| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- López Riego, Milagrosa, 1992-
(författare)
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Biomarkers of ionising radiation relevant to carcinogenesis : Dose, dose rate and LET dependency of the responses
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A better understanding of the relationship between ionising radiation (IR) dose, dose rate and radiation quality, and the risk of stochastic effects would improve risk extrapolation from atomic bomb survivors’ data. Owing to insufficient statistical power of epidemiological studies to detect excess incidence of cancer following low doses of IR delivered at low dose rates (LDLDR), as typically encountered in most common human exposure scenarios, radiobiological experiments are fundamental to describe the biological effectiveness of LDLDR and to define the underlying molecular mechanisms. DNA damage and downstream effects are major contributors to radiation carcinogenesis, and as such, these processes have been investigated in the context of plausible mechanisms of radiation-induced health effects in the studies compiled in this thesis, using different cell models and appropriate radiation sources. In Paper I, we characterized the energy, activity and dose rate of new low activity gamma and alpha sources of IR, expected to promote small-scale radiation protection research, and used to demonstrate that LDLDR led to an increased micronucleus frequency, a marker of DNA damage, in human osteosarcoma cells as compared to control cells. In Paper II, we used blood from patients undergoing radiological imaging procedures, i.e. PET-CT and scintigraphy, to investigate whether candidate IR biomarkers, i.e. ROS, γH2AX, and expression of a panel of radiation-responsive genes, are altered following in vivo low dose exposure as compared to control samples obtained before the diagnostic procedure. We showed that radiological imaging generally induced weak γH2AX, ROS, and gene expression fold changes at the selected timepoints, although few donors presented stronger responses. The observed mild increase in DNA damage was, nevertheless, coherent with a subsequent DNA damage response. This study also indicated that owing to the heterogeneity of the response across individuals, the discrimination of exposed samples might be complicated in the absence of a control for low dose exposures. The current risk assessment approach for mixed beam exposures, as encountered in space and other exposure scenarios, assumes additivity of effects of each radiation quality component, but some reports, which show synergistic effects instead, are in conflict with this assumption and indicate a potential underestimation of the corresponding cancer risk. In Paper III, we investigated the consistency of the interaction between low and high LET IR in two healthy donors who presented the largest inter- and intra-donor variability following mixed beam exposure in a previous study. Based on nine biological replicates, this study confirmed that combined alpha particles and photon radiation led to a higher cytogenetic damage and gene expression responses than those expected based on simple additivity of effects, but that the interaction was prone to seasonal intra-donor and inter-donor variation for both endpoints. This study additionally showed that IR exposure modified alternative transcription of FDXR and MDM2 in a radiation quality-dependent manner, albeit alternative transcription did not coincide with the mode of interaction between the different radiation qualities. In light of these results, we suggest that the possible interaction between low and high LET IR should be considered in calculating uncertainty of risk for mixed exposures. In Paper IV, we investigated the early- and long-term biological effects of LDLDR gamma radiation as compared to the same doses delivered acutely in human AHH-1 lymphoblasts, using relevant endpoints related to carcinogenesis, i.e. cell viability, clonogenic survival, chromosomal aberrations, cell growth and global gene expression. The results presented in this study are coherent with a potential detrimental effect of 100 mGy, delivered either chronically or acutely, with a clear dose rate effect for chromosomal aberrations and gene expression, which may modulate cancer risk by dose rate-dependent mechanisms.
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| 6. |
- López Riego, Milagrosa, 1992-, et al.
(författare)
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The DNA damage response to radiological imaging : from ROS and γH2AX foci induction to gene expression responses in vivo
- 2023
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Ingår i: Radiation and Environmental Biophysics. - 0301-634X .- 1432-2099. ; 62:3, s. 371-393
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Tidskriftsartikel (refereegranskat)abstract
- Candidate ionising radiation exposure biomarkers must be validated in humans exposed in vivo. Blood from patients undergoingpositron emission tomography–computed tomography scan (PET-CT) and skeletal scintigraphy (scintigraphy) was drawnbefore (0 h) and after (2 h) the procedure for correlation analyses of the response of selected biomarkers with radiation doseand other available patient information. FDXR, CDKN1A, BBC3, GADD45A, XPC, and MDM2 expression was determinedby qRT-PCR, DNA damage (γH2AX) by flow cytometry, and reactive oxygen species (ROS) levels by flow cytometry usingthe 2′, 7′—dichlorofluorescein diacetate test in peripheral blood mononuclear cells (PBMC). For ROS experiments, 0- and2-h samples were additionally exposed to UVA to determine whether diagnostic irradiation conditioned the response tofurther oxidative insult. With some exceptions, radiological imaging induced weak γH2AX foci, ROS and gene expressionfold changes, the latter with good coherence across genes within a patient. Diagnostic imaging did not influence oxidativestress in PBMC successively exposed to UVA. Correlation analyses with patient characteristics led to low correlation coefficientvalues. γH2AX fold change, which correlated positively with gene expression, presented a weak positive correlationwith injected activity, indicating a radiation-induced subtle increase in DNA damage and subsequent activation of the DNAdamage response pathway. The exposure discrimination potential of these biomarkers in the absence of control samples asfrequently demanded in radiological emergencies, was assessed using raw data. These results suggest that the variability ofthe response in heterogeneous populations might complicate identifying individuals exposed to low radiation doses.
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| 7. |
- Płódowska, Magdalena, et al.
(författare)
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Small is beautiful : low activity alpha and gamma sources for small-scale radiation protection research experiments
- 2021
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 97:4, s. 541-552
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Uncertainties regarding the magnitude of health effects following exposure to low dosesof ionizing radiation remain a matter of concern both for professionals and for the public. There isconsensus within the international radiation research community that more research is requiredon biological effects of radiation doses below 100 mGy applied at low dose rates. Moreover, thereis a demand for increasing education and training of future radiation researchers and regulators.Research, education and training is primarily carried out at universities but university-based radiationresearch is often hampered by limited access to radiation sources. The aim of the presentreport is to describe small and cost-effective low activity gamma and alpha sources that can easilybe installed and used in university laboratories.Methods and results: A gamma radiation source was made from an euxenite-(Y) rock(Y,Ca,Ce,U,Th)(Nb,Ta,Ti)2O6) that was found in an abandoned mine in Sweden. It allows exposingcells grown in culture dishes to radiation at a dose rate of 50 μGy/h and lower. Three alpha sourceswere custom-made and yield a dose rate of 1 mGy/h each. The construction, dosimetry andcellular effects of the sources are described.Conclusions: We hope that the report will stimulate research and training activities in the lowdose field by facilitating access to radiation sources.
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