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Cellular responses to combined irradiation with alpha particles and X-rays
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholms University. (Wojcik)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mixed radiation fields, where different ionizing particles act together, are very important in radiobiology and in radiation protection. Mixed beams are not only the most common form of radiation exposure, but the prediction of their biological effect is also full of uncertainties. Currently, prediction of the biological damage of exposure to mixed radiation fields is based on the default assumption of simple additivity between the effects of all the radiation in the field. This assumption has been proven to be incorrect. Indeed, the simultaneous effect of different radiation qualities has been shown to be greater than additive, namely synergistic. This implicates that, for instance, the predicted cancer risk for astronauts, that remain a prolonged time in space, is currently underestimated as well as the risk of developing secondary cancer for radiotherapy patients.

This thesis aims at understanding the mechanisms behind the cellular response to simultaneous exposure to alpha particles and X-rays (that is referred as mixed beam).

Paper I describes the cell killing and the mutagenic effect of mixed beam exposure in human lymphoblastoid wild type and in cells with impaired capacity to repair oxidative DNA damage .We found that oxidative DNA damage plays an important role in the lethal, synergistic effect of mixed beams.

Paper II and III investigates whether mixed beams exposure leads to an augmented DNA double strand breaks (DSB) induction or to an altered response of the cellular DSB repair machinery. We found that mixed irradiation resulted in synergistic induction of DSB, and that those lesions were repaired with slow kinetics.

Paper IV focuses on the effect of mixed beams at the level of DNA damage in normal cells. Induction and repair of DNA lesions such as DSB, single strand breaks and apurinic sites was quantified using the alkaline comet assay. We found that alpha particles and X-rays interacted in inducing DNA damage. Moreover, although mixed beam exposure resulted in strong activation of the DNA damage response, it resulted in delayed repair.

Although more research is needed to fully elucidate the mechanisms behind the detected synergistic effects, our results strongly suggest that an overwhelmed DNA-repair system causes delay in repair of damage.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2017. , 88 p.
Keyword [en]
Radiation, DNA damage, mutations, alpha particles, X-rays, mixed beam
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-145509ISBN: 978-91-7649-887-3 (print)ISBN: 978-91-7649-888-0 (electronic)OAI: oai:DiVA.org:su-145509DiVA: diva2:1129909
Public defence
2017-09-27, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Radiation Safety Authority
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Accepted. Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2017-09-04 Created: 2017-08-07 Last updated: 2017-08-28Bibliographically approved
List of papers
1. Interaction of low and high LET radiation in TK6 cells-mechanistic aspects and significance for radiation protection
Open this publication in new window or tab >>Interaction of low and high LET radiation in TK6 cells-mechanistic aspects and significance for radiation protection
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2016 (English)In: Journal of Radiological Protection, ISSN 0952-4746, E-ISSN 1361-6498, Vol. 36, no 4, 721-735 p.Article in journal (Refereed) Published
Abstract [en]

Most environmental, occupational and medical exposures to ionising radiation are associated with a simultaneous action of different radiation types. An open question remains whether radiations of different qualities interact with each other to yield effects stronger than expected based on the assumption of additivity. It is possible that DNA damage induced by high linear energy transfer (LET) radiation will lead to an opening of the chromatin structure making the DNA more susceptible to attack by reactive oxygen species (ROS) generated by the low LET radiation. In such case, the effect of mixed beams should be strongly expressed in cells that are sensitive to ROS. The present investigation was carried out to test if cells with an impaired capacity to handle oxidative stress are particularly sensitive to the effect of mixed beams of alpha particles and x-rays. Clonogenic cell survival curves and mutant frequencies were analysed in TK6 wild type (wt) cells and in TK6 cells with a knocked down hMYH glycosylase. The results showed a synergistic effect of mixed beams on clonogenic cell survival of TK6(wt) but not TK6(MYH)-cells. The frequencies of mutants showed a high degree of interexperimental variability without any indications for synergistic effects of mixed beams. TK6(MYH)-cells were generally more tolerant to radiation exposure with respect to clonogenic cell survival but showed a strong increase in mutant frequency. The results demonstrate that exposure of wt cells to a mixed beam of alpha particles and x-rays leads to a detrimental effect which is stronger than expected based on the assumption of additivity. The role of oxidative stress in the reaction of cells to mixed beams remains unclear.

Keyword
mutations, cell survival, low LET radiation, high LET radiation, mixed beams, risk assessment
National Category
Cell Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-136254 (URN)10.1088/0952-4746/36/4/721 (DOI)000385422900001 ()
Available from: 2016-12-08 Created: 2016-12-01 Last updated: 2017-08-10Bibliographically approved
2. Alpha particles and X-rays interact in inducing DNA damage in U2OS cells
Open this publication in new window or tab >>Alpha particles and X-rays interact in inducing DNA damage in U2OS cells
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2017 (English)In: Radiation Research, ISSN 0033-7587, E-ISSN 1938-5404Article in journal (Refereed) Accepted
Abstract [en]

The survivors of atomic bomb explosions in Hiroshima and Nagasaki are monitored for health effect within the Life Span Study (LSS). The LSS results represent the most important source of knowledge about cancer effects of ionizing radiation and they form the basis for the radiation protection system. One uncertainty connected to deriving universal risk factors from these results is related to the problem of mixed radiation qualities. The atomic bomb explosions generated a mixed beam of the sparsely ionizing gamma radiation and densely ionizing neutrons and what is not taken into consideration is the problem of a possible interaction of the two radiation types in inducing biological effects. The existence of such interaction would suggest that the application of risk factors derived from the LSS to predict cancer effects after exposure to pure gamma radiation (such as in the Fukushima prefecture) leads to an overestimation of risk.In order to analyze the possible interaction of radiation types a mixed beam exposure facility was constructed where cells can be exposed to sparsely ionizing X-rays and densely ionizing alpha particles. U2OS cells were used, which are stably transfected with a plasmid coding for the DNA repair gene 53BP1 coupled to a gene coding for the green fluorescent protein GFP. Induction and repair of DNA damage which are known to be related to cancer induction were analyzed. The results suggest that alpha particles and X-rays interact, leading to cellular, and possibly cancer effects not predictable based on assuming simple additivity of the individual mixed beam components.

Keyword
Mixed beam, radiation, DNA damage, Double srtand breaks, 53BP1, alpha particles, X-rays
National Category
Cell Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-145502 (URN)
Funder
Swedish Radiation Safety Authority
Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2017-08-10
3. Live dynamics of 53BP1 DNA damage foci induced by a combination of clustered and dispersed double strand breaks
Open this publication in new window or tab >>Live dynamics of 53BP1 DNA damage foci induced by a combination of clustered and dispersed double strand breaks
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cells react differently to alpha particle-induced clustered DNA damage and X-ray induced dispersed DNA damage. Little is known about how they cope when both types of damage are induced simultaneously. We used live cell microscopy to analyse the formation and motion of the DNA double strand break (DSB) repair factor 53BP1-GFP foci in U2OS cells exposed to a mixed beam of alpha particles and X-rays. We observed that the kinetics of appearance and decline of mixed beam-induced foci resemble that of after alpha particle irradiation. They showed a fast increase and almost no repair during the imaging time, while X-ray-exposed cells showed a strong increase and decline over the observation interval. Secondly, focus sizes were similar to X-ray-induced foci, with both being significantly smaller than those induced by alpha particles; thirdly, they showed a high mean pixel intensity already at early time point after irradiation. Finally, focus mobility was reduced relative to alpha particle and X-ray-induced foci, which was correlated with a delayed appearance of a fraction of mixed beam-induced foci. These results suggest that cells react to a simultaneous induction of clustered and dispersed DNA damage by sequestering the 53BP1 protein in selected foci, possibly at sites of clustered DNA damage. This happens at the cost of foci forming at chromatin areas containing dispersed, simple damage. Our results highlight that the DNA damage response to a combination of dispersed and clustered DNA damage in the same cell differs significantly from the response to exposure with single radiation types.

Keyword
Mixed beam, radiation, DNA damage, 53BP1 foci
National Category
Cell Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-145503 (URN)
Funder
Swedish Radiation Safety Authority
Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2017-08-10Bibliographically approved
4. Comet assay reveals an interaction of DNA lesions and impairment of DNA repair in peripheral blood lymphocytes simultaneously exposed to alpha particles and X-rays
Open this publication in new window or tab >>Comet assay reveals an interaction of DNA lesions and impairment of DNA repair in peripheral blood lymphocytes simultaneously exposed to alpha particles and X-rays
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The biological effectiveness of ionising radiation is related to the ionisation density which is defined by the linear energy transfer LET. Radiation quality factors are applied to calculate the equivalent dose in the field of radiation protection and the biologically effective dose in the field of radiotherapy. Additivity is assumed in exposure scenarios where radiations of different qualities are mixed. We have carried out a series of studies on the cytogenetic effect of exposing human peripheral blood lymphocytes to a mixed beam of the high LET alpha radiation and low LET X-rays and could demonstrate that both radiations interact in producing more chromosomal aberrations than expected based on additivity. The aim of the present investigation was to look at the mechanism of the interaction, especially with respect to the question if it is due to an augmented level of initial damage or impaired DNA repair. The level of DNA damage and the kinetics of damage repair was quantified by the alkaline comet assay. The levels of phosphorylated, key DNA damage response (DDR) proteins were also measured by Western blotting. The results revealed that alpha particles and X-rays interact in inducing DNA damage above the level predicted by assuming additivity and that the repair of damage occurs with a delay. Moreover, the activation levels of the key DDR proteins ATM, p53 and DNA PK were highest in cells exposed to mixed beams substantiating the idea exposure to mixed beams presents a challenge to the cellular DNA damage response system. 

Keyword
Mixed radiation fields, DNA damage, Comet assay
National Category
Cell Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-145505 (URN)
Funder
Swedish Radiation Safety Authority
Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2017-08-10Bibliographically approved

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