| 1. |
- Acheva, Anna, et al.
(författare)
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Presence of Stromal Cells Enhances Epithelial-to-Mesenchymal Transition (EMT) Induction in Lung Bronchial Epithelium after Protracted Exposure to Oxidative Stress of Gamma Radiation
- 2019
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Ingår i: Oxidative Medicine and Cellular Longevity. - : Hindawi Limited. - 1942-0900 .- 1942-0994. ; 2019
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Tidskriftsartikel (refereegranskat)abstract
- The aim of the study was to investigate the role of a microenvironment in the induction of epithelial-to-mesenchymal transition (EMT) as a sign of early stages of carcinogenesis in human lung epithelial cell lines after protracted low-dose rate gamma-radiation exposures. BEAS-2B and HBEC-3KT lung cell lines were irradiated with low-dose rate gamma-rays (Cs-137, 1.4 or 14 mGy/h) to 0.1 or 1 Gy with or without adding TGF-beta. TGF-beta-treated samples were applied as positive EMT controls and tested in parallel to find out if the radiation has a potentiating effect on the EMT induction. To evaluate the effect of the stromal component, the epithelial cells were irradiated in cocultures with stromal MRC-9 lung fibroblasts. On day 3 post treatment, the EMT markers: alpha-SMA, vimentin, fibronectin, and E-cadherin, were analyzed. The oxidative stress levels were evaluated by 8-oxo-dG analysis in both epithelial and fibroblast cells. The protracted exposure to low Linear Energy Transfer (LET) radiation at the total absorbed dose of 1 Gy was able to induce changes suggestive of EMT. The results show that the presence of the stromal component and its signaling (TGF-beta) in the cocultures enhances the EMT. Radiation had a minor cumulative effect on the TGF-beta-induced EMT with both doses. The oxidative stress levels were higher than the background in both epithelial and stromal cells post chronic irradiation (0.1 and 1 Gy); as for the BEAS-2B cell line, the increase was statistically significant. We suggest that the induction of EMT in bronchial epithelial cells by radiation requires more than single acute exposure and the presence of stromal component might enhance the effect through free radical production and accumulation.
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| 2. |
- Andreassi, Maria Grazia, et al.
(författare)
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A Longitudinal Study of Individual Radiation Responses in Pediatric Patients Treated with Proton and Photon Radiotherapy, and Interventional Cardiology : Rationale and Research Protocol of the HARMONIC Project
- 2023
-
Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24:9, s. 8416-
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Tidskriftsartikel (refereegranskat)abstract
- The Health Effects of Cardiac Fluoroscopy and Modern Radiotherapy (photon and proton) in Pediatrics (HARMONIC) is a five-year project funded by the European Commission that aimed to improve the understanding of the long-term ionizing radiation (IR) risks for pediatric patients. In this paper, we provide a detailed overview of the rationale, design, and methods for the biological aspect of the project with objectives to provide a mechanistic understanding of the molecular pathways involved in the IR response and to identify potential predictive biomarkers of individual response involved in long-term health risks. Biological samples will be collected at three time points: before the first exposure, at the end of the exposure, and one year after the exposure. The average whole-body dose, the dose to the target organ, and the dose to some important out-of-field organs will be estimated. State-of-the-art analytical methods will be used to assess the levels of a set of known biomarkers and also explore high-resolution approaches of proteomics and miRNA transcriptomes to provide an integrated assessment. By using bioinformatics and systems biology, biological pathways and novel pathways involved in the response to IR exposure will be deciphered.
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| 3. |
- Averbeck, Dietrich, et al.
(författare)
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Establishing mechanisms affecting the individual response to ionizing radiation
- 2020
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 96:3, s. 297-323
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Forskningsöversikt (refereegranskat)abstract
- Purpose: Humans are increasingly exposed to ionizing radiation (IR). Both low (<100 mGy) and high doses can cause stochastic effects, including cancer; whereas doses above 100 mGy are needed to promote tissue or cell damage. 10-15% of radiotherapy (RT) patients suffer adverse reactions, described as displaying radiosensitivity (RS). Sensitivity to IR's stochastic effects is termed radiosusceptibility (RSu). To optimize radiation protection we need to understand the range of individual variability and underlying mechanisms. We review the potential mechanisms contributing to RS/RSu focusing on RS following RT, the most tractable RS group.Conclusions: The IR-induced DNA damage response (DDR) has been well characterized. Patients with mutations in the DDR have been identified and display marked RS but they represent only a small percentage of the RT patients with adverse reactions. We review the impacting mechanisms and additional factors influencing RS/RSu. We discuss whether RS/RSu might be genetically determined. As a recommendation, we propose that a prospective study be established to assess RS following RT. The study should detail tumor site and encompass a well-defined grading system. Predictive assays should be independently validated. Detailed analysis of the inflammatory, stress and immune responses, mitochondrial function and life style factors should be included. Existing cohorts should also be optimally exploited.
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| 4. |
- Babini, Gabriele, et al.
(författare)
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A systems radiation biology approach to unravel the role of chronic low-dose-rate gamma-irradiation in inducing premature senescence in endothelial cells
- 2022
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 17:3
-
Tidskriftsartikel (refereegranskat)abstract
- PurposeThe aim of this study was to explore the effects of chronic low-dose-rate gamma-radiation at a multi-scale level. The specific objective was to obtain an overall view of the endothelial cell response, by integrating previously published data on different cellular endpoints and highlighting possible different mechanisms underpinning radiation-induced senescence.Materials and methodsDifferent datasets were collected regarding experiments on human umbilical vein endothelial cells (HUVECs) which were chronically exposed to low dose rates (0, 1.4, 2.1 and 4.1 mGy/h) of gamma-rays until cell replication was arrested. Such exposed cells were analyzed for different complementary endpoints at distinct time points (up to several weeks), investigating cellular functions such as proliferation, senescence and angiogenic properties, as well as using transcriptomics and proteomics profiling. A mathematical model was proposed to describe proliferation and senescence.ResultsSimultaneous ceasing of cell proliferation and senescence onset as a function of time were well reproduced by the logistic growth curve, conveying shared equilibria between the two endpoints. The combination of all the different endpoints investigated highlighted a dose-dependence for prematurely induced senescence. However, the underpinning molecular mechanisms appeared to be dissimilar for the different dose rates, thus suggesting a more complex scenario.ConclusionsThis study was conducted integrating different datasets, focusing on their temporal dynamics, and using a systems biology approach. Results of our analysis highlight that different dose rates have different effects in inducing premature senescence, and that the total cumulative absorbed dose also plays an important role in accelerating endothelial cell senescence.
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| 5. |
- Barregård, Lars, 1948, et al.
(författare)
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Human and Methodological Sources of Variability in the Measurement of Urinary 8-Oxo-7,8-dihydro-2 '-deoxyguanosine
- 2013
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Ingår i: Antioxidants and Redox Signaling. - : Mary Ann Liebert Inc. - 1523-0864 .- 1557-7716. ; 18:18, s. 2377-2391
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Tidskriftsartikel (refereegranskat)abstract
- Aims: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (r(p) 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended.
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| 6. |
- Beltran-Pardo, Eliana A., et al.
(författare)
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Sequence analysis of the DNA-repair gene rad51 in the tardigrades Milnesium cf. tardigradum, Hypsibius dujardini and Macrobiotus cf. harmsworthi
- 2013
-
Ingår i: Journal of limnology. - : PAGEPress Publications. - 1129-5767 .- 1723-8633. ; 72, s. 80-91
-
Tidskriftsartikel (refereegranskat)abstract
- Tardigrades are known for being resistant to extreme conditions, including tolerance to ionising and UV radiation in both the hydrated and the dehydrated state. It is known that these factors may cause damage to DNA. It has recently been shown that single and double DNA strand breaks occur when tardigrades are maintained for a long time in the anhydrobiotic state. This may suggest that perhaps tardigrades rely on efficient DNA repair mechanisms. Among all proteins that comprise the DNA repair system, recombinases such as RecA or Rad51 have a very important function: DNA exchange activity. This enzyme is used in the homologous recombination and allows repair of the damaged strand using homologous non-damaged strands as a template. In this study, Rad51 induction was evaluated by western blot in Milnesium cf. tardigradum, after exposure to gamma radiation. The Rad51 protein was highly induced by radiation, when compared to the control. The rad51 genes were searched in three tardigrades: Milnesium cf. tardigradum, Hypsibius dujardini and Macrobiotus cf. harmsworthi. The gene sequences were obtained by preparing and sequencing transcriptome libraries for H. dujardini and M. cf. harmsworthi and designing rad51 degenerate primers specific for M. cf. tardigradum. Comparison of Rad51 putative proteins from tardigrades with other organisms showed that they are highly similar to the corresponding sequence from the nematode Trichinella spiralis. A structure-based sequence alignment from tardigrades and other organisms revealed that putative Rad51 predicted proteins from tardigrades contain the expected motifs for these important recombinases. In a cladogram tree based on this alignment, tardigrades tend to cluster together suggesting that they have selective differences in these genes that make them diverge between species. Predicted Rad51 structures from tardigrades were also compared with crystalline structure of Rad51 in Saccharomyces cerevisiae. These results reveal that S. cerevisiae Rad51 structure is very similar to that of the three analysed tardigrades. On the other hand the predicted structure of Rad51 from M. cf. harmsworthi and H. dujardini are closer related to each other, than each of them to that of M. cf. tardigradum.
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| 7. |
- Beltrán-Pardo, Eliana, et al.
(författare)
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Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development
- 2013
-
Ingår i: PLOS ONE. - 1932-6203. ; 8:9, s. e72098-
-
Tidskriftsartikel (refereegranskat)abstract
- Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies havedocumented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differentialeffects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effectof gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from theeutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, andlate stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses,while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival withhigher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might havetaken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and latestage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that thetolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this patternare unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of responsemechanisms to damaged DNA in the course of development.
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| 8. |
- Beltran-Pardo, Eliana, et al.
(författare)
-
Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development
- 2013
-
Ingår i: PLoS ONE. - : Public Library of Science. - 1932-6203. ; 8:9
-
Tidskriftsartikel (refereegranskat)abstract
- Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies havedocumented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differentialeffects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effectof gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from theeutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, andlate stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses,while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival withhigher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might havetaken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and latestage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that thetolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this patternare unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of responsemechanisms to damaged DNA in the course of development.
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| 9. |
- Beltran-Pardo, Eliana, et al.
(författare)
-
Sequence analysis of the DNA-repair gene rad51 in the tardigrades Milnesium cf. tardigradum, Hypsibius dujardini and Macrobiotus cf. harmsworthi
- 2013
-
Ingår i: Journal of limnology. - 1129-5767 .- 1723-8633. ; 72:s1, s. 80-91
-
Tidskriftsartikel (refereegranskat)abstract
- Tardigrades are known for being resistant to extreme conditions, including tolerance to ionising and UV radiation in both the hydratedand the dehydrated state. It is known that these factors may cause damage to DNA. It has recently been shown that single and double DNAstrand breaks occur when tardigrades are maintained for a long time in the anhydrobiotic state. This may suggest that perhaps tardigrades rely on efficient DNA repair mechanisms. Among all proteins that comprise the DNA repair system, recombinases such as RecA or Rad51 have a very important function: DNA exchange activity. This enzyme is used in the homologous recombination and allows repair of thedamaged strand using homologous non-damaged strands as a template. In this study, Rad51 induction was evaluated by western blot in Milnesium cf. tardigradum, after exposure to gamma radiation. The Rad51 protein was highly induced by radiation, when compared to the control. The rad51 genes were searched in three tardigrades: Milnesium cf. tardigradum, Hypsibius dujardini and Macrobiotus cf. harmsworthi. The gene sequences were obtained by preparing and sequencing transcriptome libraries for H. dujardini and M. cf. harmsworthi and designing rad51 degenerate primers specific for M. cf. tardigradum. Comparison of Rad51 putative proteins from tardigrades with other organisms showed that they are highly similar to the corresponding sequence from the nematode Trichinella spiralis. A structure-based sequence alignment from tardigrades and other organisms revealed that putative Rad51 predicted proteins from tardigrades contain the expected motifs for these important recombinases. In a cladogram tree based on this alignment, tardigrades tend to cluster together suggesting that they have selective differences in these genes that make them diverge between species. Predicted Rad51 structures from tardigrades were also compared with crystalline structure of Rad51 in Saccharomyces cerevisiae. These results reveal that S. cerevisiae Rad51 structure is very similar to that of the three analysed tardigrades. On the other hand the predicted structure of Rad51 from M. cf. harmsworthi and H. dujardini are closer related to each other, than each of them to that of M. cf. tardigradum.
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| 10. |
- Beltran-Pardo, Eliana, et al.
(författare)
-
Tolerance to Gamma Radiation in the Tardigrade Hypsibius dujardini from Embryo to Adult Correlate Inversely with Cellular Proliferation
- 2015
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:7
-
Tidskriftsartikel (refereegranskat)abstract
- Tardigrades are highly tolerant to desiccation and ionizing radiation but the mechanisms of this tolerance are not well understood. In this paper, we report studies on dose responses of adults and eggs of the tardigrade Hypsibius dujardini exposed to gamma radiation. In adults the LD50/48h for survival was estimated at similar to 4200 Gy, and doses higher than 100 Gy reduced both fertility and hatchability of laid eggs drastically. We also evaluated the effect of radiation (doses 50 Gy, 200 Gy, 500 Gy) on eggs in the early and late embryonic stage of development, and observed a reduced hatchability in the early stage, while no effect was found in the late stage of development. Survival of juveniles from irradiated eggs was highly affected by a 500 Gy dose, both in the early and the late stage. Juveniles hatched from eggs irradiated at 50 Gy and 200 Gy developed into adults and produced offspring, but their fertility was reduced compared to the controls. Finally we measured the effect of low temperature during irradiation at 4000 Gy and 4500 Gy on survival in adult tardigrades, and observed a slight delay in the expressed mortality when tardigrades were irradiated on ice. Since H. dujardini is a freshwater tardigrade with lower tolerance to desiccation compared to limno-terrestrial tardigrades, the high radiation tolerance in adults, similar to limno-terrestrial tardigrades, is unexpected and seems to challenge the idea that desiccation and radiation tolerance rely on the same molecular mechanisms. We suggest that the higher radiation tolerance in adults and late stage embryos of H. dujardini (and in other studied tardigrades) compared to early stage embryos may partly be due to limited mitotic activity, since tardigrades have a low degree of somatic cell division (eutely), and dividing cells are known to be more sensitive to radiation.
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| 11. |
- Brehwens, Karl, et al.
(författare)
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A new device to expose cells to changing dose rates of ionising radiation
- 2012
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Ingår i: Radiation Protection Dosimetry. - : Oxford University Press. - 0144-8420 .- 1742-3406. ; 148:3, s. 366-371
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Tidskriftsartikel (refereegranskat)abstract
- In many exposure scenarios to ionising radiation, the dose rate is not constant. Despite this, most in vitro studies aimed at investigating the effects of ionising radiation are carried out exposing samples at constant dose rates. Consequently, very little data exist on the biological effects of exposures to changing dose rates. This may be due to technical limitations of standard irradiation facilities, but also to the fact that the importance of research in this area has not been appreciated. We have recently shown that cells exposed to a decreasing dose rate suffer higher levels of cytogenetic damage than do cells exposed to an increasing or a constant dose rate. To further study the effects of changing dose rates, a new device was constructed that permits the exposure of cell samples in tubes, flasks or Petri dishes to changing dose rates of X-rays. This report presents the technical data, performance and dosimetry of this novel device.
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| 12. |
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| 13. |
- Brehwens, Karl, et al.
(författare)
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Cytogenetic damage in cells exposed to ionizing radiation under conditions of a changing dose rate
- 2010
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Ingår i: Radiation Research. - 0033-7587 .- 1938-5404. ; 173:3, s. 283-9
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Tidskriftsartikel (refereegranskat)abstract
- The current international paradigm on the biological effects of radiation is based mainly on the effects of dose with some consideration for the dose rate. No allowance has been made for the potential influence of a changing dose rate (second derivative of dose), and the biological effects of exposing cells to changing dose rates have never been analyzed. This paper provides evidence that radiation effects in cells may depend on temporal changes in the dose rate. In these experiments, cells were moved toward or away from an X-ray source. The speed of movement, the time of irradiation, and the temperature during exposure were controlled. Here we report the results of the first experiments with TK6 cells that were exposed at a constant dose rate, at an increasing dose rate, or at a decreasing dose rate. The average dose rate and the total dose were same for all samples. Micronuclei were scored as the end point. The results show that the level of cytogenetic damage was higher in cells exposed to a decreasing dose rate compared to both an increasing and a constant dose rate. This finding may suggest that the second derivative of dose may influence radiation risk estimates, and the results should trigger further studies on this issue.
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| 14. |
- Brehwens, Karl, 1983-
(författare)
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In vitro and in vivo aspects of intrinsic radiosensitivity
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis focuses on how physical and biological factors influence the outcome of exposures to γ/X-rays. That the dose rate changes during real life exposure scenarios is well-known, but radiobiological data from exposures performed at increasing or decreasing dose rates is lacking. In paper I, it was found that an exposure where the dose rate decreases exponentially induces significantly higher levels of micronuclei in TK6 cells than exposures at an increasing or constant dose rate. Paper II describes the construction and validation of novel exposure equipment used to further study this “decreasing dose rate effect”, which is described in paper III. In paper I we also observed a radioprotective effect when cells were exposed on ice. This “temperature effect” (TE) has been known for decades but it is still not fully understood how hypothermia acts in a radioprotective manner. This was investigated in paper IV, where a multiparametric approach was used to investigate the underlying mechanisms. In paper V the aim was to investigate the role of biomarkers and clinical parameters as possible risk factors for late adverse effects to radiotherapy (RT). This was studied in a rare cohort of head-and-neck cancer patients that developed mandibular osteoradionecrosis (ORN) as a severe late adverse effect of RT. Biomarker measurements and clinical factors were then subjected to multivariate analysis in order to identify ORN risk factors. The results suggest that the patient’s oxidative stress response is an important factor in ORN pathogenesis, and support the current view that patient-related factors constitute the largest source of variation seen in the frequency of late adverse effects to RT.In summary, this thesis provides new and important insights into the roles of biological and physical factors in determining the consequences of γ/X-ray exposures.
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| 15. |
- Brehwens, Karl, 1983-, et al.
(författare)
-
Micronucleus frequencies and clonogenic cell survival in TK6 cells exposed to changing dose rates under controlled temperature conditions
- 2014
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 90:3, s. 241-247
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: In most exposure scenarios the dose rate of exposure is not constant. Despite this, very little information exists on the possible biological effects of exposing cells to radiation under the conditions of a changing dose rate. The current study highlights interesting effects following exposure under these conditions.Materials and methods: We constructed a new exposure facility that allows exposing cells inside an incubator and used it to irradiate human lymphoblastoid TK6 cells both after a moderate (0.48 Gy) and a high (1.1 Gy) dose, where the change in dose rate was, respectively, ≈ 17-fold change (2.2 - 37 mGy/min) and ≈ 39-fold (2.7 - 106 mGy/min). Clonogenic survival and micronuclei (MN) induction were the chosen endpoints.Results: The obtained results confirm the outcome of our first study that TK6 cells exposed to a decreasing dose rate express more MN than cells exposed to an increasing or constant dose rate. The effect was not seen after the moderate dose of 0.48 Gy or detectable at the level of clonogenic cell survival.Conclusions: We speculate that the high level of MN is probably related to a delayed elimination of damaged cells by interphase death, as opposed to mechanisms relating to DNA damage and repair.
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| 16. |
- Chandna, Sudhir, et al.
(författare)
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Agarose overlay selectively improves macrocolony formation and radiosensitivity assessment in primary fibroblasts
- 2014
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 90:5, s. 401-406
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Primary fibroblasts are not suitable for in vitro macrocolony assay due to their inability to form distinct colonies. Here we present a modification of agarose overlay that yielded extensive improvement in their colony formation and assessment of radiosensitivity. Materials and methods: Macrocolony formation was assessed in primary human fibroblasts VH10 and HDFn with or without overlay using 0.5% agarose in growth medium at 24 h post-seeding. Malignant human cell lines (A549, U87) and transformed nonmalignant fibroblasts (AA8 hamster, MRC5 human) were used for comparison. Results: Agarose overlay caused significant improvement marked by early appearance (one week) of distinct colonies with high cell density and multifold higher plating efficiency than conventional macrocolony assay in VH10 and HDFn human fibroblasts. Compared to conventional assay or feeder cell supplementation, agarose overlay resulted in broader cell morphology due to improved adherence, and yielded more compact colonies. Gamma-radiation dose-response survival curves could be successfully generated for both fibroblast cell lines using this method, which yielded no such effects in the transformed/malignant cell lines tested. Conclusion: This easy and inexpensive 'agarose overlay technique' significantly and selectively improves the fibroblast plating efficiency, thus considerably reducing time and effort to greatly benefit the survival studies on primary fibroblasts.
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| 17. |
- Cheng, Lei, et al.
(författare)
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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
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 18. |
- Cheng, Lei, 1981-
(författare)
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Factors modifying cellular response to ionizing radiation
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many physical factors influence the biological effect of exposure to ionizing radiation, including radiation quality, dose rate and temperature. This thesis focuses on how these factors influence the outcome of exposure and the mechanisms behind the cellular response. Mixed beam exposure, which is the combination of different ionizing radiations, occurs in many situations and the effects are important to understand for radiation protection and effect prediction. Recently, studies show that the effect of simultaneous irradiation with different qualities is greater than simple additivity of single radiation types, which is called a synergistic effect. But its mechanism is unclear. In Paper I, II and III, alpha particles and X-rays were used to study the effect of mixed beams. Paper I shows that mixed exposure induced a synergistic effect in generating double strand breaks (DSB), and these DSB were repaired by slow kinetics in U2OS cells. In Paper II, alkaline comet assay was applied to investigate the induction and repair of DNA lesions including DSB, single strand breaks and alkali labile sites in peripheral blood lymphocytes (PBL). We demonstrate that mixed beams interact in inducing DNA damage and influencing DNA damage response (DDR), which result in a delay of DNA repair. Both in Paper I and II, mixed beams showed a capability in inducing higher activity of DDR proteins than expected from additivity. Paper III investigates selected DDR-related gene expression levels after exposure to mixed beams in PBL from 4 donors. Synergy was present for all donors but the results suggested individual variability in the response to mixed beams, most likely due to life style changes.Low temperature at exposure is radioprotective at the level of cytogenetic damage. In Paper IV, data indicate that this effect is through promotion of DNA repair, which leads to reduced transformation of DNA damage into chromosomal aberrations. Paper V aims to compare the biological effectiveness of gamma radiation delivered at a very high dose rate (VHDR) with that of a high dose rate (HDR) in order to optimize chronic exposure risk prediction based on the data of atomic bomb survivors. The results suggest that VHDR gamma radiation is more effective in inducing DNA damage than HDR.
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| 19. |
- Cheng, Lei, et al.
(författare)
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Modulation of radiation-induced cytogenetic damage in human peripheral blood lymphocytes by hypothermia
- 2015
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Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis. - : Elsevier BV. - 1383-5718 .- 1879-3592. ; 793:SI, s. 96-100
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Recent studies have shown that low temperature (hypothermia) at exposure can act in a radioprotective manner at the level of cytogenetic damage. The mechanisms of this phenomenon are not understood, but it was suggested to be due to hypothermia-induced perturbations of the cell cycle. The purpose of the present study was to detect whether a reduced frequency of micronuclei is observed in peripheral blood lymphocytes (PBL) irradiated at low temperature and harvested sequentially at 3 time points. Additionally, the level of apoptosis was estimated by microscopic analysis of the MN slides. Materials and methods: Experiments were carried out with blood drawn from three donors at the Stockholm University and from three donors at the Jan Kochanowski University. Prior to irradiation, blood samples were incubated for 20 mm and irradiated at the respective temperature (0 degrees C and 37 degrees C) with gamma rays. Whole blood cultures were set up, cytochalasin B was added after 44h of irradiation and the samples were harvested after 72,96 and 120 h of incubation time. Results and conclusions: The frequency of micronuclei was markedly lower in PBL harvested at 72h, 96 h and 120 h following irradiation at 0 degrees C as compared to 37 degrees C. This indicates that the temperature effect observed in peripheral blood lymphocytes after irradiation is not related to a temporary perturbation of the cell cycle. Also, it is not due to selective elimination of damaged cells by apoptosis.
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| 20. |
- Cheng, Lei, et al.
(författare)
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Simultaneous induction of dispersed and clustered DNA lesions compromises DNA damage response in human peripheral blood lymphocytes
- 2018
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 13:10
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Tidskriftsartikel (refereegranskat)abstract
- Due to its ability to induce DNA damage in a space and time controlled manner, ionising radiation is a unique tool for studying the mechanisms of DNA repair. The biological effectiveness of ionising radiation is related to the ionisation density which is defined by the linear energy transfer (LET). Alpha particles are characterised by high LET, while X-rays by low LET values. An interesting question is how cells react when exposed to a mixed beam of high and low LET radiation. In an earlier study carried out with human peripheral blood lymphocytes (PBL) we could demonstrate that alpha radiation X-rays 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. PBL were exposed to various doses of alpha particles, X-rays and mixed beams. 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 ATM, p53 and DNA-PK were measured by Western blotting and mRNA levels of 6 damage-responsive genes were measured by qPCR. 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. The activation levels of DDR proteins and mRNA levels of the studied genes were highest in cells exposed to mixed beams. The results substantiate the idea that exposure to mixed beams presents a challenge for the cellular DDR system.
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| 21. |
- D. V. Godoy, Paulo R., et al.
(författare)
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Targeting NRF2, Regulator of Antioxidant System, to Sensitize Glioblastoma Neurosphere Cells to Radiation-Induced Oxidative Stress
- 2020
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Ingår i: Oxidative Medicine and Cellular Longevity. - : Hindawi Limited. - 1942-0900 .- 1942-0994. ; 2020
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Tidskriftsartikel (refereegranskat)abstract
- The presence of glioma stem cells (GSCs), which are enriched in neurospheres, may be connected to the radioresistance of glioblastoma (GBM) due to their enhanced antioxidant defense and elevated DNA repair capacity. The aim was to evaluate the responses to different radiation qualities and to reduce radioresistance of U87MG cells, a GBM cell line. U87MG cells were cultured in a 3D model and irradiated with low (24 mGy/h) and high (0.39 Gy/min) dose rates of low LET gamma and high LET carbon ions (1-2 Gy/min). Thereafter, expression of proteins related to oxidative stress response, extracellular 8-oxo-dG, and neurospheres were determined. LD50 for carbon ions was significantly lower compared to LD50 of high and low dose rate gamma radiation. A significantly higher level of 8-oxo-dG was detected in the media of cells exposed to a low dose rate as compared to a high dose rate of gamma or carbon ions. A downregulation of oxidative stress proteins was also observed (NRF2, hMTH1, and SOD1). The NRF2 gene was knocked down by CRISPR/Cas9 in neurosphere cells, resulting in less self-renewal, more differentiated cells, and less proliferation capacity after irradiation with low and high dose rate gamma rays. Overall, U87MG glioma neurospheres presented differential responses to distinct radiation qualities and NRF2 plays an important role in cellular sensitivity to radiation.
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| 22. |
- Dang, Li, et al.
(författare)
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Radioprotective effect of hypothermia on cells - a multiparametric approach to delineate the mechanisms
- 2012
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 88:7, s. 507-514
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Low temperature (hypothermia) during irradiation of cells has been reported to have a radioprotective effect. The mechanisms are not fully understood. This study further investigates the possible mechanisms behind hypothermia-mediated radioprotection. Materials and methods: Human lymphoblastoid TK6 cells were incubated for 20 min at 0.8 or 37 degrees C and subsequently exposed to 1 Gy of gamma- or X-rays. The influence of ataxia telangiectasia mutated (ATM)-mediated double-strand break signalling and histone deacetylase-dependent chromatin condensation was investigated using the micronucleus assay. Furthermore, the effect of hypothermia was investigated at the level of phosphorylated histone 2AX (gamma H2AX) foci, clonogenic cell survival and micronuclei in sequentially-harvested cells. Results: The radioprotective effect of hypothermia (called the temperature effect [TE]) was evident only at the level of micronuclei at a single fixation time, was not influenced by the inhibition of ATM kinase activity and completely abolished by the histone deacetylase inhibition. No TE was seen at the level of gamma H2AX foci and cell survival. Conclusions: We suggest that low temperature during irradiation can induce a temporary cell cycle shift, which could lead to a reduced micronucleus frequency. Future experiments focused on cell cycle progression are needed to confirm this hypothesis.
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| 23. |
- Danielsson, Daniel, et al.
(författare)
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Influence of genetic background and oxidative stress response on risk of mandibular osteoradionecrosis after radiotherapy of head and neck cancer
- 2016
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Ingår i: Head and Neck. - : Wiley. - 1043-3074 .- 1097-0347. ; 38:3, s. 387-393
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Tidskriftsartikel (refereegranskat)abstract
- Background: Osteoradionecrosis (ORN) of the mandible is a severe complication of head and neck radiotherapy (RT) treatment, where the impact of individual radiosensitivity has been a suggested explanation. Methods: A cohort of patients with stage II/III ORN was compared to matched controls. Blood was collected and irradiated in vitro to study the capacity to handle radiation-induced oxidative stress. Patients were also genotyped for 8 single-nucleotide polymorphisms (SNPs) in genes involved in the oxidative stress response. Results: A difference in 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dG) levels was found between the patient cohorts (p = 0.01). The SNP rs1695 in glutathione s-transferase p1 (GSTP1) was also found to be more frequent in the patients with ORN (p = .02). Multivariate analysis of the clinical and biological factors revealed concomitant brachytherapy plus the 2 biomarkers to be significant factors which influense risk of mandibular osteoradionecrosis after radiotherapy of head and neck cancer. Conclusion: The current study indicates that oxidative stress response contributes to individual radiosensitivity and healthy tissue damage caused by RT and may be predicted by biomarker analysis.
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| 24. |
- Danielsson, D., et al.
(författare)
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Osteoradionecrosis, an increasing indication for microvascular head and neck reconstruction
- 2020
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Ingår i: International Journal of Oral and Maxillofacial Surgery. - : Elsevier BV. - 0901-5027 .- 1399-0020. ; 49:1, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- Better cancer treatment has led to a steadily growing population of cancer survivors suffering from late adverse effects after cancer treatment. The aim of this study was to investigate whether there has been an increase in free flap reconstruction due to osteoradionecrosis (ORN). A retrospective review was conducted to identify all consecutive head and neck free flap reconstructions performed over an 18-year period (1995-2012) at Karolinska University Hospital. A total of 235 free flaps were identified. Cases were divided into two groups: head and neck cancer reconstructions and ORN reconstructions. A comparison between the two groups showed longer survival (P < 0.001) and higher rates of late complications (P < 0.001) among ORN cases. ORN as an indication for reconstruction increased over time, from 7.0% of the total number of free flaps performed in 1995-2000, to 15.2% during the period 2001-2006, and to 27.3% in 2007-2012 (P < 0.001). This, in accordance with the results of other studies, highlights the importance of the appropriate allocation of resources within the healthcare system to treat this patient group within the steadily increasing population of cancer survivors.
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| 25. |
- D'Auria Vieira de Godoy, Paulo Roberto, et al.
(författare)
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Effect of dose and dose rate of gamma irradiation on the formation of micronuclei in bone marrow cells isolated from whole-body-irradiated mice
- 2021
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Ingår i: Environmental and Molecular Mutagenesis. - : Wiley. - 0893-6692 .- 1098-2280. ; 62:7, s. 422-427
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Tidskriftsartikel (refereegranskat)abstract
- It is well-known that the cytotoxicity and mutagenic effects of high dose rate (HDR) ionizing radiation (IR) are increased by increasing the dose but less is known about the effects of chronic low dose rate (LDR). In vitro, we have shown that in addition to the immediate interaction of IR with DNA (the direct and indirect effects), low doses and chronic LDR exposure induce endogenous oxidative stress. During elevated oxidative stress, reactive oxygen species (ROS) react with DNA modifying its structure. Here, BL6 mice were exposed to IR at LDR and HDR and were then sacrificed 3 hours and 3 weeks after exposure to examine early and late effects of IR. The levels of micronuclei, MN, were determined in bone marrow cells. Our data indicate that the effects of 200 mGy on MN-induction are transient, but 500 and 1000 mGy (both HDR and LDR) lead to increased levels of MN up to 3 weeks after the exposure.
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