEs occur at very high frequency with biological response to radiation not just restricted to the irradiated cells and are heterogeneous (non-clonal) in their expression(1, 33) for most end points including DNA damage, cell killing, chromosome aberrations, mutations and transformation(5, 52). Although these types of biological damage overlap with effects that are well known to occur in directly irradiated cells, there are differences. For example, DNA damage responses in bystander cells are known to be different to the dominant response observed in directly irradiated cells, and typically involving ataxia telangiectasia and Rad3 related protein-dependant signalling from stalled replication forks, instead of direct ataxia telangiectasia-mediated signalling(13, 53). Transmission of information is NOT one way, and biological functionality is multilevel As discussed earlier, several signalling molecules have been identified that mediate communication between irradiated and bystander populations(8). All together, these studies led to the realisation that signal production and response in the receptor are separate processes with separate mechanisms. Since then, much work has been carried out on the precise mechanisms involved in both parts of the pathway. The response has now been well characterised and is reviewed by Hei et al.(13). Rather than radiation inducing a single unique molecule and pathway, the initiation of BEs is much more likely to result from perturbation of existing intercellular signalling pathways between cells; these may include multiple pathways that may also include feedback from the unirradiated cells(40) and will depend on the cell type and microenvironment. NTE: IMPLICATIONS FOR RISK The phenomenon of GI has the potential to play a major role in radiation-induced cancer. It is observed
Page 4 of 7
NON-TARGETED EFFECTS OF RADIATION
at low doses as a high-frequency event resulting in an ongoing rise in the rate of mutations and chromosome aberrations in the progeny of irradiated populations (including irradiated and bystander cells). GI is a well-known feature of many tumours and has been described as an ‘enabling characteristic’ of cancer, increasing the probability of accumulation of rare genetic changes required for cells to acquire the ‘hallmarks of cancer’ during the multistep development of human tumours(4). GI is also increasingly being incorporated into carcinogenesis models to fit human cancer data(54). As a result of NTE having a non-conventional dose response, they do not conform to the conventional linear no-threshold (LNT) response. These effects, therefore, have the potential to produce significant deviations from the standard expectation when extrapolating to low doses from the risk estimates evaluated at high doses, where conventional direct effects are likely to dominate. However, the relative role of NTE in comparison with conventional effects with respect to risk is poorly understood, especially as consequences of the BE have the potential to be either detrimental or beneficial depending on the end point studied. Therefore, with the large uncertainties associated with epidemiological data at low doses, at this stage, it would be premature to incorporate into lowdose risk determinations. CONCLUSION The paradigm of genetic alterations being restricted to direct DNA damage after exposure to IR has been challenged by the so-called non-targeted effects (NTE) with responses observed similar to those characteristically associated with directly irradiated cells. NTE include observations in which effects of IR arise in cells that themselves receive no radiation exposure or where DNA is not the initial target. Additionally, NTE also include effects initiated in the descendants of an irradiated cell population (radiation-induced GI) originating from either directly irradiated or nonirradiated bystander cells. Bystander signals may be transmitted by direct intercellular communication through gap junctions, or by diffusible factors, with several signalling molecules having been identified, including exosomes that have the potential to illicit effects over larger distance than normally associated with BE. Rather than radiation eliciting unique signalling pathways, radiation can be considered as perturbing existing signalling within and between cells. Radiation-induced GI and BE may reflect interrelated mechanisms and are likely be a consequence of intercellular signalling, including the production of cytokines, exosomes and free radical generation. Although NTE have the potential to play a major role in cancer induction at low doses and potentially lead to deviations from the LNT model, at present,
direct evidence of the role of NTE to human risk is lacking. Therefore, future work needs to address the relevance of NTE to human risk along with a greater mechanistic understanding of these processes involved.
ACKNOWLEDGEMENTS The authors would like to thank Miss Kim Chapman and Dr Scott Bright for their help in finalising the document. REFERENCES 1. Blyth, B. J. and Sykes, P. J.: Radiation-induced bystander effects: what are they, and how relevant are they to human radiation exposures? Radiat. Res. 176, 139–157 (2011). 2. Morgan, W. F. and Sowa, M. B. Non-targeted effects induced by ionizing radiation: mechanisms and potential impact on radiation induced health effects. Cancer Lett. 356(1), 17– 21 (2013). 3. Kadhim, M., Salomaa, S., Wright, E., Hildebrandt, G., Belyakov, O. V., Prise, K. M. and Little, M. P. Non-targeted effects of ionising radiation-implications for low dose risk. Mutat. Res. 752, 84– 98 (2013). 4. Hanahan, D. and Weinberg, R. A. Hallmarks of cancer: the next generation. Cell. 144, 646– 674 (2011). 5. Morgan, W. F. Non-targeted and delayed effects of exposure to ionizing radiation: I. Radiation-induced genomic instability and bystander effects in vitro. Radiat. Res. 159, 567–580 (2003). 6. Kadhim, M. A., Moore, S. R. and Goodwin, E. H. Interrelationships amongst radiation-induced genomic instability, bystander effects, and the adaptive response. Mutat. Res. 568, 21– 32 (2004). 7. Grønbaek, K., Hother, C. and Jones, P. Epigenetic changes in cancer. APMIS. 115, 1039–1059 (2007). 8. Bonassi, S. et al. Chromosomal aberration frequency in lymphocytes predicts the risk of cancer: results from a pooled cohort study of 22 358 subjects in 11 countries. Carcinogenesis. 29, 1178– 1183 (2008). 9. Suzuki, K., Ojima, M., Kodama, S. and Watanabe, M. Radiation-induced DNA damage and delayed induced genomic instability. Oncogene. 22, 6988–6993 (2003). 10. Butterworth, K., McGarry, C., Trainor, C., O’Sullivan, J., Hounsell, A. and Prise, K. Out-of-field cell survival following exposure to intensity-modulated radiation fields. Int. J. Radiat. Oncol. Biol. Phys. 79, 1516– 1522 (2011). 11. Little, M. P., Vineis, P. and Li, G. A stochastic carcinogenesis model incorporating multiple types of genomic instability fitted to colon cancer data. J. Theor. Biol. 254, 229–238 (2008). 12. Mothersill, C. and Seymour, C. Changing paradigms in radiobiology. Mutat. Res. 750, 85– 95 (2012). 13. Hei, T. K., Zhou, H., Ivanov, V. N., Hong, M., Lieberman, H. B., Brenner, D. J., Amundson, S. A. and Geard, C. R. Mechanism of radiation-induced bystander effects: a unifying model. J. Pharm. Pharmacol. 60, 943–950 (2008). 14. Chai, Y. and Hei, T. Radiation induced bystander effect in vivo. Acta Med. Nagasaki. 2008, S65–S69 (2008). 15. Coates, P. J., Rundle, J. K., Lorimore, S. A. and Wright, E. G. Indirect macrophage responses to ionizing
Page 5 of 7
M. A. KADHIM AND M. A. HILL
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27. 28.
29. 30.
radiation: implications for genotype-dependent bystander signaling. Cancer Res. 68, 450–456 (2008). Lehnert, B. and Goodwin, E. Extracellular factor(s) following exposure to a particles can cause sister chromatid exchanges in normal human cells. Cancer Res. 57, 2164–2171 (1997). Azzam, E. I., de Toledo, S. M. and Little, J. B. Direct evidence for the participation of gap junction-mediated intercellular communication in the transmission of damage signals from alpha -particle irradiated to nonirradiated cells. Proc. Natl. Acad. Sci. USA. 98, 473–478 (2001). Hei, T., Zhou, H., Chai, Y., Ponnaiya, B. and Ivanov, V. Radiation induced non-targeted response: mechanism and potential clinical implications. Curr. Mol. Pharmacol. 4, 96–105 (2011). Shao, C., Lyng, F. M., Folkard, M. and Prise, K. M. Calcium fluxes modulate the radiation-induced bystander responses in targeted glioma and fibroblast cells. Radiat. Res. 166, 479– 487 (2006). Lyng, F., Maguire, P., McClean, B., Seymour, C. and Mothersill, C. The involvement of calcium and MAP kinase signaling pathways in the production of radiationinduced bystander effects. Radiat. Res. 165, 400– 409 (2006). Hamada, N., Matsumoto, H., Hara, T. and Kobayashi, Y. Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects. J. Radiat. Res. 48, 87–95 (2007). Lorimore, S. and Wright, E. Radiation-induced genomic instability and bystander effects: related inflammatorytype responses to radiation-induced stress and injury? A review. Int. J. Radiat. Biol. 79, 15–25 (2003). Albanese, J. and Dainiak, N. Modulation of intercellular communication mediated at the cell surface and on extracellular, plasma membrane-derived vesicles by ionizing radiation. Exp. Hematol. 31, 455–464 (2003). Al-Mayah, A. H., Irons, S. L., Pink, R. C., Carter, D. R. and Kadhim, M. A. Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation. Radiat. Res. 177, 539–545 (2012). Record, M., Subra, C., Silvente-Poirot, S. and Poirot, M. Exosomes as intercellular signalosomes and pharmacological effectors. Biochem. Pharmacol. 81, 1171–1182 (2011). Lane, R., Anderson, W., Broom, M. and Korbie, D. Izon Science Limited, White Paper: Tunable resistive pulse sensing for detailed characterization of extracellular vesicles (2013). Fevrier, B. and Raposo, G. Exosomes: endosomalderived vesicles shipping extracellular messages. Curr. Opin. Cell Biol. 16, 415– 421 (2004). Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J. J. and Lotvall, J. O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007). Taylor, D. D. and Gercel-Taylor, C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol. Oncol. 110, 13– 21 (2008). Ji, H., Chen, M., Greening, D., He, W., Rai, A., Zhang, W. and Simpson, R. Deep sequencing of RNA from three different extracellular vesicle (EV) subtypes released from the human LIM1863 colon cancer cell line uncovers
31. 32.
33.
34.
35.
36. 37.
38. 39.
40.
41.
42.
43. 44.
45.
distinct Mirna-enrichment signatures. PLoS One. 9, e110314 (2014). van der Grein, S. and Nolte-’t Hoen, E. “Small talk” in the innate immune system via RNA-containing extracellular vesicles. Front. Immunol. 5, 542 (2014). Lorimore, S. A., Kadhim, M. A., Pocock, D. A., Papworth, D., Stevens, D. L., Goodhead, D. T. and Wright, E. G. Chromosomal instability in the descendants of unirradiated surviving cells after alpha-particle irradiation. Proc. Natl. Acad. Sci. USA. 95, 5730–5733 (1998). Bowler, D. A., Moore, S. R., Macdonald, D. A., Smyth, S. H., Clapham, P. and Kadhim, M. A. Bystandermediated genomic instability after high LET radiation in murine primary haemopoietic stem cells. Mutat. Res. 597, 50–61 (2006). Ponnaiya, B., Suzuki, M., Tsuruoka, C., Uchihori, Y., Wei, Y. and Hei, T. K. Detection of chromosomal instability in bystander cells after Si490-ion irradiation. Radiat. Res. 176, 280–290 (2011). Sawant, S. G., Randers-Pehrson, G., Geard, C. R., Brenner, D. J. and Hall, E. J. The bystander effect in radiation oncogenesis: I. Transformation in C3H 10T1/2 cells in vitro can be initiated in the unirradiated neighbors of irradiated cells. Radiat. Res. 155, 397 –401 (2001). Shao, C., Folkard, M., Michael, B. D. and Prise, K. M. Targeted cytoplasmic irradiation induces bystander responses. Proc. Natl. Acad. Sci. USA. 101, 13495–13500 (2004). Baverstock, K. Radiation-induced genomic instability: a paradigm-breaking phenomenon and its relevance to environmentally induced cancer. Mutat. Res. 454, 89– 109 (2000). Kadhim, M. A., Hill, M. A. and Moore, S. R. Genomic instability and the role of radiation quality. Radiat. Prot. Dosim. 122, 221– 227 (2006). Kadhim, M. A., Macdonald, D. A., Goodhead, D. T., Lorimore, S. A., Marsden, S. J. and Wright, E. G. Transmission of chromosomal instability after plutonium alpha-particle irradiation. Nature. 355, 738 – 740 (1992). Portess, D. I., Bauer, G., Hill, M. A. and O’Neill, P. Lowdose irradiation of nontransformed cells stimulates the selective removal of precancerous cells via intercellular induction of apoptosis. Cancer Res. 67, 1246–1253 (2007). Seymour, C. B. and Mothersill, C. Relative contribution of bystander and targeted cell killing to the low-dose region of the radiation dose-response curve. Radiat. Res. 153, 508–511 (2000). Schettino, G., Folkard, M., Michael, B. D. and Prise, K. M. Low-dose binary behavior of bystander cell killing after microbeam irradiation of a single cell with focused c(k) x rays. Radiat. Res. 163, 332– 336 (2005). Bouffler, S. D., Blasco, M. A., Cox, R. and Smith, P. J. Telomeric sequences, radiation sensitivity and genomic instability. Int. J. Radiat. Biol. 77, 995–1005 (2001). Dugan, L. C. and Bedford, J. S. Are chromosomal instabilities induced by exposure of cultured normal human cells to low- or high-LET radiation? Radiat. Res. 159, 301–311 (2003). Rithidech, K., Udomtanakunchai, C., Honikel, L. and Whorton, E. No evidence for the in vivo induction of genomic instability by low doses of 137CS gamma rays in bone marrow cells of BALB/CJ and C57BL/6J mice. Dose Response. 10, 11–36 (2012).
Page 6 of 7
NON-TARGETED EFFECTS OF RADIATION 46. Kadhim, M. A., Lorimore, S. A., Hepburn, M. D., Goodhead, D. T., Buckle, V. J. and Wright, E. G. Alphaparticle-induced chromosomal instability in human bone marrow cells. Lancet. 344, 987–988 (1994). 47. Natarajan, M., Gibbons, C. F., Mohan, S., Moore, S. and Kadhim, M. A. Oxidative stress signalling: a potential mediator of tumour necrosis factor alpha-induced genomic instability in primary vascular endothelial cells. Br. J. Radiol. 80, S13– S22 (2007). 48. Sowa, M., Goetz, W., Baulch, J., Pyles, D., Dziegielewski, J., Yovino, S., Snyder, A., de Toledo, S., Azzam, E. and Morgan, W. Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells. Int. J. Radiat. Biol. 86, 102–113 (2010). 49. Wright, E. G. Inherited and inducible chromosomal instability: a fragile bridge between genome integrity mechanisms and tumourigenesis. J. Pathol. 187, 19–27 (1999).
50. Fox, E. J. and Loeb, L. A. Lethal mutagenesis: targeting the mutator phenotype in cancer. Semin. Cancer Biol. 20, 353–359 (2010). 51. Rugo, R. E., Mutamba, J. T., Mohan, K. N., Yee, T., Chaillet, J. R., Greenberger, J. S. and Engelward, B. P. Methyltransferases mediate cell memory of a genotoxic insult. Oncogene. 30, 751–756 (2011). 52. UNSCEAR. 2006 Report, Annex C, Non-Targeted and Delayed Effects of Exposure to Ionizing Radition (2009). 53. Burdak-Rothkamm, S., Rothkamm, K. and Prise, K. ATM acts downstream of ATR in the DNA damage response signalling of bystander cells. Cancer Res. 68, 7059–7065 (2008). 54. Jacob, P., Meckbach, R., Kaiser, J. C. and Sokolnikov, M. Possible expressions of radiation-induced genomic instability, bystander effects or low-dose hypersensitivity in cancer epidemiology. Mutat. Res. 687, 34–39 (2010).
Page 7 of 7
doi:10.1093/rpd/ncv167
NON-TARGETED EFFECTS OF RADIATION EXPOSURE: RECENT ADVANCES AND IMPLICATIONS M. A. Kadhim1,* and M. A. Hill2 1 Genomic Instability Group, Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, UK 2 CRUK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, ORCRB Roosevelt Drive, Oxford OX3 7DQ, UK *Corresponding author: [email protected] The target theory of radiation-induced effects has been challenged by numerous studies, which indicate that in addition to biological effects resulting from direct DNA damage within the cell, a variety of non-DNA targeted effects (NTE) may make important contributions to the overall outcome. Ionising radiation induces complex, global cellular responses, such as genomic instability (GI) in both irradiated and never-irradiated ‘bystander’ cells that receive molecular signals produced by irradiated cells. GI is a well-known feature of many cancers, increasing the probability of cells to acquire the ‘hallmarks of cancer’ during the development of tumours. Although epidemiological data include contributions of both direct and NTE, they lack (i) statistical power at low dose where differences in dose response for NTE and direct effects are likely to be more important and (ii) heterogeneity of non-targeted responses due to genetic variability between individuals. In this article, NTE focussing on GI and bystander effects were critically examined, the specific principles of NTE were discussed and the potential influence on human health risk assessment from low-dose radiation was considered.
INTRODUCTION The interpretation of radiation-induced damage and associated consequences has followed a classical target theory for many years, implying that biological effects produced after radiation such as cell death, chromosomal aberrations, mutation and ultimately cancer induction are a direct result of energy deposition from ionising radiation (IR) in the effected cell. Specifically, in assessing radiation effects, it is assumed that DNA is the primary target with biological consequences relating to radiation interactions in the DNA and its immediate environment leading to DNA damage. However, evidence of non-targeted effects (NTE) emerged in the early 1990s to challenge conventional target theory and substantial work in this area has been extensively discussed in recent reviews(1, 2). NTE of radiation exposure are characterised by cellular responses that occur in (i) progeny of irradiated somatic/germ cells and (ii) cells influenced by energy deposition in neighbouring irradiated cells as a result of intercellular signalling. Distinct classes of NTE include genomic instability (GI) and bystander effects (BEs) (Figure 1), the latter includes abscopal effects and bystander-mediated adaptive response (AR). Here, the discussion is limited to effects of low dose on the induction of GI and associated BE responses in normal tissue encountered from occupational, environmental and medical diagnostic exposures. GENOMIC INSTABILITY GI is defined by its novel biological effects, i.e. delayed gene mutations, gene amplifications, chromosomal
damage, micronucleus formation and de novo chromosomal aberrations that occur in the progeny of cells many generations after the original radiation event(3) and is a well-known characteristic of many tumours assisting the cells transition from normal to oncogenic(4). GI does not support the conventional target theory of IR as it occurs at a higher frequency than can be explained by mutation of specific genes (discussed later). It is also heterogeneous arising non-clonally within the clonal descendants multiple generations from the irradiated population and germ cells resulting in transgenerational GI(3). The induction of GI follows a non-conventional dose response compared with that seen for biological effects initiated by radiation-induced DNA damage and influenced by several factors such as genetic predisposition, cell type and radiation quality. Although the multiple phenotypes produced are well characterised, the initiating molecular, biochemical and cellular events are still not fully understood(3, 5).
Mechanisms of genomic instability Evidence suggests that radiation-induced DNA damage is not the initiating factor of GI(6) but are likely to be associated with epigenetic mechanisms, where small adjustments are made to individual DNA bases modulating gene expression and irregularities in these processes have also been associated with cancer(7). The unique properties and mechanisms of the GI phenotype have been recently reviewed by Kadhim et al. (3). Mechanistic studies implicate elevated intracellular oxidative stress, as the potential
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
M. A. KADHIM AND M. A. HILL
Figure 1. Paradigms of radiation exposure effects.
dominant candidate for initiating GI. This could involve direct interaction of reactive oxygen species (ROS)(8) with DNA or effects of ROS on intracellular compartments and organelles(9). BYSTANDER EFFECTS Radiation-induced BEs are generally defined as biological effects observed in un-irradiated cells as a consequence of cellular communication with irradiated cells; however, similar intercellular signalling across dose gradients has also been observed to modify the response of irradiated cells(10). BEs have been observed in a range of cell types for several biological end points, for external beam as well as radionuclide exposures(11 – 13). The in vitro and in vivo evidence, and proposed mechanisms, has been extensively reviewed(1, 5, 14). Possibly related to these in vitro observations of BE are abscopal, out-of-field effects observed in vivo and cohort effects between irradiated cells within an irradiated volume(1, 15). The interrelationship between these ‘long-range’ BE and shortrange cellular effects has not been clearly defined but are probably manifestations of an integrated stress response to localised irradiation.
Mechanisms of bystander effects There are two main mechanisms by which radiationinduced BE signalling molecules can be transmitted between cells (1) via gap junction intercellular communication (GJIC) and (2) via soluble factors within cells/tissue’s microenvironment and/or medium(1, 12, 16). Gap junction channels permit the transfer of small-molecular-weight molecules and ROS, and their importance in mediating the bystander and cell population responses as an integrated unit was demonstrated by Azzam et al.(17), who showed that bystander-induced DNA damage was eliminated when using GJIC-deficient cultures. The role of media-borne signals has been demonstrated using a range of experimental techniques including media transfer from irradiated cells, coculture insert systems, partial shielding or microbeams. Some types of transmissible signals produced from irradiated cells have been shown to play a role in BE (reviewed by Hei et al.(18)); these include ROS, nitric oxide species (NOS)(19), cytokines(13), calcium fluxes(20), plasma membrane-bound lipid rafts(21), signals associated with the inflammatory response(22) and also microvesicles such as exosomes(23, 24). Albanese and Dainiak(23) showed that irradiated cells shed vesicles such as exosomes that affect the
Page 2 of 7
NON-TARGETED EFFECTS OF RADIATION
recipient cells(25). Exosomes are small (,150 nm)(26) membrane-bound vesicles containing cargo material from endosomes, cytosol, plasma membrane and reportedly microRNA molecules and released by a number of non-cancer and cancer cells into both the extracellular microenvironment (27) and body fluids, such as blood plasma. Exosomes can be taken up by recipient cells, resulting in the delivery of their protein and RNA cargo(28). They have been shown to play roles in a variety of biological responses including cell –cell communication, and their function is determined by cell type and conditions of release; in particular, increased blood exosome levels have been reported in cancer patients and may mediate signalling of the cancer ‘field effect’(29). Recent work(24) has demonstrated that exosomes released from irradiated cells are responsible in part for BE in MCF7 breast cancer cells. MCF7 cells were irradiated with 2 Gy of X rays, and the extracellular media was harvested. RNase treatment abrogated the ability of media to induce early and late chromosomal damages in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the BE, and GI, are at least in part mediated by exosomes and implicate a role for RNA(27, 30, 31). The transportation of exosomes through body fluids and their relative stability means that they have the potential to induce biological effects over greater distances than normally associated with BE and therefore has the potential to play a role in abscopal effects.
models of radiation effects. These principals include the following: (1) DNA is NOT the sole direct target. (2) NTE do not follow conventional dose dependency. (3) NTE are not universally expressed due to influencing factors (e.g. genetic predisposition, cell/ tissue type, radiation dose and quality). (4) NTE response results in non-clonal aberrations and heterogeneity within the progeny of the exposed cell population. (5) NTE are induced at higher frequency than expected for mutation in a single gene: epigenetic mechanism. (6) Transmission of information is NOT one way, and biological functionality is multilevel. DNA is NOT the sole direct target NTE represent a paradigm shift from the ‘DNA centric’ view that IR only elicits biological effects and subsequent health consequences as a result of energy deposition in the cell nucleus and more specifically the DNA. Cytoplasmic irradiation using microbeams is not only capable of inducing genotoxic effects in the irradiated cells, i.e. mutation and transformation(35), but also in neighbouring un-irradiated bystander cells(36). NTE responses (radiation-induced GI) are exhibited by the descendants of irradiated cells or by cells that have communicated with irradiated cells (BE). NTE do not follow conventional dose dependency
LINK BETWEEN BYSTANDER EFFECT AND GENOMIC INSTABILITY (32 – 34)
and share important BE and GI are interlinked phenotypes (see Figure 1), e.g. micronuclei formation, increased mutation and chromosomal rearrangements and up-regulation of oxidative stress; these are expressed at high frequency, and there is generally an absence of conventional dose response. This link has been demonstrated experimentally using various different approaches such as the grid technique(32) and co-culture system(33) in the haemopoietic cell system. Results from these experiments show that most of the progeny demonstrating GI were from the un-irradiated cell population resulting in the conclusion that the induction of this damage was caused by intercellular communication, i.e. a bystander mechanism. Furthermore, in other studies(34), the up-regulation of oxidative stress in bystander cells is similar to that observed in radiation-induced GI. PRINCIPLES OF NTE Many of the principles of NTE are unique and do not conform to the expectations of the conventional
Another notable feature is the non-conventional dose dependence of induction of GI and BE. In general, there is a tendency for the instability response to reach a plateau at low doses(37) and in some cases even decrease at high doses, contrasting to the steadily increasing responses conventionally observed. For low linear energy transfer (LET) X rays, the GI response in the progeny of irradiated cells was high for a relatively low dose of 0.1 Gy. However, in the case of CBA/H haemopoietic stem cells, the response was observed to reduce at higher doses and at 3 Gy was not significantly different to the control level(38). While for low to medium doses of high-LET particles, the induction of GI appears to be relatively dose independent (39). Therefore, radiation-induced GI could profoundly impact the interpretation of dose– response relationships in two ways: firstly, an increased level of response at low doses in comparison with that expected by a conventional linear or linear-quadratic extrapolation from higher doses, and secondly, the uncertainties associated with consequences of delayed non-clonal expression of damage and therefore the potential to play a major role in radiation-induced carcinogenesis. Also, as with GI, BE has been observed
Page 3 of 7
M. A. KADHIM AND M. A. HILL
at very low doses for low- and high-LET radiations, e.g. Portess et al. (40) showed significant BE for 2 mGy g-rays and 0.3 mGy a-particles with response saturation at low dose (above 25 mGy) with no additional increase with increasing dose, similar dose responses have been observed for a range of end points and radiation types. Work from Mothersill’s lab using media transfer methods also showed that bystander cells have decreased survival relative to un-irradiated controls; the magnitude of BE depended on the number of cells irradiated but was dose independent with 0.02–5 Gy(41). In situations where single cells are traversed following microbeam exposure, there is evidence that the event that triggers the emission of the bystander signal may be a binary, all-or-nothing response, with the probability of effect increasing with radiation dose to the single cell irradiated(42). NTE are not universally expressed due to influencing factors As expected from any biological process, there are likely to be multiple pathways to a particular end point associated with NTE and as such there are some inconsistencies in NTE studies, as reviewed by Morgan and Sowa(2). Some studies showed that following irradiation, GI is not universally expressed in mammalian cells in vivo or in vitro (43 – 45). In other studies, whether animal or human, its expression has been reported to depend on the genotype of the irradiated cell(46), with considerable inter-individual variation even in those genotypes that may express high levels of instability(47). Conflicting issues and observations may be attributed to cell type, genotype or other, poorly understood factors. Similarly, as with GI, BE is not universally expressed(48), these conflicts confirm that biological/experimental systems, radiation type and dose, dose rate, genotype, biological end points/time points should all be considered in comparing results of different experiments. All together, these findings clearly indicate a need for caution in drawing generalised conclusions from limited data or attempting to extract a simple coherent picture from the literature in general. NTE response results in non-clonal aberrations and heterogeneity within the progeny of the exposed populations As a result of radiation-induced GI, responses may be observed in the progeny of both irradiated and unirradiated cells, multiple generations after the initial insult (Figure 1). Multiple end points have been observed including chromosomal alterations, micronucleus formation, gene mutations and amplifications, microsatellite instabilities and/or decreased plating efficiency. These effects are heterogeneous and expressed non-clonally(5).
NTE induced at higher frequency than expected for mutation in a single gene: epigenetic mechanism Expression of GI is observed in a larger proportion of cells irradiated with low to medium doses (about 10– 20 %) than can be explained by specific/single gene mutations from conventional targeted effects (typically ,1024 per cell per Gy)(49). Rather than a genetic mutation, instability might arise through epigenetic mechanisms, for example, induced expression of a mutator gene(50). The observed high frequency of instability responses, as well as the lack of evidence of involvement of DNA double-strand breaks per se in instability initiation, has led Baverstock(37) to speculate that alterations in gene expression that disrupt cellular homeostasis may underlie induced instability. Rugo et al.(51) demonstrated the role of methylation in GI transmission in embryonic stem cells, showing that elimination of DNA cytosine methylation genes, Dmmt1 and Dmmt3a, completely eliminated transmission of GI. Like GI, BEs occur at very high frequency with biological response to radiation not just restricted to the irradiated cells and are heterogeneous (non-clonal) in their expression(1, 33) for most end points including DNA damage, cell killing, chromosome aberrations, mutations and transformation(5, 52). Although these types of biological damage overlap with effects that are well known to occur in directly irradiated cells, there are differences. For example, DNA damage responses in bystander cells are known to be different to the dominant response observed in directly irradiated cells, and typically involving ataxia telangiectasia and Rad3 related protein-dependant signalling from stalled replication forks, instead of direct ataxia telangiectasia-mediated signalling(13, 53). Transmission of information is NOT one way, and biological functionality is multilevel As discussed earlier, several signalling molecules have been identified that mediate communication between irradiated and bystander populations(8). All together, these studies led to the realisation that signal production and response in the receptor are separate processes with separate mechanisms. Since then, much work has been carried out on the precise mechanisms involved in both parts of the pathway. The response has now been well characterised and is reviewed by Hei et al.(13). Rather than radiation inducing a single unique molecule and pathway, the initiation of BEs is much more likely to result from perturbation of existing intercellular signalling pathways between cells; these may include multiple pathways that may also include feedback from the unirradiated cells(40) and will depend on the cell type and microenvironment. NTE: IMPLICATIONS FOR RISK The phenomenon of GI has the potential to play a major role in radiation-induced cancer. It is observed
Page 4 of 7
NON-TARGETED EFFECTS OF RADIATION
at low doses as a high-frequency event resulting in an ongoing rise in the rate of mutations and chromosome aberrations in the progeny of irradiated populations (including irradiated and bystander cells). GI is a well-known feature of many tumours and has been described as an ‘enabling characteristic’ of cancer, increasing the probability of accumulation of rare genetic changes required for cells to acquire the ‘hallmarks of cancer’ during the multistep development of human tumours(4). GI is also increasingly being incorporated into carcinogenesis models to fit human cancer data(54). As a result of NTE having a non-conventional dose response, they do not conform to the conventional linear no-threshold (LNT) response. These effects, therefore, have the potential to produce significant deviations from the standard expectation when extrapolating to low doses from the risk estimates evaluated at high doses, where conventional direct effects are likely to dominate. However, the relative role of NTE in comparison with conventional effects with respect to risk is poorly understood, especially as consequences of the BE have the potential to be either detrimental or beneficial depending on the end point studied. Therefore, with the large uncertainties associated with epidemiological data at low doses, at this stage, it would be premature to incorporate into lowdose risk determinations. CONCLUSION The paradigm of genetic alterations being restricted to direct DNA damage after exposure to IR has been challenged by the so-called non-targeted effects (NTE) with responses observed similar to those characteristically associated with directly irradiated cells. NTE include observations in which effects of IR arise in cells that themselves receive no radiation exposure or where DNA is not the initial target. Additionally, NTE also include effects initiated in the descendants of an irradiated cell population (radiation-induced GI) originating from either directly irradiated or nonirradiated bystander cells. Bystander signals may be transmitted by direct intercellular communication through gap junctions, or by diffusible factors, with several signalling molecules having been identified, including exosomes that have the potential to illicit effects over larger distance than normally associated with BE. Rather than radiation eliciting unique signalling pathways, radiation can be considered as perturbing existing signalling within and between cells. Radiation-induced GI and BE may reflect interrelated mechanisms and are likely be a consequence of intercellular signalling, including the production of cytokines, exosomes and free radical generation. Although NTE have the potential to play a major role in cancer induction at low doses and potentially lead to deviations from the LNT model, at present,
direct evidence of the role of NTE to human risk is lacking. Therefore, future work needs to address the relevance of NTE to human risk along with a greater mechanistic understanding of these processes involved.
ACKNOWLEDGEMENTS The authors would like to thank Miss Kim Chapman and Dr Scott Bright for their help in finalising the document. REFERENCES 1. Blyth, B. J. and Sykes, P. J.: Radiation-induced bystander effects: what are they, and how relevant are they to human radiation exposures? Radiat. Res. 176, 139–157 (2011). 2. Morgan, W. F. and Sowa, M. B. Non-targeted effects induced by ionizing radiation: mechanisms and potential impact on radiation induced health effects. Cancer Lett. 356(1), 17– 21 (2013). 3. Kadhim, M., Salomaa, S., Wright, E., Hildebrandt, G., Belyakov, O. V., Prise, K. M. and Little, M. P. Non-targeted effects of ionising radiation-implications for low dose risk. Mutat. Res. 752, 84– 98 (2013). 4. Hanahan, D. and Weinberg, R. A. Hallmarks of cancer: the next generation. Cell. 144, 646– 674 (2011). 5. Morgan, W. F. Non-targeted and delayed effects of exposure to ionizing radiation: I. Radiation-induced genomic instability and bystander effects in vitro. Radiat. Res. 159, 567–580 (2003). 6. Kadhim, M. A., Moore, S. R. and Goodwin, E. H. Interrelationships amongst radiation-induced genomic instability, bystander effects, and the adaptive response. Mutat. Res. 568, 21– 32 (2004). 7. Grønbaek, K., Hother, C. and Jones, P. Epigenetic changes in cancer. APMIS. 115, 1039–1059 (2007). 8. Bonassi, S. et al. Chromosomal aberration frequency in lymphocytes predicts the risk of cancer: results from a pooled cohort study of 22 358 subjects in 11 countries. Carcinogenesis. 29, 1178– 1183 (2008). 9. Suzuki, K., Ojima, M., Kodama, S. and Watanabe, M. Radiation-induced DNA damage and delayed induced genomic instability. Oncogene. 22, 6988–6993 (2003). 10. Butterworth, K., McGarry, C., Trainor, C., O’Sullivan, J., Hounsell, A. and Prise, K. Out-of-field cell survival following exposure to intensity-modulated radiation fields. Int. J. Radiat. Oncol. Biol. Phys. 79, 1516– 1522 (2011). 11. Little, M. P., Vineis, P. and Li, G. A stochastic carcinogenesis model incorporating multiple types of genomic instability fitted to colon cancer data. J. Theor. Biol. 254, 229–238 (2008). 12. Mothersill, C. and Seymour, C. Changing paradigms in radiobiology. Mutat. Res. 750, 85– 95 (2012). 13. Hei, T. K., Zhou, H., Ivanov, V. N., Hong, M., Lieberman, H. B., Brenner, D. J., Amundson, S. A. and Geard, C. R. Mechanism of radiation-induced bystander effects: a unifying model. J. Pharm. Pharmacol. 60, 943–950 (2008). 14. Chai, Y. and Hei, T. Radiation induced bystander effect in vivo. Acta Med. Nagasaki. 2008, S65–S69 (2008). 15. Coates, P. J., Rundle, J. K., Lorimore, S. A. and Wright, E. G. Indirect macrophage responses to ionizing
Page 5 of 7
M. A. KADHIM AND M. A. HILL
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27. 28.
29. 30.
radiation: implications for genotype-dependent bystander signaling. Cancer Res. 68, 450–456 (2008). Lehnert, B. and Goodwin, E. Extracellular factor(s) following exposure to a particles can cause sister chromatid exchanges in normal human cells. Cancer Res. 57, 2164–2171 (1997). Azzam, E. I., de Toledo, S. M. and Little, J. B. Direct evidence for the participation of gap junction-mediated intercellular communication in the transmission of damage signals from alpha -particle irradiated to nonirradiated cells. Proc. Natl. Acad. Sci. USA. 98, 473–478 (2001). Hei, T., Zhou, H., Chai, Y., Ponnaiya, B. and Ivanov, V. Radiation induced non-targeted response: mechanism and potential clinical implications. Curr. Mol. Pharmacol. 4, 96–105 (2011). Shao, C., Lyng, F. M., Folkard, M. and Prise, K. M. Calcium fluxes modulate the radiation-induced bystander responses in targeted glioma and fibroblast cells. Radiat. Res. 166, 479– 487 (2006). Lyng, F., Maguire, P., McClean, B., Seymour, C. and Mothersill, C. The involvement of calcium and MAP kinase signaling pathways in the production of radiationinduced bystander effects. Radiat. Res. 165, 400– 409 (2006). Hamada, N., Matsumoto, H., Hara, T. and Kobayashi, Y. Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects. J. Radiat. Res. 48, 87–95 (2007). Lorimore, S. and Wright, E. Radiation-induced genomic instability and bystander effects: related inflammatorytype responses to radiation-induced stress and injury? A review. Int. J. Radiat. Biol. 79, 15–25 (2003). Albanese, J. and Dainiak, N. Modulation of intercellular communication mediated at the cell surface and on extracellular, plasma membrane-derived vesicles by ionizing radiation. Exp. Hematol. 31, 455–464 (2003). Al-Mayah, A. H., Irons, S. L., Pink, R. C., Carter, D. R. and Kadhim, M. A. Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation. Radiat. Res. 177, 539–545 (2012). Record, M., Subra, C., Silvente-Poirot, S. and Poirot, M. Exosomes as intercellular signalosomes and pharmacological effectors. Biochem. Pharmacol. 81, 1171–1182 (2011). Lane, R., Anderson, W., Broom, M. and Korbie, D. Izon Science Limited, White Paper: Tunable resistive pulse sensing for detailed characterization of extracellular vesicles (2013). Fevrier, B. and Raposo, G. Exosomes: endosomalderived vesicles shipping extracellular messages. Curr. Opin. Cell Biol. 16, 415– 421 (2004). Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J. J. and Lotvall, J. O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007). Taylor, D. D. and Gercel-Taylor, C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol. Oncol. 110, 13– 21 (2008). Ji, H., Chen, M., Greening, D., He, W., Rai, A., Zhang, W. and Simpson, R. Deep sequencing of RNA from three different extracellular vesicle (EV) subtypes released from the human LIM1863 colon cancer cell line uncovers
31. 32.
33.
34.
35.
36. 37.
38. 39.
40.
41.
42.
43. 44.
45.
distinct Mirna-enrichment signatures. PLoS One. 9, e110314 (2014). van der Grein, S. and Nolte-’t Hoen, E. “Small talk” in the innate immune system via RNA-containing extracellular vesicles. Front. Immunol. 5, 542 (2014). Lorimore, S. A., Kadhim, M. A., Pocock, D. A., Papworth, D., Stevens, D. L., Goodhead, D. T. and Wright, E. G. Chromosomal instability in the descendants of unirradiated surviving cells after alpha-particle irradiation. Proc. Natl. Acad. Sci. USA. 95, 5730–5733 (1998). Bowler, D. A., Moore, S. R., Macdonald, D. A., Smyth, S. H., Clapham, P. and Kadhim, M. A. Bystandermediated genomic instability after high LET radiation in murine primary haemopoietic stem cells. Mutat. Res. 597, 50–61 (2006). Ponnaiya, B., Suzuki, M., Tsuruoka, C., Uchihori, Y., Wei, Y. and Hei, T. K. Detection of chromosomal instability in bystander cells after Si490-ion irradiation. Radiat. Res. 176, 280–290 (2011). Sawant, S. G., Randers-Pehrson, G., Geard, C. R., Brenner, D. J. and Hall, E. J. The bystander effect in radiation oncogenesis: I. Transformation in C3H 10T1/2 cells in vitro can be initiated in the unirradiated neighbors of irradiated cells. Radiat. Res. 155, 397 –401 (2001). Shao, C., Folkard, M., Michael, B. D. and Prise, K. M. Targeted cytoplasmic irradiation induces bystander responses. Proc. Natl. Acad. Sci. USA. 101, 13495–13500 (2004). Baverstock, K. Radiation-induced genomic instability: a paradigm-breaking phenomenon and its relevance to environmentally induced cancer. Mutat. Res. 454, 89– 109 (2000). Kadhim, M. A., Hill, M. A. and Moore, S. R. Genomic instability and the role of radiation quality. Radiat. Prot. Dosim. 122, 221– 227 (2006). Kadhim, M. A., Macdonald, D. A., Goodhead, D. T., Lorimore, S. A., Marsden, S. J. and Wright, E. G. Transmission of chromosomal instability after plutonium alpha-particle irradiation. Nature. 355, 738 – 740 (1992). Portess, D. I., Bauer, G., Hill, M. A. and O’Neill, P. Lowdose irradiation of nontransformed cells stimulates the selective removal of precancerous cells via intercellular induction of apoptosis. Cancer Res. 67, 1246–1253 (2007). Seymour, C. B. and Mothersill, C. Relative contribution of bystander and targeted cell killing to the low-dose region of the radiation dose-response curve. Radiat. Res. 153, 508–511 (2000). Schettino, G., Folkard, M., Michael, B. D. and Prise, K. M. Low-dose binary behavior of bystander cell killing after microbeam irradiation of a single cell with focused c(k) x rays. Radiat. Res. 163, 332– 336 (2005). Bouffler, S. D., Blasco, M. A., Cox, R. and Smith, P. J. Telomeric sequences, radiation sensitivity and genomic instability. Int. J. Radiat. Biol. 77, 995–1005 (2001). Dugan, L. C. and Bedford, J. S. Are chromosomal instabilities induced by exposure of cultured normal human cells to low- or high-LET radiation? Radiat. Res. 159, 301–311 (2003). Rithidech, K., Udomtanakunchai, C., Honikel, L. and Whorton, E. No evidence for the in vivo induction of genomic instability by low doses of 137CS gamma rays in bone marrow cells of BALB/CJ and C57BL/6J mice. Dose Response. 10, 11–36 (2012).
Page 6 of 7
NON-TARGETED EFFECTS OF RADIATION 46. Kadhim, M. A., Lorimore, S. A., Hepburn, M. D., Goodhead, D. T., Buckle, V. J. and Wright, E. G. Alphaparticle-induced chromosomal instability in human bone marrow cells. Lancet. 344, 987–988 (1994). 47. Natarajan, M., Gibbons, C. F., Mohan, S., Moore, S. and Kadhim, M. A. Oxidative stress signalling: a potential mediator of tumour necrosis factor alpha-induced genomic instability in primary vascular endothelial cells. Br. J. Radiol. 80, S13– S22 (2007). 48. Sowa, M., Goetz, W., Baulch, J., Pyles, D., Dziegielewski, J., Yovino, S., Snyder, A., de Toledo, S., Azzam, E. and Morgan, W. Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells. Int. J. Radiat. Biol. 86, 102–113 (2010). 49. Wright, E. G. Inherited and inducible chromosomal instability: a fragile bridge between genome integrity mechanisms and tumourigenesis. J. Pathol. 187, 19–27 (1999).
50. Fox, E. J. and Loeb, L. A. Lethal mutagenesis: targeting the mutator phenotype in cancer. Semin. Cancer Biol. 20, 353–359 (2010). 51. Rugo, R. E., Mutamba, J. T., Mohan, K. N., Yee, T., Chaillet, J. R., Greenberger, J. S. and Engelward, B. P. Methyltransferases mediate cell memory of a genotoxic insult. Oncogene. 30, 751–756 (2011). 52. UNSCEAR. 2006 Report, Annex C, Non-Targeted and Delayed Effects of Exposure to Ionizing Radition (2009). 53. Burdak-Rothkamm, S., Rothkamm, K. and Prise, K. ATM acts downstream of ATR in the DNA damage response signalling of bystander cells. Cancer Res. 68, 7059–7065 (2008). 54. Jacob, P., Meckbach, R., Kaiser, J. C. and Sokolnikov, M. Possible expressions of radiation-induced genomic instability, bystander effects or low-dose hypersensitivity in cancer epidemiology. Mutat. Res. 687, 34–39 (2010).
Page 7 of 7
