Radiation Protection Dosimetry Advance Access published January 6, 2011 Radiation Protection Dosimetry (2011), pp. 1–4
doi:10.1093/rpd/ncq487
NO EVIDENCE FOR A LOW LINEAR ENERGY TRANSFER ADAPTIVE RESPONSE IN IRRADIATED RKO CELLS M. B. Sowa1,*, W. Goetz2, J. E. Baulch2, A. J. Lewis1 and W. F. Morgan1 1 Cell Biology and Biochemistry, Pacific Northwest National Laboratory, PO BOX 999, MS J4-02, Richland, WA 99354, USA 2 Department of Radiation Oncology, University of Maryland Medical School, Baltimore, MD 21201, USA *Corresponding author:
[email protected]
INTRODUCTION There has been much interest in recent years in radiation-induced non-targeted effects, i.e. those effects observed in cells not directly hit by radiation. Such effects could have significant implications for risk assessment as they indicate that the area of response is potentially greater than the volume irradiated (reviewed in the studies of Hamada(1), Hei et al.(2) and Morgan et al.(3)). Non-targeted effects have been observed in a variety of cell types, in tissue constructs and for a variety of radiation qualities(4 – 11). However and not surprisingly, non-targeted effects have not been universally observed(12 – 18). Here we will review the discrepancies that have begun to appear in the literature, and propose possible causes for the variations observed between different experimental conditions.
BYSTANDER EFFECTS The radiation-induced bystander effect is a non-targeted response where a signal produced by a directly irradiated cell can elicit a response in an unirradiated cell. A variety of exposure strategies have been used to study bystander effects. These include co-culture of irradiated and non-irradiated cells(11, 19 – 22), the use of low fluences of alpha particles(4, 23), charged particle microbeam irradiations(24 – 26) and media transfer from irradiated to non-irradiated cells(27). The bystander effect has been observed to have contributions from both diffusible factors and gap junction intercellular communication, with the latter playing a larger role in the observed response(5, 28). Using similar endpoints and techniques, some cell lines have failed to display any response that can be associated with a bystander effect (12 – 16, 27).
Mothersill and co-workers have found that radiation-induced bystander effects are highly variable and dependent on the individual donor or cell line origin(27, 29, 30). From the literature, there appears to be consistent evidence that alpha-particle irradiation can produce a bystander effect (4, 23, 31). However, there are now reports that the responses may not be universally observed for all types of radiation(12 – 18). Role of linear energy transfer in the production of bystander effects We have recently conducted an extensive study of the low-linear energy transfer (LET) radiation-induced bystander effect, including multiple cell lines and endpoints, various radiation sources and exposure scenarios(16). In no instance did we find evidence of a low-LET radiation-induced bystander effect that is mediated by a secreted factor. Nor did we see the evidence of a high-LET, iron ion (1 GeV/n) radiation-induced bystander effect. However, direct comparison with 3.2 MeV alpha-particle (122 keV mm21) exposures showed a statistically significant mediatransfer bystander effect for this high-LET radiation(16). These results clearly showed that alpha particles are capable of producing a bystander effect in cases where electrons, gamma rays, X-rays and Fe ions do not. Few studies have made direct comparisons on the effects of different radiation qualities, however Shao and co-workers(21) have done so for the media-borne bystander effect. Using a co-culture system, they observed increased cell proliferation and micronucleus frequency in human salivary gland tumour cells, which were bystanders to direct exposures. The resulting response was much more robust for highLET exposures than for low LET. These studies
# The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email:
[email protected]
Downloaded from rpd.oxfordjournals.org at Pacific North West Libraries on January 7, 2011
It has become increasingly evident from reports in the literature that there are many confounding factors capable of modulating radiation-induced non-targeted responses, such as the bystander effect and the adaptive response. In this paper, we examine recent data which suggest that the observation of non-targeted responses may not be universally observable for differing radiation qualities. We have conducted a study of the adaptive response following low-linear energy transfer exposures for human colon carcinoma cells and failed to observe adaption for the endpoints of clonogenic survival or micronucleus formation.
SOWA ET AL.
suggest that the low-LET radiation-induced bystander response is less robust than that induced by high-LET radiation. However, in a recent publication, Groesser et al.(14) failed to find evidence of a high-LET bystander effect in multiple cell lines and with multiple radiation sources when a medium transfer protocol was used. ADAPTIVE RESPONSE
Low LET-induced adaptive response To determine if the RKO36 cell line was capable of exhibiting such an adaptive response following low-dose exposure, we irradiated confluent cultures with a low dose, 1 or 10 cGy, of 50 keV electrons (2 keV mm21) using a microbeam(16, 36). The electron microbeam was used to allow comparison with bystander experiments previously reported(16), however in this instance, all cells in the population were exposed to a priming dose. After 4 h, the cells were given a challenge dose of X-rays. Figure 1 shows the effect of adaption on clonogenic survival (a) and micronuclei frequency (b). For clonogenic survival, we see a characteristic decrease in survival with increasing dose. For all doses, the exposure to a low dose of electrons prior to X-ray exposure was not found to significantly enhance survival. Similarly, the production of micronuclei increased with increasing dose but again no statistical decrease for a given dose was found following the low-dose pre-exposure. Studies were also performed where cells were exposed to an X-ray priming dose followed by an X-ray challenge dose. No observable adaptive response was evident with the Xray priming dose (data not shown). We conclude that RKO36 cells do not exhibit a radio-adaptive response as measured by these endpoints. Does the adaptive response also depend on LET? Both detrimental and adaptive bystander effects have been observed following medium transfer(28, 31). Iyer
Figure 1. Adaptive response of RKO36 cells as measured by clonogenic survival and micronucleus formation. Cell cultures were exposed to 50 keV electrons (MB) at a dose of 0, 1 or 10 cGy, white, light grey and dark grey, respectively. After 4 h, the same cultures were exposed to an X-ray challenge dose of 1.5, 4 or 6 Gy. (a) Clonogenic survival was measured 10– 14 days post-challenge exposure. (b) The per cent cells with radiation-induced micronuclei were evaluated using the cytochalasin B micronucleus assay 48 h post-irradiation. Each bar represents the mean+S.E. of four independent experiments. No statistically significant (P 0.05) adaptive response was observed relative to controls. See Sowa et al.(16) for full experimental details.
and Lenhert (31) found that normal fibroblasts did adapt when exposed to a priming dose of alpha particles, however they did not observe a cytotoxic
Page 2 of 4
Downloaded from rpd.oxfordjournals.org at Pacific North West Libraries on January 7, 2011
The radio-adaptive response describes the ability of a low dose of radiation to induce cellular changes that alter the level of subsequent radiation-induced damage(32, 33). Preliminary data for a radio-adaptive bystander effect were presented by Iyer and Lehnert (31) who found that an adaptive response could be induced in unirradiated cells by a transmissible factor(s) present in the supernatants of cells exposed to low-dose gamma rays. Both radiationinduced bystander effects(27, 29, 30) and adaptive responses(34, 35) have been found to be highly variable-dependent on the individual donors or cell lines tested. It remains to be seen how relevant adaptive responses will be for human exposures.
NO LOW LET ADAPTIVE RESPONSE
bystander effect, rather they saw an increase in the number of colonies following medium transfer. Similar to the work presented here (Figure 1), Mitchell and co-workers(28) found no evidence of increased clonogenic survival for 2 cGy pre-treatment of C3H10T1/2 cells exposed to a subsequent 4 Gy challenge dose of X-rays. We have not performed an extensive study of the LET dependence of the adaptive response, but we do not find evidence of adaption for the RKO36 cell line following low LET exposures (Figure 1).
It is unclear why bystander effects are not always observed in the various experiments reported in the literature even when similar experimental conditions are employed(12 – 18). We do not dispute a wealth of published data and there must be a plausible explanation for inter-laboratory differences. A possible explanation is that the bystander response is dependent on particular experimental conditions, such as factors in the media, serum batch, etc. Mothersill and co-workers(37) have recently reported that the levels of serotonin in serum can determine both the magnitude and the type of bystander effect observed following medium transfer. Their results could explain much of the inter-laboratory variability that has been appearing in the literature of late. However, this then begs the question as to whether there really is a bystander effect following radiation exposure or if it is a consequence of factors in the culture medium.
CONCLUSIONS Here we discussed the apparent discrepancies in the literature concerning the universality or robustness of radiation-induced non-targeted effects. The recent publications that fail to find an effect raises an important point regarding the bias of the scientific literature towards positive results and suggests the observation of negative results, i.e. the lack of a nontargeted effect may be underestimated.
FUNDING This work was supported by the Biological and Environmental Research programme (BER), U.S. Department of Energy [DE-AC06-76RLO (M.B.S. and W.F.M.)] and the National Aeronautics and Space Administration [NNX07AT42G (J.E.B.)].
1. Hamada, N. Recent insights into the biological action of heavy-ion radiation. J. Radiat. Res. (Tokyo) 50, 1– 9 (2009). 2. 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). 3. Morgan, W. F. and Sowa, M. B. Non-targeted effects of ionizing radiation: implications for risk assessment and the radiation dose response profile. Health Phys. 97, 426–432 (2009). 4. Azzam, E. I., de Toledo, S. M., Gooding, T. and Little, J. B. Intercellular communication is involved in the bystander regulation of gene expression in human cells exposed to very low fluences of alpha particles. Radiat. Res. 150, 497– 504 (1998). 5. 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). 6. Belyakov, O. V., Mitchell, S. A., Parikh, D., RandersPehrson, G., Marino, S. A., Amundson, S. A., Geard, C. R. and Brenner, D. J. Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away. Proc. Natl Acad. Sci. USA 102, 14203– 14208 (2005). 7. Morgan, W. F. and Sowa, M. B. Effects of ionizing radiation in nonirradiated cells. Proc. Natl Acad. Sci. USA 102, 14127– 14128 (2005). 8. Mothersill, C. and Seymour, C. Radiation-induced bystander effects, carcinogenesis and models. Oncogene 22, 7028– 7033 (2003). 9. Prise, K. M., Folkard, M. and Michael, B. D. Bystander responses induced by low LET radiation. Oncogene 22, 7043– 7049 (2003). 10. Yang, H., Anzenberg, V. and Held, K. D. Effects of heavy ions and energetic protons on normal skin fibroblasts. Radiat. Biol. Radiecol. 47, 302– 306 (2007). 11. Yang, H., Asaad, N. and Held, K. D. Mediummediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts. Oncogene 24, 2096–2103 (2005). 12. Durante, M. Bystander response measured at the GSI microbeam. J. Radiat. Res. 50, A117 (2009). 13. Fournier, C., Barberet, P., Pouthier, T., Ritter, S., Fischer, B., Voss, K. O., Funayama, T., Hamada, N., Kobayashi, Y. and Taucher-Scholz, G. No evidence for DNA and early cytogenetic damage in bystander cells after heavy-ion microirradiation at two facilities. Radiat. Res. 171, 530– 540 (2009). 14. Groesser, T., Cooper, B. and Rydberg, B. Lack of bystander effects from high-LET radiation for early cytogenetic end points. Radiat. Res. 170, 794– 802 (2008). 15. Morgan, W. F., Goetz, W. and Sowa, M. B. No bystander effect after irradiation of mammalian cells with a variable energy electron microbeam. J. Radiat. Res. 50, A91 (2009). 16. Sowa, M. B., Goetz, W., Baulch, J. E., Pyles, D. N., Dziegielewski, J., Yovino, S., Snyder, A. R., de Toledo,
Page 3 of 4
Downloaded from rpd.oxfordjournals.org at Pacific North West Libraries on January 7, 2011
POSSIBLE CAUSES OF VARIATION IN THE OBSERVATION OF NON-TARGETED EFFECTS
REFERENCES
SOWA ET AL.
17.
18. 19.
21.
22.
23. 24.
25.
26.
27. Mothersill, C. and Seymour, C. Medium from irradiated human epithelial cells but not human fibroblasts reduces the clonogenic survival of unirradiated cells. Int. J. Radiat. Biol. 71, 421–427 (1997). 28. Mitchell, S. A., Randers-Pehrson, G., Brenner, D. J. and Hall, E. J. The bystander response in C3H 10T1/2 cells: the influence of cell-to-cell contact. Radiat. Res. 161, 397–401 (2004). 29. Mothersill, C., Rea, D., Wright, E. G., Lorimore, S. A., Murphy, D., Seymour, C. B. and O’Malley, K. Individual variation in the production of a ‘bystander signal’ following irradiation of primary cultures of normal human urothelium. Carcinogenesis 22, 1465–1471 (2001). 30. Mothersill, C. and Seymour, C. Radiation-induced bystander effects: past history and future directions. Radiat. Res. 155, 759– 767 (2001). 31. Iyer, R. and Lehnert, B. E. Alpha-particle-induced increases in the radioresistance of normal human bystander cells. Radiat. Res. 157, 3 –7 (2002). 32. Wolff, S. The adaptive response in radiobiology: evolving insights and implications. Environ. Health Perspect. 106(Suppl. 1), 277– 283 (1998). 33. Tapio, S. and Jacob, V. Radioadaptive response revisited. Radiat. Environ. Biophys. 46, 1– 12 (2007). 34. Bosi, A. and Olivieri, G. Variability of the adaptive response to ionizing radiations in humans. Mutat. Res. 211, 13–17 (1989). 35. Raaphorst, G. P. and Boyden, S. Adaptive response and its variation in human normal and tumour cells. Int. J. Radiat. Biol. 75, 865–873 (1999). 36. Sowa, M. B., Murphy, M. K., Miller, J. H., McDonald, J. C., Strom, D. J. and Kimmel, G. A. A variable-energy electron microbeam: a unique modality for targeted low-LET radiation. Radiat. Res. 164, 695– 700 (2005). 37. Mothersill, C., Saroya, R., Smith, R. W., Singh, H. and Seymour, C. Serum serotonin levels determine the magnitude and type of bystander effects in medium transfer experiments. Radiat. Res. 174, 119– 123 (2010).
Page 4 of 4
Downloaded from rpd.oxfordjournals.org at Pacific North West Libraries on January 7, 2011
20.
S. M., Azzam, E. I. and Morgan, W. F. 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). Nagar, S., Smith, L. E. and Morgan, W. F. Characterization of a novel epigenetic effect of ionizing radiation: the death-inducing effect. Cancer Res. 63, 324– 328 (2003). Cherubini, R. personal communication. Gerashchenko, B. I. and Howell, R. W. Cell proximity is a prerequisite for the proliferative response of bystander cells co-cultured with cells irradiated with gammarays. Cytometry A 56, 71– 80 (2003). Shao, C., Aoki, M. and Furusawa, Y. Bystander effect on cell growth stimulation in neoplastic HSGc cells induced by heavy-ion irradiation. Radiat. Environ. Biophys. 42, 183– 187 (2003). Shao, C., Furusawa, Y., Aoki, M., Matsumoto, H. and Ando, K. Nitric oxide-mediated bystander effect induced by heavy-ions in human salivary gland tumour cells. Int. J. Radiat. Biol. 78, 837– 844 (2002). Yang, H., Anzenberg, V. and Held, K. D. The time dependence of bystander responses induced by iron-ion radiation in normal human skin fibroblasts. Radiat. Res. 168, 292–298 (2007). Nagasawa, H. and Little, J. B. Induction of sister chromatid exchanges by extremely low doses of alphaparticles. Cancer Res. 52, 6394– 6396 (1992). Folkard, M., Vojnovic, B., Prise, K. M., Bowey, A. G., Locke, R. J., Schettino, G. and Michael, B. D. A charged-particle microbeam: I. Development of an experimental system for targeting cells individually with counted particles. Int. J. Radiat. Biol. 72, 375–385 (1997). Randers-Pehrson, G., Geard, C. R., Johnson, G., Elliston, C. D. and Brenner, D. J. The Columbia University single-ion microbeam. Radiat. Res. 156, 210– 214 (2001). Sowa Resat, M. and Morgan, W. F. Microbeam developments and applications: a low linear energy transfer perspective. Cancer Metastasis Rev. 23, 323–331 (2004).
