| 1. |
- Abend, M., et al.
(författare)
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Inter-laboratory comparison of gene expression biodosimetry for protracted radiation exposures as part of the RENEB and EURADOS WG10 2019 exercise
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Large-scale radiation emergency scenarios involving protracted low dose rate radiation exposure (e.g. a hidden radioactive source in a train) necessitate the development of high throughput methods for providing rapid individual dose estimates. During the RENEB (Running the European Network of Biodosimetry) 2019 exercise, four EDTA-blood samples were exposed to an Iridium-192 source (1.36 TBq, Tech-Ops 880 Sentinal) at varying distances and geometries. This resulted in protracted doses ranging between 0.2 and 2.4 Gy using dose rates of 1.5-40 mGy/min and exposure times of 1 or 2.5 h. Blood samples were exposed in thermo bottles that maintained temperatures between 39 and 27.7 degrees C. After exposure, EDTA-blood samples were transferred into PAXGene tubes to preserve RNA. RNA was isolated in one laboratory and aliquots of four blinded RNA were sent to another five teams for dose estimation based on gene expression changes. Using an X-ray machine, samples for two calibration curves (first: constant dose rate of 8.3 mGy/min and 0.5-8 h varying exposure times; second: varying dose rates of 0.5-8.3 mGy/min and 4 h exposure time) were generated for distribution. Assays were run in each laboratory according to locally established protocols using either a microarray platform (one team) or quantitative real-time PCR (qRT-PCR, five teams). The qRT-PCR measurements were highly reproducible with coefficient of variation below 15% in >= 75% of measurements resulting in reported dose estimates ranging between 0 and 0.5 Gy in all samples and in all laboratories. Up to twofold reductions in RNA copy numbers per degree Celsius relative to 37 degrees C were observed. However, when irradiating independent samples equivalent to the blinded samples but increasing the combined exposure and incubation time to 4 h at 37 degrees C, expected gene expression changes corresponding to the absorbed doses were observed. Clearly, time and an optimal temperature of 37 degrees C must be allowed for the biological response to manifest as gene expression changes prior to running the gene expression assay. In conclusion, dose reconstructions based on gene expression measurements are highly reproducible across different techniques, protocols and laboratories. Even a radiation dose of 0.25 Gy protracted over 4 h (1 mGy/min) can be identified. These results demonstrate the importance of the incubation conditions and time span between radiation exposure and measurements of gene expression changes when using this method in a field exercise or real emergency situation.
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| 2. |
- Abend, M., et al.
(författare)
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RENEB Inter-Laboratory Comparison 2021 : The Gene Expression Assay
- 2023
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Ingår i: Radiation Research. - 0033-7587 .- 1938-5404. ; 199:6, s. 598-615
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Tidskriftsartikel (refereegranskat)abstract
- Early and high-throughput individual dose estimates are essential following large-scale radiation exposure events. In the context of the Running the European Network for Biodosimetry and Physical Dosimetry (RENEB) 2021 exercise, gene expression assays were conducted and their corresponding performance for dose-assessment is presented in this publication. Three blinded, coded whole blood samples from healthy donors were exposed to 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 1 Gy/min) using the X-ray source Yxlon. These exposures correspond to clinically relevant groups of unexposed, low dose (no severe acute health effects expected) and high dose exposed individuals (requiring early intensive medical health care). Samples were sent to eight teams for dose estimation and identification of clinically relevant groups. For quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray analyses, samples were lysed, stored at 20°C and shipped on wet ice. RNA isolations and assays were run in each laboratory according to locally established protocols. The time-to-result for both rough early and more precise later reports has been documented where possible. Accuracy of dose estimates was calculated as the difference between estimated and reference doses for all doses (summed absolute difference, SAD) and by determining the number of correctly reported dose estimates that were defined as ±0.5 Gy for reference doses <2.5 Gy and ±1.0 Gy for reference doses >3 Gy, as recommended for triage dosimetry. We also examined the allocation of dose estimates to clinically/diagnostically relevant exposure groups. Altogether, 105 dose estimates were reported by the eight teams, and the earliest report times on dose categories and estimates were 5 h and 9 h, respectively. The coefficient of variation for 85% of all 436 qRT-PCR measurements did not exceed 10%. One team reported dose estimates that systematically deviated several-fold from reported dose estimates, and these outliers were excluded from further analysis. Teams employing a combination of several genes generated about two-times lower median SADs (0.8 Gy) compared to dose estimates based on single genes only (1.7 Gy). When considering the uncertainty intervals for triage dosimetry, dose estimates of all teams together were correctly reported in 100% of the 0 Gy, 50% of the 1.2 Gy and 50% of the 3.5 Gy exposed samples. The order of dose estimates (from lowest to highest) corresponding to three dose categories (unexposed, low dose and highest exposure) were correctly reported by all teams and all chosen genes or gene combinations. Furthermore, if teams reported no exposure or an exposure >3.5 Gy, it was always correctly allocated to the unexposed and the highly exposed group, while low exposed (1.2 Gy) samples sometimes could not be discriminated from highly (3.5 Gy) exposed samples. All teams used FDXR and 78.1% of correct dose estimates used FDXR as one of the predictors. Still, the accuracy of reported dose estimates based on FDXR differed considerably among teams with one team's SAD (0.5 Gy) being comparable to the dose accuracy employing a combination of genes. Using the workflow of this reference team, we performed additional experiments after the exercise on residual RNA and cDNA sent by six teams to the reference team. All samples were processed similarly with the intention to improve the accuracy of dose estimates when employing the same workflow. Re-evaluated dose estimates improved for half of the samples and worsened for the others. In conclusion, this inter-laboratory comparison exercise enabled (1) identification of technical problems and corrections in preparations for future events, (2) confirmed the early and high-throughput capabilities of gene expression, (3) emphasized different biodosimetry approaches using either only FDXR or a gene combination, (4) indicated some improvements in dose estimation with FDXR when employing a similar methodology, which requires further research for the final conclusion and (5) underlined the applicability of gene expression for identification of unexposed and highly exposed samples, supporting medical management in radiological or nuclear scenarios.
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| 3. |
- Akuwudike, Pamela, et al.
(författare)
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Cell Type-Specific Patterns in the Accumulation of DNA Damage Following Multifractional Radiation Exposure
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:21
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Tidskriftsartikel (refereegranskat)abstract
- Predicting the risk of second malignant neoplasms is complicated by uncertainties regarding the shape of the dose–response relationship at high doses. Limited understanding of the competitive relationship between cell killing and the accumulation of DNA lesions at high doses, as well as the effects of other modulatory factors unique to radiation exposure during radiotherapy, such as dose heterogeneity across normal tissue and dose fractionation, contribute to these uncertainties. The aim of this study was to analyze the impact of fractionated irradiations on two cell systems, focusing on the endpoints relevant for cancer induction. To simulate the heterogeneous dose distribution across normal tissue during radiotherapy, exponentially growing VH10 fibroblasts and AHH-1 lymphoblasts were irradiated with 9 and 12 fractions (VH10) and 10 fractions (AHH-1) at 0.25, 0.5, 1, or 2 Gy per fraction. The effects on cell growth, cell survival, radiosensitivity and the accumulation of residual DNA damage lesions were analyzed as functions of dose per fraction and the total absorbed dose. Residual γH2AX foci and other DNA damage markers (micronuclei, nuclear buds, and giant nuclei) were accumulated at high doses in both cell types, but in a cell type-dependent manner. The competitive relationship between cell killing and the accumulation of carcinogenic DNA damage following multifractional radiation exposure is cell type-specific.
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| 4. |
- Akuwudike, Pamela, 1987-, et al.
(författare)
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Impact of fractionated cisplatin and radiation treatment on cell growth and accumulation of DNA damage in two normal cell types differing in origin
- 2023
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Ingår i: Scientific Reports. - 2045-2322. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Evidence on the impact of chemotherapy on radiotherapy-induced second malignant neoplasms is controversial. We estimated how cisplatin modulates the in vitro response of two normal cell types to fractionated radiation. AHH-1 lymphoblasts and VH10 fibroblasts were irradiated at 1 Gy/fraction 5 and 3 times per week during 12 and 19 days, respectively, and simultaneously treated with 0.1, 0.2, 0.4, 0.8, 1.7 and 3.3 µM of cisplatin twice a week. Cell growth during treatment was monitored. Cell growth/cell death and endpoints related to accumulation of DNA damage and, thus, carcinogenesis, were studied up to 21 days post treatment in cells exposed to radiation and the lowest cisplatin doses. Radiation alone significantly reduced cell growth. The impact of cisplatin alone below 3.3 µM was minimal. Except the lowest dose of cisplatin in VH10 cells, cisplatin reduced the inhibitory effect of radiation on cell growth. Delayed cell death was highest in the combination groups while the accumulation of DNA damage did not reveal a clear pattern. In conclusion, fractionated, concomitant exposure to radiation and cisplatin reduces the inhibitory effect of radiation on cell proliferation of normal cells and does not potentiate delayed effects resulting from accumulation of DNA damage.
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| 5. |
- Akuwudike, Pamela, 1987-, et al.
(författare)
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Mechanistic insights from high resolution DNA damage analysis to understand mixed radiation exposure
- 2023
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Ingår i: DNA Repair. - 1568-7864 .- 1568-7856. ; 130
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Tidskriftsartikel (refereegranskat)abstract
- Cells exposed to densely ionising high and scattered low linear energy transfer (LET) radiation (50 % dose of each) react more strongly than to the same dose of each separately. The relationship between DNA double strand break location inside the nucleus and chromatin structure was evaluated, using high-resolution transmission electron microscopy (TEM) in breast cancer MDA-MB-231 cells at 30 min post 5 Gy. Additionally, response to high and/or low LET radiation was assessed using single (1 ×1.5 Gy) versus fractionated dose delivery (5 ×0.3 Gy). By TEM analysis, the highest total number of γH2AX nanobeads were found in cells irradiated with alpha radiation just prior to gamma radiation (called mixed beam), followed by alpha, then gamma radiation. γH2AX foci induced by mixed beam radiation tended to be surrounded by open chromatin (lighter TEM regions), yet foci containing the highest number of beads, i.e. larger foci representing complex damage, remained in the heterochromatic areas. The γH2AX large focus area was also greater in mixed beam-treated cells when analysed by immunofluorescence. Fractionated mixed beams given daily induced the strongest reduction in cell viability and colony formation in MDA-MB-231 and osteosarcoma U2OS cells compared to the other radiation qualities, as well as versus acute exposure. This may partially be explained by recurring low LET oxidative DNA damage by every fraction together with a delay in recompaction of chromatin after high LET, demonstrated by low levels of heterochromatin marker H3K9me3 at 2 h after the last mixed beam fraction in MDA-MB-231. In conclusion, early differences in response to complex DNA damage may lead to a stronger cell kill induced by fractionated exposure, which suggest a therapeutic potential of combined high and low LET irradiation.
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| 6. |
- Akuwudike, Pamela, 1987-, et al.
(författare)
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Short- and long-term effects of radiation exposure at low dose and low dose rate in normal human VH10 fibroblasts
- 2023
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Ingår i: Frontiers In Public Health. - 2296-2565. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Experimental studies complement epidemiological data on the biological effects of low doses and dose rates of ionizing radiation and help in determining the dose and dose rate effectiveness factor.Methods: Human VH10 skin fibroblasts exposed to 25, 50, and 100 mGy of 137Cs gamma radiation at 1.6, 8, 12 mGy/h, and at a high dose rate of 23.4 Gy/h, were analyzed for radiation-induced short- and long-term effects. Two sample cohorts, i.e., discovery (n = 30) and validation (n = 12), were subjected to RNA sequencing. The pool of the results from those six experiments with shared conditions (1.6 mGy/h; 24 h), together with an earlier time point (0 h), constituted a third cohort (n = 12).Results: The 100 mGy-exposed cells at all abovementioned dose rates, harvested at 0/24 h and 21 days after exposure, showed no strong gene expression changes. DMXL2, involved in the regulation of the NOTCH signaling pathway, presented a consistent upregulation among both the discovery and validation cohorts, and was validated by qPCR. Gene set enrichment analysis revealed that the NOTCH pathway was upregulated in the pooled cohort (p = 0.76, normalized enrichment score (NES) = 0.86). Apart from upregulated apical junction and downregulated DNA repair, few pathways were consistently changed across exposed cohorts. Concurringly, cell viability assays, performed 1, 3, and 6 days post irradiation, and colony forming assay, seeded just after exposure, did not reveal any statistically significant early effects on cell growth or survival patterns. Tendencies of increased viability (day 6) and reduced colony size (day 21) were observed at 12 mGy/h and 23.4 Gy/min. Furthermore, no long-term changes were observed in cell growth curves generated up to 70 days after exposure.Discussion: In conclusion, low doses of gamma radiation given at low dose rates had no strong cytotoxic effects on radioresistant VH10 cells.
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| 7. |
- Applegate, K. E., et al.
(författare)
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Individual response of humans to ionising radiation : governing factors and importance for radiological protection
- 2020
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Ingår i: Radiation and Environmental Biophysics. - : Springer Science and Business Media LLC. - 0301-634X .- 1432-2099. ; 59:2, s. 185-209
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Forskningsöversikt (refereegranskat)abstract
- Tissue reactions and stochastic effects after exposure to ionising radiation are variable between individuals but the factors and mechanisms governing individual responses are not well understood. Individual responses can be measured at different levels of biological organization and using different endpoints following varying doses of radiation, including: cancers, non-cancer diseases and mortality in the whole organism; normal tissue reactions after exposures; and, cellular endpoints such as chromosomal damage and molecular alterations. There is no doubt that many factors influence the responses of people to radiation to different degrees. In addition to the obvious general factors of radiation quality, dose, dose rate and the tissue (sub)volume irradiated, recognized and potential determining factors include age, sex, life style (e.g., smoking, diet, possibly body mass index), environmental factors, genetics and epigenetics, stochastic distribution of cellular events, and systemic comorbidities such as diabetes or viral infections. Genetic factors are commonly thought to be a substantial contributor to individual response to radiation. Apart from a small number of rare monogenic diseases such as ataxia telangiectasia, the inheritance of an abnormally responsive phenotype among a population of healthy individuals does not follow a classical Mendelian inheritance pattern. Rather it is considered to be a multi-factorial, complex trait.
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| 8. |
- Brzozowska, Beata, et al.
(författare)
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Monte Carlo Modeling of DNA Lesions and Chromosomal Aberrations Induced by Mixed Beams of Alpha Particles and X-Rays
- 2020
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Ingår i: Frontiers in Physics. - : Frontiers Media SA. - 2296-424X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Prediction of health risks associated with exposure to mixed beams of high- and low-linear energy transfer ionizing radiation is based on the assumption that the biological effect caused by mixed radiation equals the sum of effects resulting from the action of individual beam components. Experimental studies have demonstrated that the cellular effects in cells exposed to mixed radiations are higher than that calculated based on the assumption of additivity. The present work contains a comparative analysis of published results on chromosomal aberrations in human peripheral blood lymphocytes exposed to mixed beams of alpha particles and X-rays with computer simulations using the PARTRAC program based on Monte Carlo methods. PARTRAC was used to calculate the levels of DNA single-strand breaks (SSB) and double-strand breaks (DSB—both complex and simple) and the level of chromosomal aberrations. SSB and DSB yields were found to be additive. A synergistic effect was obtained at the level of chromosomal aberrations, being in good agreement with the experimental results. This result demonstrates that the synergistic action of mixed beams results from processing of SSB and DSB and not from their initial frequencies. The level of synergy was dependent on the composition of the mixed beam, with highest level at 50:50 ratio of alpha particles and X-rays.
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| 9. |
- Chiaka Akuwudike, Pamela, 1987-
(författare)
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Cellular effects of ionizing radiation : Relevant for understanding cancer risk after medical and environmental radiation exposures
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Radiation-induced cancers are stochastic and delayed effects of exposure to ionizing radiation. The dose-response relationship for radiation-induced cancers at both low dose/low dose rates and high doses (doses encountered during radiotherapy) remains unclear. Uncertainties observed in epidemiological studies at low doses and dose rates hamper cancer risk estimation at this dose level. Assessing dose-response relationships for radiotherapy-induced cancers is also complicated due to the inherent difficulty in assessing the doses absorbed by tissues at the site of tumours. In addition, the modulatory effect of chemotherapy on the incidence of radiotherapy-induced cancer risk has been debated. Although included as a modifying factor of the incidence of radiotherapy-induced cancers, results from epidemiological studies do not provide sufficient evidence to support this claim. This thesis summarizes studies conducted to improve the understanding of the association of cancer incidence and radiation dose at clinically relevant (high) doses, low doses and low dose rates, as well as the modulatory role of platinum-based chemotherapy on radiation-induced carcinogenesis. In Paper I, we investigated the competitive relationship between cell killing and the accumulation of DNA damage and genomic instability using two normal cell types (VH10 fibroblasts and AHH-1 lymphoblasts). Dose fractionation schemes were designed based on the cell growth characteristics of each cell type. Cells were irradiated at 0.25, 0.5, 1.0, or 2 Gy per fraction, representing the various dose levels within a radiation field, to simulate the heterogeneous dose distribution across normal tissue during radiotherapy. Following fractionated radiation exposure, the effects on cell growth, cell survival, radiosensitivity, and the accumulation of residual DNA damage and genomic instability were analyzed as a function of dose per fraction and the total absorbed dose. The accumulation of DNA damage and markers of genomic instability associated with DNA damage depended on cell type-specific factors.In Paper II, we investigated the modulatory effects of combining cisplatin and radiation on the accumulation of micronuclei (a biomarker of DNA damage and carcinogenesis) in peripheral blood lymphocytes of patients receiving treatment for gynaecological cancers. We also determined the modulatory effects of the combination of both agents on cell death and cell proliferation, by scoring the frequency of apoptotic and binucleated cells. We compared the frequency of these markers between patients receiving treatment with radiotherapy alone and a combination of cisplatin and radiotherapy. There was a decline in the frequency of micronuclei in patients receiving a combination of cisplatin and radiotherapy.We conducted in vitro experiments in Paper III using AHH-1 and VH10 cells. We investigated the effects of the concurrent combination of cisplatin treatment and multifractionated radiation exposure at 1 Gy per fraction on cell growth, cell survival, cell death, changes in radiosensitivity, accumulation of DNA damage, and other markers of genomic instability as well as the expression of cancer stem cell markers. We also investigated the interaction between cisplatin and radiation exposure in our schedule. The concurrent combination of cisplatin and radiation did not increase the accumulation of markers of genomic instability.In Paper IV, we investigated the short and long-term effects of radiation exposure at low doses and low dose rates on global gene expression, cell growth and cell survival of VH10 fibroblasts to identify unique dose rate signatures that could be useful biomarkers in determining if the application of DDREF is accurate. Except for the differential expression of DMXL2, the long-term effects of LDLDR exposure on global gene expression, cell growth and cell survival of VH10 fibroblasts were negligible. These results suggest that the accumulation of DNA damage and other markers of genomic instability is regulated by cell type-specific factors at these dose levels.
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| 10. |
- Chylenski, Maciej, et al.
(författare)
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Patrilocality and hunter-gatherer-related ancestry of populations in East-Central Europe during the Middle Bronze Age
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- The demographic history of East-Central Europe after the Neolithic period remains poorly explored, despite this region being on the confluence of various ecological zones and cultural entities. Here, the descendants of societies associated with steppe pastoralists form Early Bronze Age were followed by Middle Bronze Age populations displaying unique characteristics. Particularly, the predominance of collective burials, the scale of which, was previously seen only in the Neolithic. The extent to which this re-emergence of older traditions is a result of genetic shift or social changes in the MBA is a subject of debate. Here by analysing 91 newly generated genomes from Bronze Age individuals from present Poland and Ukraine, we discovered that Middle Bronze Age populations were formed by an additional admixture event involving a population with relatively high proportions of genetic component associated with European hunter-gatherers and that their social structure was based on, primarily patrilocal, multigenerational kin-groups. By analysing 91 Bronze Age genomes from East-Central Europe, the authors discovered that Middle Bronze Age populations were formed by an admixture event involving hunter-gatherers and that the social structure of resulting population was primarily patrilocal.
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