RESUMEN
Increased radiological and nuclear threats require preparedness. Our earlier work identified a set of four genes (DDB2, FDXR, POU2AF1 and WNT3), which predicts severity of the hematological acute radiation syndrome (H-ARS) within the first three days postirradiation In this study of 41 Rhesus macaques (Macaca mulatta, 27 males, 14 females) irradiated with 5.8-7.2 Gy (LD29-50/60), including some treated with gamma-tocotrienol (GT3, a radiation countermeasure) we independently validated these genes as predictors in both sexes and examined them after three days. At the Armed Forces Radiobiology Research Institute/Uniformed Services University of the Health Sciences, peripheral whole blood (1 ml) of Rhesus macaques was collected into PAXgene® Blood RNA tubes pre-irradiation after 1, 2, 3, 35 and 60 days postirradiation, stored at -80°C for internal experimental analyses. Leftover tubes from these already ongoing studies were kindly provided to Bundeswehr Institute of Radiobiology. RNA was isolated (QIAsymphony), converted into cDNA, and for further gene expression (GE) studies quantitative RT-PCR was performed. Differential gene expression (DGE) was measured relative to the pre-irradiation Rhesus macaques samples. Within the first three days postirradiation, we found similar results to human data: 1. FDXR and DDB2 were up-regulated, FDXR up to 3.5-fold, and DDB2 up to 13.5-fold in the median; 2. POU2AF1 appeared down regulated around tenfold in nearly all Rhesus macaques; 3. Contrary to human data, DDB2 was more up-regulated than FDXR, and the difference of the fold change (FC) ranged between 2.4 and 10, while the median fold changes of WNT3, except days 1 and 35, were close to 1. Nevertheless, 46% of the Rhesus macaques showed down-regulated WNT3 on day one postirradiation, which decreased to 12.2% on day 3 postirradiation. Considering the extended phase, there was a trend towards decreased fold changes at day 35, with median-fold changes ranging from 0.7 for DDB2 to 0.1 for POU2AF1, and on day 60 postirradiation, DGE in surviving animals was close to pre-exposure values for all four genes. In conclusion, the diagnostic significance for radiation-induced H-ARS severity prediction of FDXR, DDB2, and POU2AF1 was confirmed in this Rhesus macaques model. However, DDB2 showed higher GE values than FDXR. As shown in previous studies, the diagnostic significance of WNT3 could not be reproduced in Rhesus macaques; this could be due to the choice of animal model and methodological challenges.
Asunto(s)
Síndrome de Radiación Aguda , Macaca mulatta , Animales , Masculino , Femenino , Síndrome de Radiación Aguda/sangre , Síndrome de Radiación Aguda/genéticaRESUMEN
In times of war, radiological/nuclear emergency scenarios have become a reemphasized threat. However, there are challenges in transferring whole-blood samples to laboratories for specialized diagnostics using RNA. This project aims to miniaturize the process of unwieldy conventional RNA extraction with its stationed technical equipment using a microfluidic-based slide (MBS) for point-of-care diagnostics. The MBS is thought to be a preliminary step toward the development of a so-called lab-on-a-chip microfluidic device. A MBS would enable early and fast field care combined with gene expression (GE) analysis for the prediction of hematologic acute radiation syndrome (HARS) severity or identification of RNA microbes. Whole blood samples from ten healthy donors were irradiated with 0, 0.5 and 4 Gy, simulating different ARS severity degrees. RNA quality and quantity of a preliminary MBS was compared with a conventional column-based (CB) RNA extraction method. GE of four HARS severity-predicting radiation-induced genes (FDXR, DDB2, POU2AF1 and WNT3) was examined employing qRT-PCR. Compared to the CB method, twice as much total RNA from whole blood could be extracted using the MBS (6.6 ± 3.2 µg vs. 12.0 ± 5.8 µg) in half of the extraction time, and all MBS RNA extracts appeared DNA-free in contrast to the CB method (30% were contaminated with DNA). Using MBS, RNA quality [RNA integrity number equivalent (RINe)] values decreased about threefold (3.3 ± 0.8 vs. 9.0 ± 0.4), indicating severe RNA degradation, while expected high-quality RINe ≥ 8 were found using column-based method. However, normalized cycle threshold (Ct) values, as well as radiation-induced GE fold-changes appeared comparable for all genes utilizing both methods, indicating that no RNA degradation took place. In summary, the preliminary MBS showed promising features such as: 1. halving the RNA extraction time without the burden of heavy technical equipment (e.g., a centrifuge); 2. absence of DNA contamination in contrast to CB RNA extraction; 3. reduction in blood required, because of twice the biological output of RNA; and 4. equal GE performance compared to CB, thus, increasing its appeal for later semi-automatic parallel field applications.
Asunto(s)
Sistemas de Atención de Punto , ARN , Humanos , ARN/aislamiento & purificación , ARN/sangre , ARN/genética , Dispositivos Laboratorio en un Chip , Síndrome de Radiación Aguda/sangre , Síndrome de Radiación Aguda/etiología , Síndrome de Radiación Aguda/diagnóstico , Síndrome de Radiación Aguda/genéticaRESUMEN
Radiological and especially nuclear accidents and incidents pose a threat to populations. In such events, gene expression (GE) analysis of a set of 4 genes (FDXR, DDB2, POU2AF1, WNT3) is an emerging approach for early and high-throughput prediction of the later manifesting severity degrees of the hematological acute radiation syndrome (H-ARS). Validation of this gene set on radiation victims is difficult since these events are rare. However, chemotherapy (CTX) is widely used e.g., breast cancer patient treatment and pathomechanisms, as well as blood cell count changes are comparable among both exposure types. We wondered whether GE changes are similarly deregulated after CTX, which would be interpreted as a confirmation of our already identified gene set for H-ARS prediction after irradiation. We examined radiation-induced differential GE (DGE) of our gene set as a positive control using in vitro whole blood samples from ten healthy donors (6 females, 4 males, aged: 24-40 years). Blood was incubated in vitro for 8 h after X irradiation with 0 and 4 Gy (1 Gy/min). These data were compared with DGE measured in vivo in blood samples of 10 breast tumor CTX patients (10 females, aged: 39-71 years) before and 4 days after administration of cyclophosphamide and epirubicin. RNA was isolated, reverse transcribed and quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to assess DGE of FDXR, DDB2, POU2AF1 and WNT3 relative to the unexposed samples using TaqMan assays. After X irradiation, we found a significant upregulation (irrespective of sex) with mean fold changes of 21 (P < 0.001) and 7 (P < 0.001) for FDXR and DDB2 and a significant down-regulation with mean fold changes of 2.5 (P < 0.001) and 2 (P = 0.005) for POU2AF1 and WNT3, respectively. After CTX, a similar pattern was observed, although mean fold changes of up-regulated FDXR (6-fold, P < 0.001) and DDB2 (3-fold, P < 0.001) as well as down-regulated POU2AF1 (1.2-fold, P = 0.270) and WNT3 (1.3-fold, P = 0.069) appeared lower corresponding to less altered blood cell count changes observed after CTX compared to historic radiation exposure data. However, a subpopulation of CTX patients (n = 6) showed on average a significant downregulation of POU2AF1 (1.8-fold, P = 0.04) and WNT3 (2.1-fold, P = 0.008). In summary, the pattern of up-regulated GE changes observed in all CTX patients and down-regulated GE changes observed in a subgroup of CTX patients appeared comparable with an already identified gene set predictive for the radiation-induced H-ARS. This underlines the significance of in vivo GE measurements in CTX patients, employed as a surrogate model to further validate already identified radiation-induced GE changes predictive for the H-ARS.
Asunto(s)
Síndrome de Radiación Aguda , Exposición a la Radiación , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Síndrome de Radiación Aguda/genética , Relación Dosis-Respuesta en la Radiación , Perfilación de la Expresión Génica , Radiografía , ARNRESUMEN
PURPOSE: In a nuclear or radiological event, an early diagnostic or prognostic tool is needed to distinguish unexposed from low- and highly exposed individuals with the latter requiring early and intensive medical care. Radiation-induced gene expression (GE) changes observed within hours and days after irradiation have shown potential to serve as biomarkers for either dose reconstruction (retrospective dosimetry) or the prediction of consecutively occurring acute or chronic health effects. The advantage of GE markers lies in their capability for early (1-3 days after irradiation), high-throughput, and point-of-care (POC) diagnosis required for the prediction of the acute radiation syndrome (ARS). CONCLUSIONS: As a key session of the ConRad conference in 2021, experts from different institutions were invited to provide state-of-the-art information on a range of topics including: (1) Biodosimetry: What are the current efforts to enhance the applicability of this method to perform retrospective biodosimetry? (2) Effect prediction: Can we apply radiation-induced GE changes for prediction of acute health effects as an approach, complementary to and integrating retrospective dose estimation? (3) High-throughput and point-of-care diagnostics: What are the current developments to make the GE approach applicable as a high-throughput as well as a POC diagnostic platform? (4) Low level radiation: What is the lowest dose range where GE can be used for biodosimetry purposes? (5) Methodological considerations: Different aspects of radiation-induced GE related to more detailed analysis of exons, transcripts and next-generation sequencing (NGS) were reported.
Asunto(s)
Síndrome de Radiación Aguda , Radiometría , Síndrome de Radiación Aguda/genética , Biomarcadores , Expresión Génica , Humanos , Radiometría/métodos , Estudios RetrospectivosRESUMEN
Recent political unrest has highlighted the importance of understanding the short- and long-term effects of gamma-radiation exposure on human health and survivability. In this regard, effective treatment for acute radiation syndrome (ARS) is a necessity in cases of nuclear disasters. Here, we propose 20 therapeutic targets for ARS identified using a systematic approach that integrates gene coexpression networks obtained under radiation treatment in humans and mice, drug databases, disease-gene association, radiation-induced differential gene expression, and literature mining. By selecting gene targets with existing drugs, we identified potential candidates for drug repurposing. Eight of these genes (BRD4, NFKBIA, CDKN1A, TFPI, MMP9, CBR1, ZAP70, IDH3B) were confirmed through literature to have shown radioprotective effect upon perturbation. This study provided a new perspective for the treatment of ARS using systems-level gene associations integrated with multiple biological information. The identified genes might provide high confidence drug target candidates for potential drug repurposing for ARS.
Asunto(s)
Síndrome de Radiación Aguda , Bases de Datos de Ácidos Nucleicos , Sistemas de Liberación de Medicamentos , Redes Reguladoras de Genes , Factores de Transcripción , Transcriptoma , Síndrome de Radiación Aguda/tratamiento farmacológico , Síndrome de Radiación Aguda/genética , Síndrome de Radiación Aguda/metabolismo , Síndrome de Radiación Aguda/patología , Animales , Reposicionamiento de Medicamentos , Humanos , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: In a nuclear or radiological event, an early diagnostic tool is needed to distinguish the worried well from those individuals who may later develop life-threatenFing hematologic acute radiation syndrome. We examined the contribution of the peripheral blood's cell populations on radiation-induced gene expression (GE) changes. MATERIALS AND METHODS: EDTA-whole-blood from six healthy donors was X-irradiated with 0 and 4Gy and T-lymphocytes, B-lymphocytes, NK-cells and granulocytes were separated using immunomagnetic methods. GE were examined in cell populations and whole blood. RESULTS: The cell populations contributed to the total RNA amount with a ratio of 11.6 for T-lymphocytes, 1.2 for B-cells, 1.2 for NK-cells, 1.0 for granulocytes. To estimate the contribution of GE per cell population, the baseline (0Gy) and the radiation-induced fold-change in GE relative to unexposed was considered for each gene. The T-lymphocytes (74.8%/80.5%) contributed predominantly to the radiation-induced up-regulation observed for FDXR/DDB2 and the B-lymphocytes (97.1%/83.8%) for down-regulated POU2AF1/WNT3 with a similar effect on whole blood gene expression measurements reflecting a corresponding order of magnitude. CONCLUSIONS: T- and B-lymphocytes contributed predominantly to the radiation-induced up-regulation of FDXR/DDB2 and down-regulation of POU2AF1/WNT3. This study underlines the use of FDXR/DDB2 for biodosimetry purposes and POU2AF1/WNT3 for effect prediction of acute health effects.
Asunto(s)
Síndrome de Radiación Aguda/genética , Células Sanguíneas/metabolismo , Células Sanguíneas/efectos de la radiación , Perfilación de la Expresión Génica , Adulto , Humanos , Irradiación Corporal Total/efectos adversosRESUMEN
Current models to study the hematopoietic syndrome largely rely on the uniform whole-body exposures. However, in the radio-nuclear accidents or terrorist events, exposure can be non-uniform. The data available on the non-uniform exposures is limited. Thus, we have developed a mice model for studying the hematopoietic syndrome in the non-uniform or partial body exposure scenarios using the localized cobalt60 gamma radiation exposure. Femur region of Strain 'A' male mice was exposed to doses ranging from 7 to 20 Gy. The 30 day survival assay showed 19 Gy as LD100 and 17 Gy as LD50. We measured an array of cytokines and important stem cell markers such as IFN-γ, IL-3, IL-6, GM-CSF, TNF-α, G-CSF, IL-1α, IL-1ß, CD 34 and Sca 1. We found significant changes in IL-6, GM-CSF, TNF-α, G-CSF, and IL-1ß levels compared to untreated groups and amplified levels of CD 34 and Sca 1 positive population in the irradiated mice compared to the untreated controls. Overall, we have developed a mouse model of the hematopoietic acute radiation syndrome that might be useful for understanding of the non-uniform body exposure scenarios. This may also be helpful in the screening of drugs intended for individuals suffering from radiation induced hematopoietic syndrome.
Asunto(s)
Síndrome de Radiación Aguda/etiología , Modelos Animales de Enfermedad , Enfermedades Hematológicas/etiología , Exposición a la Radiación/efectos adversos , Síndrome de Radiación Aguda/genética , Síndrome de Radiación Aguda/metabolismo , Animales , Radioisótopos de Cobalto/efectos adversos , Radioisótopos de Cobalto/química , Citocinas/genética , Citocinas/metabolismo , Fémur/metabolismo , Fémur/efectos de la radiación , Rayos gamma/efectos adversos , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/metabolismo , Humanos , Masculino , RatonesRESUMEN
Exposure to acute, high-dose, whole-body ionizing radiation results in bone marrow failure (hematopoietic acute radiation syndrome with resultant infection, bleeding, anemia, and increased risk of death). Sargramostim (yeast-derived rhu GM-CSF), a yeast-derived, molecularly cloned, hematopoietic growth factor and pleiotropic cytokine supports proliferation, differentiation, maturation and survival of cells of several myeloid lineages. We evaluated the efficacy of sargramostim in non-human primates (rhesus macaques) exposed to whole-body ionizing radiation at a 50-60% lethal dose. The primary end point was day 60 survival. Non-human primates received daily subcutaneous sargramostim (7 mcg/kg/day) or control. To reflect the anticipated setting of a nuclear or radiologic event, treatment began 48 h postirradiation, and non-human primates received only moderate supportive care (no whole blood transfusions or individualized antibiotics). Sargramostim significantly increased day 60 survival to 78% (95% confidence interval, 61-90%) vs. 42% (26-59%; P = 0.0018) in controls. Neutrophil, platelet and lymphocyte recovery rates were accelerated and infection rates decreased. Improved survival when sargramostim was started 48 h postirradiation, without use of intensive supportive care, suggests sargramostim may be effective in treating humans exposed to acute, high-dose whole-body, ionizing radiation in a scenario such as a mass casualty event.
Asunto(s)
Síndrome de Radiación Aguda/tratamiento farmacológico , Células de la Médula Ósea/efectos de los fármacos , Trastornos de Fallo de la Médula Ósea/tratamiento farmacológico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Síndrome de Radiación Aguda/genética , Síndrome de Radiación Aguda/patología , Animales , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/efectos de la radiación , Trastornos de Fallo de la Médula Ósea/genética , Trastornos de Fallo de la Médula Ósea/patología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de la radiación , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Factor Estimulante de Colonias de Granulocitos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Macaca mulatta/genética , Masculino , Proteínas Recombinantes/farmacología , Irradiación Corporal Total/efectos adversosRESUMEN
Identification of medical countermeasures (MCM) to mitigate radiation damage and/or protect first responders is a compelling unmet medical need. The prostaglandin E2 (PGE2) analog, 16,16 dimethyl-PGE2 (dmPGE2), has shown efficacy as a radioprotectant and radiomitigator that can enhance hematopoiesis and ameliorate intestinal mucosal cell damage. In this study, we optimized the time of administration of dmPGE2 for protection and mitigation against mortality from the hematopoietic acute radiation syndrome (H-ARS) in young adult mice, evaluated its activity in pediatric and geriatric populations, and investigated potential mechanisms of action. Windows of 30-day survival efficacy for single administration of dmPGE2 were defined as within 3 h prior to and 6-30 h after total-body γ irradiation (TBI). Radioprotective and radio-mitigating efficacy was also observed in 2-year-old geriatric mice and 6-week-old pediatric mice. PGE2 receptor agonist studies suggest that signaling through EP4 is primarily responsible for the radioprotective effects. DmPGE2 administration prior to TBI attenuated the drop in red blood cells and platelets, accelerated recovery of all peripheral blood cell types, and resulted in higher hematopoietic and mesenchymal stem cells in survivor bone marrow. Multiplex analysis of bone marrow cytokines together with RNA sequencing of hematopoietic stem cells indicated a pro-hematopoiesis cytokine milieu induced by dmPGE2, with IL-6 and G-CSF strongly implicated in dmPGE2-mediated radioprotective activity. In summary, we have identified windows of administration for significant radio-mitigation and radioprotection by dmPGE2 in H-ARS, demonstrated survival efficacy in special populations, and gained insight into radioprotective mechanisms, information useful towards development of dmPGE2 as a MCM for first responders, military personnel, and civilians facing radiation threats.
Asunto(s)
Síndrome de Radiación Aguda/tratamiento farmacológico , Dinoprostona/farmacología , Tolerancia a Radiación/genética , Protectores contra Radiación/farmacología , Síndrome de Radiación Aguda/genética , Síndrome de Radiación Aguda/patología , Animales , Dinoprostona/análogos & derivados , Dinoprostona/genética , Relación Dosis-Respuesta en la Radiación , Rayos gamma/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Factor Estimulante de Colonias de Granulocitos/genética , Hematopoyesis/efectos de los fármacos , Hematopoyesis/efectos de la radiación , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Interleucina-6/genética , Ratones , Tolerancia a Radiación/efectos de los fármacos , Análisis de Secuencia de ARN , Irradiación Corporal TotalRESUMEN
In the event of a mass casualty radiological or nuclear scenario, it is important to distinguish between the unexposed (worried well), low-dose exposed individuals and those developing the hematological acute radiation syndrome (HARS) within the first three days postirradiation. In previous baboon studies, we identified altered gene expression changes after irradiation, which were predictive for the later developing HARS severity. Similar changes in the expression of four of these genes were observed using an in vitro human whole blood model. However, these studies have provided only limited information on the time frame of the changes after exposure in relationship to the development of HARS. In this study we analyzed the time-dependent changes in mRNA expression after in vitro irradiation of whole blood. Changes in the expression of informative mRNAs (FDXR, DBB2, POU2AF1 and WNT3) were determined in the blood of eight healthy donors (6 males, 2 females) after irradiation at 0 (control), 0.5, 2 and 4 Gy using qRT-PCR. FDXR expression was significantly upregulated (P < 0.001) 4 h after ≥0.5 Gy irradiation, with an 18-40-fold peak attained 4-12 h postirradiation which remained elevated (4-9-fold) at 72 h. DDB2 expression was upregulated after 4 h (fold change, 5-8, P < 0.001 at ≥ 0.5 Gy) and remained upregulated (3-4-fold) until 72 h (P < 0.001). The earliest time points showing a significant downregulation of POU2AF1 and WNT3 were 4 h (fold change = 0.4, P = 0.001, at 4 Gy) and 8 h (fold change = 0.3-0.5, P < 0.001, 2-4 Gy), respectively. These results indicate that the diagnostic window for detecting HARS-predictive changes in gene expression may be opened as early as 2 h for most (75%) and at 4 h postirradiation for all individuals examined. Depending on the RNA species studied this may continue for at least three days postirradiation.
Asunto(s)
Síndrome de Radiación Aguda/diagnóstico , Regulación de la Expresión Génica/efectos de la radiación , ARN Mensajero/genética , Irradiación Corporal Total/efectos adversos , Síndrome de Radiación Aguda/genética , Síndrome de Radiación Aguda/patología , Animales , Relación Dosis-Respuesta en la Radiación , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Humanos , Masculino , Papio/genética , ARN Mensajero/efectos de la radiación , Dosis de RadiaciónRESUMEN
Exposure to high-dose total body irradiation (TBI) can result in hematopoietic acute radiation syndrome (H-ARS), characterized by leukopenia, anemia, and coagulopathy. Death from H-ARS occurs from hematopoietic insufficiency and opportunistic infections. Following radiation exposure, red blood cells (RBCs) undergo hemolysis from radiation-induced hemoglobin denaturation, causing the release of iron. Free iron can have multiple detrimental biological effects, including suppression of hematopoiesis. We investigated the impact of radiation-induced iron release on the bone marrow following TBI and the potential impact of the ACE inhibitor captopril, which improves survival from H-ARS. C57BL/6J mice were exposed to 7.9 Gy, 60Co irradiation, 0.6 Gy/min (LD70-90/30). RBCs and reticulocytes were significantly reduced within 7 days of TBI, with the RBC nadir at 14-21 days. Iron accumulation in the bone marrow correlated with the time course of RBC hemolysis, with an â¼10-fold increase in bone marrow iron at 14-21 days post-irradiation, primarily within the cytoplasm of macrophages. Iron accumulation in the bone marrow was associated with increased expression of genes for iron binding and transport proteins, including transferrin, transferrin receptor 1, ferroportin, and integrin αMß2. Expression of the gene encoding Nrf2, a transcription factor activated by oxidative stress, also increased at 21 days post-irradiation. Captopril did not alter iron accumulation in the bone marrow or expression of iron storage genes, but did suppress Nrf2 expression. Our study suggests that following TBI, iron is deposited in tissues not normally associated with iron storage, which may be a secondary mechanism of radiation-induced tissue injury.
Asunto(s)
Síndrome de Radiación Aguda/metabolismo , Médula Ósea/metabolismo , Rayos gamma/efectos adversos , Hematopoyesis/efectos de la radiación , Hierro/metabolismo , Traumatismos Experimentales por Radiación/metabolismo , Síndrome de Radiación Aguda/genética , Síndrome de Radiación Aguda/patología , Animales , Médula Ósea/patología , Captopril/farmacología , Eritrocitos/metabolismo , Eritrocitos/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Ratones , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/biosíntesis , Factor 2 Relacionado con NF-E2/genética , Traumatismos Experimentales por Radiación/genética , Traumatismos Experimentales por Radiación/patologíaRESUMEN
Radiological exposure scenarios involving large numbers of people require a rapid and high-throughput method to identify the unexposed, and those exposed to low- and high-dose radiation. Those with high-dose exposure, e.g., >2 Gy and depending on host characteristics, may develop severe hematological acute radiation syndrome (HARS), requiring hospitalization and treatment. Previously, we identified a set of genes that discriminated these clinically relevant groups. In the current work, we examined the utility of gene expression changes to classify 1,000 split blood samples into HARS severity scores of H0, H1 and H2-4, with the latter indicating likely hospitalization. In several previous radiation dose experiments, we determined that these HARS categories corresponded, respectively, to doses of 0 Gy (unexposed), 0.5 Gy and 5 Gy. The main purpose of this work was to assess the rapidity of blood sample processing using targeted next-generation sequencing (NGS). Peripheral blood samples from two healthy donors were X-ray irradiated in vitro and incubated at 37°C for 24 h. A total of 1,000 samples were evaluated by laboratory personnel blinded to the radiation dose. Changes in gene expression of FDXR, DDB2, POU2AF1 and WNT3 were examined with qRT-PCR as positive controls. Targeted NGS (TREX) was used on all samples for the same four genes. Agreement using both methods was almost 78%. Using NGS, all 1,000 samples were processed within 30 h. Classification of the HARS severity categories corresponding to radiation dose had an overall agreement ranging between 90-97%. Depending on the end point, either a combination of all genes or FDXR alone (H0 HARS or unexposed) provided the best classification. Using this optimized automated methodology, we assessed 100× more samples approximately three times faster compared to standard cytogenetic studies. We showed that a small set of genes, rather than a complex constellation of genes, provided robust positive (97%) and negative (97%) predictive values for HARS categories and radiation doses of 0, 0.5 and 5 Gy. The findings of this study support the potential utility of early radiation-induced gene expression changes for high-throughput biodosimetry and rapid identification of irradiated persons in need of hospitalization.
Asunto(s)
Síndrome de Radiación Aguda/diagnóstico , Síndrome de Radiación Aguda/genética , Perfilación de la Expresión Génica , Exposición a la Radiación/efectos adversos , Triaje/métodos , Síndrome de Radiación Aguda/sangre , Síndrome de Radiación Aguda/etiología , Adulto , Reacciones Falso Positivas , Femenino , Humanos , Masculino , Factores de Tiempo , Adulto JovenRESUMEN
Genomes are mostly protected from constant DNA-damaging threats, either internal or external, which ultimately sustain the organism. Herein, we report that AIMP3, a previously demonstrated tumour suppressor, plays an essential role in maintaining genome integrity in adult mice. Upon induction of the temporal systemic deletion of AIMP3 by tamoxifen in adult mice, the animals developed an acute radiation syndrome-like phenotype, typified by scleroderma, hypotrophy of haematopoietic cells and organs, and intestinal failure. Induction of γH2AX, an early marker of DNA double-strand breaks, was observed in the spleen, intestine, and the highly replicating embryonic cortex. In addition, sub-lethal irradiation of AIMP3 mKO mice dramatically affected organ damage and survival. Using isolated MEFs from conditional KO mice or AIMP3 knockdown cells, we confirmed the presence of spontaneously occurring DNA double-strand breaks by COMET assay and γH2AX induction. Furthermore, γH2AX removal was delayed, and homologous DNA repair activity was significantly reduced. Reduction of RPA foci formation and subsequent Rad51 foci formation probably underlie the significant reduction in homologous recombination activity in the absence of AIMP3. Together, our data demonstrate that AIMP3 plays a role in genome stability through the DNA repair process.
Asunto(s)
Síndrome de Radiación Aguda/genética , Histonas/genética , Factores de Elongación de Péptidos/genética , Recombinasa Rad51/genética , Síndrome de Radiación Aguda/patología , Animales , Ensayo Cometa , Roturas del ADN de Doble Cadena/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Fibroblastos/efectos de la radiación , Inestabilidad Genómica/efectos de la radiación , Recombinación Homóloga/efectos de la radiación , Humanos , Ratones , Ratones Noqueados , Fenotipo , Radiación , Radiación Ionizante , Proteínas Supresoras de Tumor/genéticaRESUMEN
Threats of nuclear terrorism coupled with potential unintentional ionizing radiation exposures have necessitated the need for large-scale response efforts of such events, including high-throughput biodosimetry for medical triage. Global metabolomics utilizing mass spectrometry (MS) platforms has proven an ideal tool for generating large compound databases with relative quantification and structural information in a short amount of time. Determining metabolite panels for biodosimetry requires experimentation to evaluate the many factors associated with compound concentrations in biofluids after radiation exposures, including temporal changes, pre-existing conditions, dietary intake, partial- vs. total-body irradiation (TBI), among others. Here, we utilize a nonhuman primate (NHP) model and identify metabolites perturbed in serum after 7.2 Gy TBI without supportive care [LD70/60, hematologic (hematopoietic) acute radiation syndrome (HARS) level H3] at 24, 36, 48 and 96 h compared to preirradiation samples with an ultra-performance liquid chromatography quadrupole time-of-flight (UPLC-QTOF) MS platform. Additionally, we document changes in cytokine levels. Temporal changes observed in serum carnitine, acylcarnitines, amino acids, lipids, deaminated purines and increases in pro-inflammatory cytokines indicate clear metabolic dysfunction after radiation exposure. Multivariate data analysis shows distinct separation from preirradiation groups and receiver operator characteristic curve analysis indicates high specificity and sensitivity based on area under the curve at all time points after 7.2 Gy irradiation. Finally, a comparison to a 6.5 Gy (LD50/60, HARS level H2) cohort after 24 h postirradiation revealed distinctly increased separations from the 7.2 Gy cohort based on multivariate data models and higher compound fold changes. These results highlight the utility of MS platforms to differentiate time and absorbed dose after a potential radiation exposure that may aid in assigning specific medical interventions and contribute as additional biodosimetry tools.
Asunto(s)
Síndrome de Radiación Aguda/sangre , Metaboloma/efectos de la radiación , Metabolómica , Primates/sangre , Síndrome de Radiación Aguda/genética , Síndrome de Radiación Aguda/fisiopatología , Aminoácidos/sangre , Animales , Carnitina/análogos & derivados , Carnitina/sangre , Citocinas/sangre , Humanos , Lípidos/sangre , Macaca mulatta/sangre , Espectrometría de Masas , Metaboloma/genética , Purinas/sangre , Radiación Ionizante , Irradiación Corporal TotalRESUMEN
In previous studies we determined a gene expression signature in baboons for predicting the severity of hematological acute radiation syndrome. We subsequently validated a set of eight of these genes in leukemia patients undergoing total-body irradiation. In the current study, we addressed the effect of intra-individual variability on the basal level of expression of those eight radiation-responsive genes identified previously, by examining baseline levels in 200 unexposed healthy human donors (122 males and 88 females with an average age of 46 years) using real-time PCR. In addition to the eight candidate genes ( DAGLA, WNT3, CD177, PLA2G16, WLS, POU2AF1, STAT4 and PRF1), we examined two more genes ( FDXR and DDB2) widely used in ex vivo whole blood experiments. Although significant sex- (seven genes) and age-dependent (two genes) differences in expression were found, the fold changes ranged only between 1.1-1.6. These were well within the twofold differences in gene expression generally considered to represent control values. Age and sex contributed less than 20-30% to the complete inter-individual variance, which is calculated as the fold change between the lowest (reference) and the highest Ct value minimum-maximum fold change (min-max FC). Min-max FCs ranging between 10-17 were observed for most genes; however, for three genes, min-max FCs of complete inter-individual variance were found to be 37.1 ( WNT3), 51.4 ( WLS) and 1,627.8 ( CD177). In addition, to determine whether discrimination between healthy and diseased baboons might be altered by replacing the published gene expression data of the 18 healthy baboons with that of the 200 healthy humans, we employed logistic regression analysis and calculated the area under the receiver operating characteristic (ROC) curve. The additional inter-individual variance of the human data set had either no impact or marginal impact on the ROC area, since up to 32-fold change gene expression differences between healthy and diseased baboons were observed.
Asunto(s)
Síndrome de Radiación Aguda/genética , Regulación de la Expresión Génica/efectos de la radiación , Biosíntesis de Proteínas/efectos de la radiación , Síndrome de Radiación Aguda/fisiopatología , Adulto , Animales , Relación Dosis-Respuesta en la Radiación , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Papio/genética , Biosíntesis de Proteínas/genética , Triaje , Irradiación Corporal TotalRESUMEN
Exposure to high doses of ionizing radiation can cause lethal injury to normal tissue, thus inducing acute radiation syndrome. Acute radiation syndrome is caused by depletion of bone marrow cells (hematopoietic syndrome) and irreparable damage to the epithelial cells in the gastrointestinal tract (gastrointestinal syndrome). Although radiation initiates apoptosis in the hematopoietic and gastrointestinal compartments within the first few hours after exposure, alternative mechanisms of cell death may contribute to injury in these radiosensitive tissues. In this study, we utilized mice lacking a critical regulator of necroptosis, receptor interacting protein 3 (RIP3) kinase, to characterize the role of RIP3 in normal tissue toxicity after irradiation. Our results suggest that RIP3-mediated signaling is not a critical driver of acute radiation syndrome.
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Síndrome de Radiación Aguda/enzimología , Síndrome de Radiación Aguda/genética , Técnicas de Inactivación de Genes , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Síndrome de Radiación Aguda/patología , Animales , Apoptosis/efectos de la radiación , Hematopoyesis/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Necrosis/etiología , Transducción de Señal/efectos de la radiaciónAsunto(s)
Síndrome de Radiación Aguda/diagnóstico , MicroARNs/genética , Traumatismos por Radiación/diagnóstico , Protectores contra Radiación/farmacología , gamma-Tocoferol/farmacología , Síndrome de Radiación Aguda/genética , Síndrome de Radiación Aguda/patología , Síndrome de Radiación Aguda/prevención & control , Animales , Biomarcadores/sangre , Modelos Animales de Enfermedad , Relación Dosis-Respuesta en la Radiación , Regulación de la Expresión Génica , Humanos , Ratones , MicroARNs/sangre , Traumatismos por Radiación/genética , Traumatismos por Radiación/patología , Traumatismos por Radiación/prevención & control , Radiación Ionizante , Liberación de Radiactividad Peligrosa , Irradiación Corporal TotalRESUMEN
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.
Asunto(s)
Síndrome de Radiación Aguda/genética , Expresión Génica , MicroARNs/genética , Pancitopenia/etiología , ARN Mensajero/genética , Animales , Modelos Animales de Enfermedad , Expresión Génica/efectos de la radiación , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Papio , Tolerancia a RadiaciónRESUMEN
For effective medical management of radiation-exposed persons after a radiological/nuclear event, blood-based screening measures in the first few days that could predict hematologic acute radiation syndrome (HARS) are needed. For HARS severity prediction, we used microRNA (miRNA) expression changes measured on days one and two after irradiation in a baboon model. Eighteen baboons underwent different patterns of partial or total body irradiation, corresponding to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts (BCC) the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. In a two Stage study design we screened 667 miRNAs using a quantitative real-time polymerase chain reaction (qRT-PCR) platform. In Stage II we validated candidates where miRNAs had to show a similar regulation (up- or down-regulated) and a significant 2-fold miRNA expression difference over H0. Seventy-two candidate miRNAs (42 for H1-2 and 30 for H2-3) were forwarded for validation. Forty-two of the H1-2 miRNA candidates from the screening phase entered the validation step and 20 of them showed a statistically significant 2-4 fold up-regulation relative to the unexposed reference (H0). Fifteen of the 30 H2-3 miRNAs were validated in Stage II. All miRNAs appeared 2-3 fold down-regulated over H0 and allowed an almost complete separation of HARS categories; the strongest candidate, miR-342-3p, showed a sustained and 10-fold down-regulation on both days 1 and 2. In summary, our data support the medical decision making of the HARS even within the first two days after exposure where diagnostic tools for early medical decision are required but so far missing. The miRNA species identified and in particular miR-342-3p add to the previously identified mRNAs and complete the portfolio of identified mRNA and miRNA transcripts for HARS prediction and medical management.
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Síndrome de Radiación Aguda/diagnóstico , Síndrome de Radiación Aguda/genética , MicroARNs/genética , Papio/genética , Síndrome de Radiación Aguda/sangre , Animales , Perfilación de la Expresión Génica , Masculino , MicroARNs/metabolismo , ARN/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Exposición a la Radiación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
We implemented a two-stage study to predict late occurring hematologic acute radiation syndrome (HARS) in a baboon model based on gene expression changes measured in peripheral blood within the first two days after irradiation. Eighteen baboons were irradiated to simulate different patterns of partial-body and total-body exposure, which corresponded to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. Blood samples taken before irradiation served as unexposed control (H0, n = 17). For stage I of this study, a whole genome screen (mRNA microarrays) was performed using a portion of the samples (H0, n = 5; H1-2, n = 4; H2-3, n = 5). For stage II, using the remaining samples and the more sensitive methodology, qRT-PCR, validation was performed on candidate genes that were differentially up- or down-regulated during the first two days after irradiation. Differential gene expression was defined as significant (P < 0.05) and greater than or equal to a twofold difference above a H0 classification. From approximately 20,000 genes, on average 46% appeared to be expressed. On day 1 postirradiation for H2-3, approximately 2-3 times more genes appeared up-regulated (1,418 vs. 550) or down-regulated (1,603 vs. 735) compared to H1-2. This pattern became more pronounced at day 2 while the number of differentially expressed genes decreased. The specific genes showed an enrichment of biological processes coding for immune system processes, natural killer cell activation and immune response (P = 1 × E-06 up to 9 × E-14). Based on the P values, magnitude and sustained differential gene expression over time, we selected 89 candidate genes for validation using qRT-PCR. Ultimately, 22 genes were confirmed for identification of H1-3 classifications and seven genes for identification of H2-3 classifications using qRT-PCR. For H1-3 classifications, most genes were constantly three to fivefold down-regulated relative to H0 over both days, but some genes appeared 10.3-fold (VSIG4) or even 30.7-fold up-regulated (CD177) over H0. For H2-3, some genes appeared four to sevenfold up-regulated relative to H0 (RNASE3, DAGLA, ARG2), but other genes showed a strong 14- to 33-fold down-regulation relative to H0 (WNT3, POU2AF1, CCR7). All of these genes allowed an almost completely identifiable separation among each of the HARS categories. In summary, clinically relevant HARS can be independently predicted with all 29 irradiated genes examined in the peripheral blood of baboons within the first two days postirradiation. While further studies are needed to confirm these findings, this model shows potential relevance in the prediction of clinical outcomes in exposed humans and as an aid in the prioritizing of medical treatment.