RESUMO
PURPOSE: A reliable method for regional in vivo imaging of radiation-induced cellular damage would be of great importance for the detection of therapy-induced injury to healthy tissue and the choice of adequate treatment of radiation emergency patients in both civilian and military events. This study aimed to investigate in a mouse model if positron emission tomography (PET) imaging with proliferation and apoptosis markers is potentially suitable for this purpose. METHODS: Four groups, including twenty mice (wild-type C57BL/6) each, were whole-body irradiated with 0 Gy, 0.5 Gy, 1 Gy, and 3 Gy and examined by PET over a six-month period at defined time points. 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) and 2-(5-[18F]fluoropentyl)-2-methyl malonic acid ([18F]ML-10) were used to visualise proliferation and apoptosis. Regional standard uptake values were compared with respect to irradiation dose over time. Histologic data and peripheral blood cell values were correlated with the PET results. RESULTS: The hematopoietic bone marrow showed a significantly increased [18F]FLT signal at early time points after radiation exposure (day 3 and day 7). This correlated with blood parameters, especially leukocytes, and histological data. A significantly increased [18F]FLT signal also occurred in the gastrointestinal tract and thymus at early time points. An increased [18F]ML-10 signal related to irradiation doses was observed in the bone marrow on day 8, but there was a high variability of standard uptake values and no correlation with histological data. CONCLUSION: [18F]FLT showed potential to visualise the extent, regional distribution and recovery from radiation-induced cellular damage in the bone marrow, gastrointestinal tract and thymus. The potential of [18F]FLT imaging to assess the extent of bone marrow affected by irradiation might be especially useful to predict the subsequent severity of hematopoietic impairment and to adapt the therapy of the bone marrow reserve. [18F]ML-10 PET proved to be not sensitive enough for the reliable detection of radiation induced apoptosis.
Assuntos
Tomografia por Emissão de Pósitrons , Irradiação Corporal Total , Humanos , Camundongos , Animais , Irradiação Corporal Total/efeitos adversos , Camundongos Endogâmicos C57BL , Tomografia por Emissão de Pósitrons/métodos , Modelos Animais de Doenças , Proliferação de Células/efeitos da radiação , Apoptose , DidesoxinucleosídeosRESUMO
The combination of high and low LET radionuclides has been tested in several patient studies to improve treatment response. Radionuclide mixtures can also be released in nuclear power plant accidents or nuclear bomb deployment. This study investigated the DNA damage response and DNA double-strand break (DSB) repair in peripheral blood mononuclear cells (PBMCs) after internal exposure of blood samples of 10 healthy volunteers to either no radiation (baseline) or different radionuclide mixtures of the α- and ß-emitters [223Ra]RaCl2 and [177Lu]LuCl3, i.e., 25 mGy/75 mGy, 50 mGy/50 mGy and 75 mGy/25 mGy, respectively. DSB foci and γ-H2AX α-track enumeration directly after 1 h of exposure or after 4 h or 24 h of repair revealed that radiation-induced foci (RIF) and α-track induction in 100 cells was similar for mixed α/ß and pure internal α- or ß-irradiation, as were the repair rates for all radiation qualities. In contrast, the fraction of unrepaired RIF (Qß) in PBMCs after mixed α/ß-irradiation (50% 223Ra & 50% 177Lu: Qß = 0.23 ± 0.10) was significantly elevated relative to pure ß-irradiation (50 mGy: Qß, pure = 0.06 ± 0.02), with a similar trend being noted for all mixtures. This α-dose-dependent increase in persistent foci likely relates to the formation of complex DNA damage that remains difficult to repair.
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Dano ao DNA , Reparo do DNA , Leucócitos Mononucleares , Humanos , Leucócitos Mononucleares/efeitos da radiação , Leucócitos Mononucleares/metabolismo , Reparo do DNA/efeitos da radiação , Masculino , Adulto , Dano ao DNA/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Radioisótopos , Lutécio , Feminino , Histonas/metabolismoRESUMO
This work reports on a model that describes patient-specific absorbed dose-dependent DNA damage response in peripheral blood mononuclear cells of thyroid cancer patients during radioiodine therapy and compares the results with the ex vivo DNA damage response in these patients. Blood samples of 18 patients (nine time points up to 168 h post-administration) were analyzed for radiation-induced γ-H2AX + 53BP1 DNA double-strand break foci (RIF). A linear one-compartment model described the absorbed dose-dependent time course of RIF (Parameters: c characterizes DSB damage induction; k1 and k2 are rate constants describing fast and slow repair). The rate constants were compared to ex vivo repair rates. A total of 14 patient datasets could be analyzed; c ranged from 0.012 to 0.109 mGy-1, k2 from 0 to 0.04 h-1. On average, 96% of the damage is repaired quickly with k1 (range: 0.19-3.03 h-1). Two patient subgroups were distinguished by k1-values (n = 6, k1 > 1.1 h-1; n = 8, k1 < 0.6 h-1). A weak correlation with patient age was observed. While induction of RIF was similar among ex vivo and in vivo, the respective repair rates failed to correlate. The lack of correlation between in vivo and ex vivo repair rates and the applicability of the model to other therapies will be addressed in further studies.
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Quebras de DNA de Cadeia Dupla , Reparo do DNA , Neoplasias da Glândula Tireoide , Humanos , Neoplasias da Glândula Tireoide/radioterapia , Neoplasias da Glândula Tireoide/sangue , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Pessoa de Meia-Idade , Masculino , Feminino , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Adulto , Idoso , Dano ao DNA , Radioisótopos do Iodo/uso terapêutico , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Histonas/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos da radiação , Modelos BiológicosRESUMO
As the war in Ukraine progresses, the radiological and nuclear threat has never been as real as now. The formation of life-threatening acute radiation syndrome (ARS), in particular after the deployment of a nuclear weapon or an attack on a nuclear power station, must be considered realistic. ARS is caused by massive cell death, leading to functional organ deficits and, via systemic inflammatory responses, finally aggravates into multiple organ failure. As a deterministic effect, the severity of the disease dictates the clinical outcome. Hence, predicting ARS severity via biodosimetry or alternative approaches appears straightforward. Because the disease occurs delayed, therapy starting as early as possible has the most significant benefit. A clinically relevant diagnosis should be carried out within the diagnostic time window of about 3 days after exposure. Biodosimetry assays providing retrospective dose estimations within this time frame will support medical management decision-making. However, how closely can dose estimates be associated with the later developing ARS severity degrees when considering dose as one among other determinants of radiation exposure and cell death? From a clinical/triage point of view, ARS severity degrees can be further aggregated into unexposed, weakly diseased (no acute health effects expected), and strongly diseased patient groups, with the latter requiring hospitalization as well as an early and intensive treatment. Radiation-induced gene expression (GE) changes occur early after exposure and can be quickly quantified. GE can be used for biodosimetry purposes. Can GE be used to predict later developing ARS severity degrees and allocate individuals to the three clinically relevant groups as well?
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Estudos Retrospectivos , Humanos , Prognóstico , Expressão GênicaRESUMO
In the case of a radiological or nuclear event, biological dosimetry can be an important tool to support clinical decision-making. During a nuclear event, individuals might be exposed to a mixed field of neutrons and photons. The composition of the field and the neutron energy spectrum influence the degree of damage to the chromosomes. During the transatlantic BALANCE project, an exposure similar to a Hiroshima-like device at a distance of 1.5 km from the epicenter was simulated, and biological dosimetry based on dicentric chromosomes was performed to evaluate the participants ability to discover unknown doses and to test the influence of differences in neutron spectra. In a first step, calibration curves were established by irradiating blood samples with 5 doses in the range of 0-4 Gy at two different facilities in Germany (Physikalisch-Technische Bundesanstalt [PTB]) and the USA (the Columbia IND Neutron Facility [CINF]). The samples were sent to eight participating laboratories from the RENEB network and dicentric chromosomes were scored by each participant. Next, blood samples were irradiated with 4 blind doses in each of the two facilities and sent to the participants to provide dose estimates based on the established calibration curves. Manual and semiautomatic scoring of dicentric chromosomes were evaluated for their applicability to neutron exposures. Moreover, the biological effectiveness of the neutrons from the two irradiation facilities was compared. The calibration curves from samples irradiated at CINF showed a 1.4 times higher biological effectiveness compared to samples irradiated at PTB. For manual scoring of dicentric chromosomes, the doses of the test samples were mostly successfully resolved based on the calibration curves established during the project. For semiautomatic scoring, the dose estimation for the test samples was less successful. Doses >2 Gy in the calibration curves revealed nonlinear associations between dose and dispersion index of the dicentric counts, especially for manual scoring. The differences in the biological effectiveness between the irradiation facilities suggested that the neutron energy spectrum can have a strong impact on the dicentric counts.
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Nêutrons , Humanos , AlemanhaRESUMO
OBJECTIVES: Photon-counting computed tomography has lately found its way into clinical routine. The new technique could offer substantial improvements regarding general image quality, image noise, and radiation dose reduction. This study evaluated the first abdominal examinations in clinical routine and compared the results to conventional computed tomography. METHODS: In this single-center retrospective study, 66 patients underwent photon-counting and conventional abdominal CT. Four radiologists assessed general image quality, image noise, and image artifacts. Signal-to-noise ratio and dose properties of both techniques within the clinical application were compared. An ex vivo phantom study revealed the radiobiological impact by means of DNA double-strand break foci in peripheral blood cells by enumerating γ-H2AX+53BP1 foci. RESULTS: General image quality in accordance with the Likert scale was found superior for photon-counting CT (4.74 ± 0.46 vs. 4.25 ± 0.54; p < 0.001). Signal-to-noise ratio (p < 0.001) and also dose exposure were higher for photon-counting CT (DLP: 419.2 ± 162.2 vs. 372.3 ± 236.6 mGy*cm; p = 0.0435). CT exposure resulted in significantly increased DNA damage in comparison to sham control (p < 0.001). Investigation of the average foci per cell and radiation-induced foci numbers revealed significantly elevated numbers (p = 0.004 and p < 0.0001, respectively) after photon-counting CT. CONCLUSION: Photon-counting CT in abdominal examinations showed superior results regarding general image quality and signal-to-noise ratio in clinical routine. However, this seems to be traded for a significantly higher dose exposure and corresponding double-strand break frequency. Optimization of standard protocols in further clinical applications is required to find a compromise regarding picture quality and dose exposure. KEY POINTS: ⢠Photon-counting computed tomography promises to enhance the diagnostic potential of medical imaging in clinical routine. ⢠Retrospective single-center study showed superior general image quality accompanied by higher dose exposure in initial abdominal PCCT protocols compared to state-of-the-art conventional CT. ⢠A simultaneous ex vivo phantom study revealed correspondingly increased frequencies of DNA double-strand breaks after PCCT.
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DNA , Tomografia Computadorizada por Raios X , Humanos , Estudos Retrospectivos , Doses de Radiação , Tomografia Computadorizada por Raios X/métodos , Razão Sinal-Ruído , Imagens de FantasmasRESUMO
PURPOSE: As α-emitters for radiopharmaceutical therapies are administered systemically by intravenous injection, blood will be irradiated by α-particles that induce clustered DNA double-strand breaks (DSBs). Here, we investigated the induction and repair of DSB damage in peripheral blood mononuclear cells (PBMCs) as a function of the absorbed dose to the blood following internal ex vivo irradiation with [223Ra]RaCl2. METHODS: Blood samples of ten volunteers were irradiated by adding [223Ra]RaCl2 solution with different activity concentrations resulting in absorbed doses to the blood of 3 mGy, 25 mGy, 50 mGy and 100 mGy. PBMCs were isolated, divided in three parts and either fixed directly (d-samples) or after 4 h or 24 h culture. After immunostaining, the induced γ-H2AX α-tracks were counted. The time-dependent decrease in α-track frequency was described with a model assuming a repair rate R and a fraction of non-repairable damage Q. RESULTS: For 25 mGy, 50 mGy and 100 mGy, the numbers of α-tracks were significantly increased compared to baseline at all time points. Compared to the corresponding d-samples, the α-track frequency decreased significantly after 4 h and after 24 h. The repair rates R were (0.24 ± 0.05) h-1 for 25 mGy, (0.16 ± 0.04) h-1 for 50 mGy and (0.13 ± 0.02) h-1 for 100 mGy, suggesting faster repair at lower absorbed doses, while Q-values were similar. CONCLUSION: The results obtained suggest that induction and repair of the DSB damage depend on the absorbed dose to the blood. Repair rates were similar to what has been observed for irradiation with low linear energy transfer.
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Reparo do DNA , Leucócitos Mononucleares , DNA/efeitos da radiação , Dano ao DNA , Relação Dose-Resposta à Radiação , Humanos , Compostos RadiofarmacêuticosRESUMO
Sulfur mustard (SM) is a chemical warfare agent that can damage DNA via alkylation and oxidative stress. Because of its genotoxicity, SM is cancerogenic and the progenitor of many chemotherapeutics. Previously, we developed an SM-resistant cell line via chronic exposure of the popular keratinocyte cell line HaCaT to increasing doses of SM over a period of 40 months. In this study, we compared the genomic landscape of the SM-resistant cell line HaCaT/SM to its sensitive parental line HaCaT in order to gain insights into genetic changes associated with continuous alkylation and oxidative stress. We established chromosome numbers by cytogenetics, analyzed DNA copy number changes by means of array Comparative Genomic Hybridization (array CGH), employed the genome-wide chromosome conformation capture technique Hi-C to detect chromosomal translocations, and derived mutational signatures by whole-genome sequencing. We observed that chronic SM exposure eliminated the initially prevailing hypotetraploid cell population in favor of a hyperdiploid one, which contrasts with previous observations that link polyploidization to increased tolerance and adaptability toward genotoxic stress. Furthermore, we observed an accumulation of chromosomal translocations, frequently flanked by DNA copy number changes, which indicates a high rate of DNA double-strand breaks and their misrepair. HaCaT/SM-specific single-nucleotide variants showed enrichment of C > A and T > A transversions and a lower rate of deaminated cytosines in the CpG dinucleotide context. Given the frequent use of HaCaT in toxicology, this study provides a valuable data source with respect to the original genotype of HaCaT and the mutational signatures associated with chronic alkylation and oxidative stress.
Assuntos
Aberrações Cromossômicas/induzido quimicamente , Dano ao DNA , Queratinócitos/efeitos dos fármacos , Gás de Mostarda/toxicidade , Mutação , Radiação Ionizante , Alquilantes/farmacologia , Alquilantes/toxicidade , Linhagem Celular , Aberrações Cromossômicas/efeitos da radiação , Hibridização Genômica Comparativa , DNA/efeitos dos fármacos , DNA/metabolismo , DNA/efeitos da radiação , Adutos de DNA , Quebras de DNA de Cadeia Dupla , Humanos , Gás de Mostarda/farmacologia , Estresse OxidativoRESUMO
The accepted generic multiple-parameter and early-response biodosimetry and dosimetry assessment approach for suspected high-dose radiation (i.e. life-threatening) exposure includes measuring radioactivity associated with the exposed individual (if appropriate); observing and recording prodromal signs/symptoms; obtaining serial complete blood counts with white-blood-cell differential; sampling blood for the chromosome-aberration cytogenetic bioassay using the 'gold standard' dicentric assay (premature chromosome condensation assay for exposures >5 Gy photon acute doses equivalent), measurement of proteomic biomarkers and gene expression assays for dose assessment; bioassay sampling, if appropriate, to determine radioactive internal contamination; physical dose reconstruction, and using other available opportunistic dosimetry approaches. Biodosimetry and dosimetry resources are identified and should be setup in advance along with agreements to access additional national, regional, and international resources. This multifaceted capability needs to be integrated into a biodosimetry/dosimetry 'concept of operations' for use in a radiological emergency. The combined use of traditional biological-, clinical-, and physical-dosimetry should be use in an integrated approach to provide: (a) early-phase diagnostics to guide the development of initial medical-management strategy, and (b) intermediate and definitive assessment of radiation dose and injury. Use of early-phase (a) clinical signs and symptoms, (b) blood chemistry biomarkers, and (c) triage cytogenetics shows diagnostic utility to predict acute radiation injury severity.
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Proteômica , Lesões por Radiação , Aberrações Cromossômicas , Humanos , Lesões por Radiação/diagnóstico , Radiometria , TriagemRESUMO
A collection of powerful diagnostic tools have been developed under the umbrellas of NATO for ionising radiation dose assessment (BAT, WinFRAT) and estimate of acute health effects in humans (WinFRAT, H-Module). We assembled a database of 191 ARS cases using the medical treatment protocols for radiation accident victims (n= 167) and the system for evaluation and archiving of radiation accidents based on case histories (n= 24) for training purposes of medical personnel. From 2016 to 2019, we trained 39 participants comprising MSc level radiobiology students in an on-site teaching class. Enforced by the covid-19 pandemic in 2020 for the first time, an online teaching of nine MSc radiobiology students replaced the on-site teaching. We found that: (a) limitations of correct diagnostic decision-making based on clinical signs and symptoms were experienced unrelated to the teaching format. (b) A significant performance decrease concerning online (first number in parenthesis) versus on-site teaching (reference and second number in parenthesis) was seen regarding the estimate time (31 vs 61 cases per hour, two-fold decrease,p= 0.005). Also, the accurate assessment of response categories (89.9% vs 96.9%,p= 0.001), ARS (92.4% vs 96.7%,p= 0.002) and hospitalisation (93.5% vs 97.0%,p= 0.002) decreased by around 3%-7%. The performances of the online attendees were mainly distributed within the lower quartile performance of on-site participants and the 25%-75% interquartile range increased 3-7-fold. (c) Comparison of dose estimates performed by training participants with hematologic acute radiation syndrome (HARS) severity mirrored the known limitations of dose alone as a surrogate parameter for HARS severity at doses less than 1.5 Gy, but demonstrated correct determination of HARS 2-4 and support for clinical decision making at dose estimates >1.5 Gy, regardless of teaching format. (d) Overall, one-third of the online participants showed substantial misapprehension and insecurities of elementary course content that did not occur after the on-site teaching.
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Síndrome Aguda da Radiação , Educação a Distância , Medicina de Emergência/educação , Triagem , Síndrome Aguda da Radiação/diagnóstico , COVID-19 , Humanos , PandemiasRESUMO
In the case of a nuclear power plant accident, repetitive/prolonged radioiodine release may occur. Radioiodine accumulates in the thyroid and by irradiation enhances the risk of cancer. Large doses of non-radioactive iodine may protect the thyroid by inhibiting radioiodine uptake into the gland (iodine blockade). Protection is based on a competition at the active carrier site in the cellular membrane and the Wolff-Chaikoff effect, the latter being, however, only transient (24-48 h). Perchlorate may alternatively provide protection by a carrier competition mechanism only. Perchlorate has, however, a stronger affinity to the carrier than iodide. Based on an established biokinetic-dosimetric model developed to study iodine blockade, and after its extension to describe perchlorate pharmacokinetics and the inhibition of iodine transport through the carrier, we computed the protective efficacies that can be achieved by stable iodine or perchlorate in the case of an acute or prolonged radioiodine exposure. In the case of acute radioiodine exposure, perchlorate is less potent than stable iodine considering its ED50. A dose of 100 mg stable iodine has roughly the same protective efficacy as 1000 mg perchlorate. For prolonged exposures, single doses of protective agents, whether stable iodine or perchlorate, offer substantially lower protection than after acute radioiodine exposure, and thus repetitive administrations seem necessary. In case of prolonged exposure, the higher affinity of perchlorate for the carrier in combination with the fading Wolff-Chaikoff effect of iodine confers perchlorate a higher protective efficacy compared to stable iodine. Taking into account the frequency and seriousness of adverse effects, iodine and perchlorate at equieffective dosages seem to be alternatives in case of short-term acute radioiodine exposure, whereas preference should be given to perchlorate in view of its higher protective efficacy in the case of longer lasting radioiodine exposures.
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Radioisótopos do Iodo/toxicidade , Iodo/farmacologia , Percloratos/farmacologia , Exposição à Radiação , Protetores contra Radiação/farmacologia , Glândula Tireoide/fisiologia , HumanosRESUMO
Ionizing radiation produces reactive oxygen species (ROS) leading to cellular DNA damage. Therefore, patients undergoing radiation therapy or first responders in radiological accident scenarios could both benefit from the identification of specifically acting pharmacological radiomitigators. The synthetic triterpenoid bardoxolone-methyl (CDDO-Me) has previously been shown to exert antioxidant, anti-inflammatory and anticancer activities in several cell lines, in part by enhancing the DNA damage response. In our study, we examined the effect of nanomolar concentrations of CDDO-Me in human peripheral blood mononuclear cells (PBMC). We observed increased cellular levels of the antioxidative enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase (quinone1) and mitochondrial superoxide dismutase 2 by immunoblotting. Surprisingly, we found increased intracellular ROS-levels using imaging flow-cytometry. However, the radiation-induced DNA double-strand break (DSB) formation using the γ-H2AX + 53BP1 DSB focus assay and the cytokinesis-block micronucleus assay both revealed, that nanomolar CDDO-Me pre-treatment of PBMC for 2 h or 6 h ahead of X irradiation with 2 Gy did neither significantly affect γ-H2AX + 53BP1 DSB foci formation nor the frequency of micronuclei. CDDO-Me treatment also failed to alter the nuclear division index and the frequency of IR-induced PBMC apoptosis as investigated by Annexin V-labeled live-cell imaging. Our results indicate that pharmacologically increased cellular concentrations of antioxidative enzymes might not necessarily exert radiomitigating short-term effects in IR-exposed PBMC. However, the increase of antioxidative enzymes could also be a result of a defensive cellular mechanism towards elevated ROS levels.
Assuntos
Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/efeitos da radiação , Ácido Oleanólico/análogos & derivados , Raios X , Adulto , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Testes para Micronúcleos , Pessoa de Meia-Idade , Ácido Oleanólico/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
While ionizing radiation (IR) is a powerful tool in medical diagnostics, nuclear medicine, and radiology, it also is a serious threat to the integrity of genetic material. Mutagenic effects of IR to the human genome have long been the subject of research, yet still comparatively little is known about the genome-wide effects of IR exposure on the DNA-sequence level. In this study, we employed high throughput sequencing technologies to investigate IR-induced DNA alterations in human gingiva fibroblasts (HGF) that were acutely exposed to 0.5, 2, and 10 Gy of 240 kV X-radiation followed by repair times of 16 h or 7 days before whole-genome sequencing (WGS). Our analysis of the obtained WGS datasets revealed patterns of IR-induced variant (SNV and InDel) accumulation across the genome, within chromosomes as well as around the borders of topologically associating domains (TADs). Chromosome 19 consistently accumulated the highest SNVs and InDels events. Translocations showed variable patterns but with recurrent chromosomes of origin (e.g., Chr7 and Chr16). IR-induced InDels showed a relative increase in number relative to SNVs and a characteristic signature with respect to the frequency of triplet deletions in areas without repetitive or microhomology features. Overall experimental conditions and datasets the majority of SNVs per genome had no or little predicted functional impact with a maximum of 62, showing damaging potential. A dose-dependent effect of IR was surprisingly not apparent. We also observed a significant reduction in transition/transversion (Ti/Tv) ratios for IR-dependent SNVs, which could point to a contribution of the mismatch repair (MMR) system that strongly favors the repair of transitions over transversions, to the IR-induced DNA-damage response in human cells. Taken together, our results show the presence of distinguishable characteristic patterns of IR-induced DNA-alterations on a genome-wide level and implicate DNA-repair mechanisms in the formation of these signatures.
Assuntos
DNA/genética , DNA/efeitos da radiação , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Genoma Humano , Gengiva/citologia , Cromossomos Humanos Par 19/genética , Variações do Número de Cópias de DNA/genética , Bases de Dados Genéticas , Humanos , Mutação INDEL/genética , Translocação Genética , Raios XRESUMO
BACKGROUND: The analysis of DNA copy number variants (CNV) has increasing impact in the field of genetic diagnostics and research. However, the interpretation of CNV data derived from high resolution array CGH or NGS platforms is complicated by the considerable variability of the human genome. Therefore, tools for multidimensional data analysis and comparison of patient cohorts are needed to assist in the discrimination of clinically relevant CNVs from others. RESULTS: We developed GenomeCAT, a standalone Java application for the analysis and integrative visualization of CNVs. GenomeCAT is composed of three modules dedicated to the inspection of single cases, comparative analysis of multidimensional data and group comparisons aiming at the identification of recurrent aberrations in patients sharing the same phenotype, respectively. Its flexible import options ease the comparative analysis of own results derived from microarray or NGS platforms with data from literature or public depositories. Multidimensional data obtained from different experiment types can be merged into a common data matrix to enable common visualization and analysis. All results are stored in the integrated MySQL database, but can also be exported as tab delimited files for further statistical calculations in external programs. CONCLUSIONS: GenomeCAT offers a broad spectrum of visualization and analysis tools that assist in the evaluation of CNVs in the context of other experiment data and annotations. The use of GenomeCAT does not require any specialized computer skills. The various R packages implemented for data analysis are fully integrated into GenomeCATs graphical user interface and the installation process is supported by a wizard. The flexibility in terms of data import and export in combination with the ability to create a common data matrix makes the program also well suited as an interface between genomic data from heterogeneous sources and external software tools. Due to the modular architecture the functionality of GenomeCAT can be easily extended by further R packages or customized plug-ins to meet future requirements.
Assuntos
Variações do Número de Cópias de DNA , Software , Simulação por Computador , Genoma Humano , Genômica , Humanos , Modelos Teóricos , Reprodutibilidade dos TestesRESUMO
Based on gene expression changes measured in the peripheral blood within the first 2 days after irradiation, we predicted a pancytopenia in a baboon model. Eighteen baboons were irradiated with 2.5 or 5 Gy. According to changes in blood cell counts, the surviving baboons (n = 17) exhibited a hematological acute radiation syndrome (HARS) either with or without a pancytopenia. We used a two stage study design where stage I was a whole genome screen (microarrays) for mRNA combined with a qRT-PCR platform for simultaneous detection of 667 miRNAs using a part of the samples. Candidate mRNAs and miRNAs differentially upregulated or downregulated (>2-fold, p < 0.05) during the first 2 days after irradiation were chosen for validation in stage II using the remaining samples and using throughout more sensitive qRT-PCR. We detected about twice as many upregulated (mean 2128) than downregulated genes (mean 789) in baboons developing an HARS either with or without a pancytopenia. From 51 candidate mRNAs altogether, 11 mRNAs were validated using qRT-PCR. These mRNAs showed only significant differences between HARS groups and H0, but not between HARS groups with and without pancytopenia. Six miRNA species (e.g., miR-574-3p, p = 0.009, ROC = 0.94) revealed significant gene expression differences between HARS groups with and without pancytopenia and are known to sensitize irradiated cells. Hence, in particular, the newly identified miRNA species for prediction of pancytopenia will support the medical management decision making.
Assuntos
Irradiação Hemicorpórea , Pancitopenia/diagnóstico , Pancitopenia/etiologia , Papio , Transcriptoma , Irradiação Corporal Total , Animais , Contagem de Células Sanguíneas , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genômica/métodos , Masculino , MicroRNAs/genética , Pancitopenia/sangue , Prognóstico , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Irradiação Corporal Total/efeitos adversosRESUMO
Radiosensitivity differs in humans and likely among primates. The reasons are not well known. We examined pre-exposure gene expression in baboons (n = 17) who developed haematologic acute radiation syndrome (HARS) without pancytopenia or a more aggravated HARS with pancytopenia after irradiation. We evaluated gene expression in a two stage study design where stage I comprised a whole genome screen for messenger RNAs (mRNA) (microarray) and detection of 667 microRNAs (miRNA) (real-time quantitative polymerase chain reaction (qRT-PCR) platform). Twenty candidate mRNAs and nine miRNAs were selected for validation in stage II (qRT-PCR). None of the mRNA species could be confirmed during the validation step, but six of the nine selected candidate miRNA remained significantly different during validation. In particular, miR-425-5p (receiver operating characteristic = 0.98; p = 0.0003) showed nearly complete discrimination between HARS groups with and without pancytopenia. Target gene searches of miR-425-5p identified new potential mRNAs and associated biological processes linked with radiosensitivity. We found that one miRNA species examined in pre-exposure blood samples was associated with HARS characterized by pancytopenia and identified new target mRNAs that might reflect differences in radiosensitivity of irradiated normal tissue.
Assuntos
Síndrome Aguda da Radiação/genética , Expressão Gênica , MicroRNAs/genética , Pancitopenia/etiologia , RNA Mensageiro/genética , Animais , Modelos Animais de Doenças , Expressão Gênica/efeitos da radiação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Papio , Tolerância a RadiaçãoRESUMO
Several single-nucleotide polymorphisms (SNPs) have been associated with papillary and follicular thyroid cancer (PTC and FTC, respectively) risk, but few have replicated. After analyzing 17525 tag SNPs in 1129 candidate genes, we found associations with PTC risk in SERPINA5, FTO, HEMGN (near FOXE1) and other genes. Here, we report results from a replication effort in a large independent PTC/FTC case-control study conducted in Germany. We evaluated the best tagging SNPs from our previous PTC study and additionally included SNPs in or near FOXE1 and NKX2-1 genes, known susceptibility loci for thyroid cancer. We genotyped 422 PTC and 130 FTC cases and 752 controls recruited from three German clinical centers. We used polytomous logistic regression to simultaneously estimate PTC and FTC associations for 79 SNPs based on log-additive models. We assessed effect modification by body mass index (BMI), gender and age for all SNPs, and selected SNP by SNP interactions. We confirmed associations with PTC and SNPs in FOXE1/HEMGN, SERPINA5 (rs2069974), FTO (rs8047395), EVPL (rs2071194), TICAM1 (rs8120) and SCARB1 (rs11057820) genes. We found associations with SNPs in FOXE1, SERPINA5, FTO, TICAM1 and HSPA6 and FTC. We found two significant interactions between FTO (rs8047395) and BMI (P = 0.0321) and between TICAM1 (rs8120) and FOXE1 (rs10984377) (P = 0.0006). Besides the known associations with FOXE1 SNPs, we confirmed additional PTC SNP associations reported previously. We also found several new associations with FTC risk and noteworthy interactions. We conclude that multiple variants and host factors might interact in complex ways to increase risk of PTC and FTC.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Adenocarcinoma Folicular/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Carcinoma/genética , Fatores de Transcrição Forkhead/genética , Inibidor da Proteína C/genética , Receptores Depuradores Classe B/genética , Neoplasias da Glândula Tireoide/genética , Adenocarcinoma Folicular/patologia , Adulto , Idoso , Carcinoma/patologia , Carcinoma Papilar , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/patologiaRESUMO
The lymphocyte cytokinesis-block micronucleus (CBMN) assay is a biodosemeter for the exposure to ionizing radiation. We examined the feasibility to implement a fully automated cell harvesting system for binucleate lymphocyte (BN) fixation. We compared fully automated versus manual BN fixation and evaluated its relevance on the accuracy of dose estimates using the CBMN. First, dose-response curves based on X-ray irradiated blood samples of ten healthy donors (0-4 Gy, dose rate 1.0 Gy/min) were established. BN was either prepared manually or fully automatically using the Hanabi cell harvester system PII. Slides were finally scored following an automatic or semi-automatic approach using the Metafer4 platform. The variance was calculated per dose and separately for each of the four fixation and scoring combinations. Thereafter, a serial of 16 blood samples of unknown exposure doses (0-3.9 Gy X-ray) was analyzed. Employing the four fixation and scoring combinations, we compared the number of dose estimates lying outside the ±0.5 Gy interval and the mean absolute difference (MAD) and examined sensitivity, specificity and accuracy of doses merged into binary dose categories of clinical significance. Irrespective of the fixation procedure, we observed at doses ≤1.0 Gy about 2-4 times higher median variances for the automated scoring procedure over the semi-automated approach (p ≤ 0.03). The lowest median variance was observed for automatic fixation + semi-automated scoring (135) which was even 2 times lower relative to manual fixation + semi-automated scoring (276, p = 0.04). These differences became negligible after doses >1.0 Gy. For the automatic fixation procedure, we also observed a tendency toward borderline significant higher numbers of dose estimates falling into the ±0.5 Gy interval (25 %, p = 0.08) and lower MAD values (50 %, p = 0.09), which was predominantly caused by the accuracy of dose assessment >1.0 Gy. Regarding the discrimination of binary dose categories of clinical significance, we observed a good agreement of both fixation procedures. The implementation of the automatic cell harvesting system considerably reduces the workload and results in dose estimates with a tendency of being slightly more accurate as they are after a manual fixation.
Assuntos
Citocinese/efeitos da radiação , Linfócitos/efeitos da radiação , Coleta de Tecidos e Órgãos/métodos , Relação Dose-Resposta à Radiação , Humanos , Testes para Micronúcleos/métodos , Doses de RadiaçãoRESUMO
BACKGROUND: We aimed to better discriminate metastasized (lymphogen/occult/both combined) from non-metastasized seminoma based on post-transcriptional changes examined in the peripheral blood. METHODS: Total RNAs including small RNAs were isolated from the peripheral blood of patients suffering from metastasized testicular tumours (lymphogen, n = 5, clinical stage IIb/c; occult, n = 5, clinical stage I) and non-metastasized patients (n = 5, clinical stage I). Small RNA next generation sequencing (SOLID, Life Technologies) was employed to examine post-transcriptional changes. We searched for small RNAs showing at least 50 reads and a significant ≥ 2-fold difference using peripheral blood small RNAs of non-metastasized tumours as the reference group. Candidate small RNAs were examined in univariate logistic regression analysis and combinations of two small RNAs were further examined using support vector machines. RESULTS: On average 1.3 x 10(7), 1.2 x 10(7) and 1.2 x 10(7) small RNA reads were detectable in non-metastasized, lymphogen and occult metastasized seminoma, respectively of which 73-76% remained after trimming. From these between 80-82% represented annotated reads and 7.2-7.8% (1.6-1.7 x 10(4)) were annotated small RNA tags. Of them 137 small RNAs showed > 50 reads and a ≥ two-fold difference to the reference. In univariate analysis we detected 33-35 different small RNAs which significantly discriminated lymphogen/occult/combined metastasized from non-metastasized seminoma and among these different comparisons it were the same small RNAs in 44-79%. Many combinations of two of these small RNAs completely discriminated metastasized from non-metastasized seminoma irrespective of the metastasis subtype. CONCLUSIONS: Metastasized (either lymphogen or occult) seminoma can be completely discriminated from non-metastasized seminoma with a combination of two small RNAs measured in the peripheral blood.
Assuntos
Biomarcadores Tumorais/sangue , MicroRNAs/sangue , Metástase Neoplásica/diagnóstico , Seminoma/sangue , Neoplasias Testiculares/sangue , Adulto , Biomarcadores Tumorais/genética , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/genética , Seminoma/genética , Seminoma/patologia , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologiaRESUMO
A multiple-parameter based approach using radiation-induced clinical signs and symptoms, hematology changes, cytogenetic chromosomal aberrations, and molecular biomarkers changes after radiation exposure is used for biodosimetry-based dose assessment. In the current article, relevant milestones from Radiation Research are documented that forms the basis of the current consensus approach for diagnostics after radiation exposure. For example, in 1962 the use of cytogenetic chromosomal aberration using the lymphocyte metaphase spread dicentric assay for biodosimetry applications was first published in Radiation Research. This assay is now complimented using other cytogenetic chromosomal aberration assays (i.e., chromosomal translocations, cytokinesis-blocked micronuclei, premature chromosome condensation, γ-H2AX foci, etc.). Changes in blood cell counts represent an early-phase biomarker for radiation exposures. Molecular biomarker changes have evolved to include panels of organ-specific plasma proteomic and blood-based gene expression biomarkers for radiation dose assessment. Maturation of these assays are shown by efforts for automated processing and scoring, development of point-of-care diagnostics devices, service laboratories inter-comparison exercises, and applications for dose and injury assessments in radiation accidents. An alternative and complementary approach has been advocated with the focus to de-emphasize "dose" and instead focus on predicting acute or delayed health effects. The same biomarkers used for dose estimation (e.g., lymphocyte counts) can be used to directly predict the later developing severity degree of acute health effects without performing dose estimation as an additional or intermediate step. This review illustrates contributing steps toward these developments published in Radiation Research.