In-Utero Low-Dose Irradiation Leads to Persistent Alterations in the Mouse Heart Proteome.

Prenatal exposure to stress such as increased level of reactive oxygen species or antiviral therapy are known factors leading to adult heart defects. The risks following a radiation exposure during fetal period are unknown, as are the mechanisms of any potential cardiac damage. The aim of this study was to gather evidence for possible damage by investigating long-term changes in the mouse heart proteome after prenatal exposure to low and moderate radiation doses. Pregnant C57Bl/6J mice received on embryonic day 11 (E11) a single total body dose of ionizing radiation that ranged from 0.02 Gy to 1.0 Gy. The offspring were sacrificed at the age of 6 months or 2 years. Quantitative proteomic analysis of heart tissue was performed using Isotope Coded Protein Label technology and tandem mass spectrometry. The proteomics data were analyzed by bioinformatics and key changes were validated by immunoblotting. Persistent changes were observed in the expression of proteins representing mitochondrial respiratory complexes, redox and heat shock response, and the cytoskeleton, even at the low dose of 0.1 Gy. The level of total and active form of the kinase MAP4K4 that is essential for the embryonic development of mouse heart was persistently decreased at the radiation dose of 1.0 Gy. This study provides the first insight into the molecular mechanisms of cardiac impairment induced by ionizing radiation exposure during the prenatal period

Significantly deregulated proteins after 1.0 Gy <i>in-utero</i> dose common to time points of 6 months and 2 years.

<p>Significantly deregulated proteins after 1.0 Gy <i>in-utero</i> dose common to time points of 6 months and 2 years.</p

STRING protein networks of significantly changed proteins at prenatal (E11) radiation exposure of 0.1 Gy and 1.0 Gy.

<p>Common networks between 6-month- and 2-year-time points are shown. Mitochondrial proteins, acute phase proteins and structural proteins represent the major protein classes of proteins affected by the pre-natal irradiation.</p

Immunoblot validation of proteomics data at 1.0 Gy using antibodies against vimentin (VIM), apolipoprotein E (APOE), LIM domain-binding protein (LDB3), and peroxiredoxin 5 (PRDX5).

<p>(A) The immunoblot images of VIM, APOE (6 months), PRDX5, and LDB3 are shown. The bar charts at 6 months (B) and 2 years (C) represent the average ratios with standard deviation (SD) of relative protein expression in control and 1.0 Gy irradiated samples after background correction and normalization to ATP synthase ß. (unpaired Student´s t-test; *p ≤0.05; **p≤0.01; n = 3).</p

Significantly deregulated proteins after 1.0 Gy <i>in-utero</i> dose common to time points of 6 months and 2 years.

<p>Significantly deregulated proteins after 1.0 Gy <i>in-utero</i> dose common to time points of 6 months and 2 years.</p

Venn diagrams showing the number of all and shared deregulated proteins at doses of 0.1 Gy and 1.0 Gy.

<p>(A) The numbers of deregulated proteins at 6 months and (B) 2 years are indicated above the circles. The list of shared proteins at these time points is shown on the right.</p

Characterization of MAP4K4 in the control and 1.0 Gy-irradiated mouse heart.

<p>(A) The immunoblot images of phospho-MAP4K4 (Ser-801) in the 1.0 Gy irradiated hearts compared to the controls at 6 months and 2 years is shown. (B) Columns represent the average ratios with standard deviation (SD) of relative protein expression in control and 1.0 Gy irradiated samples after background correction and normalization to ATP synthase ß (unpaired Student´s t-test; *p ≤0.05; ns, non-significant; n = 3). (C) The total amount of MAP4K4 measured using ELISA in control and 1.0 Gy irradiated heart tissue shows significant radiation-induced decrease at 6 months and 2 years (unpaired Student´s <i>t</i>-test; *<i>p</i> ≤0.05; ns, non-significant; n = 3).</p

Total Body Exposure to Low-Dose Ionizing Radiation Induces Long-Term Alterations to the Liver Proteome of Neonatally Exposed Mice

Tens of thousands of people are being exposed daily to environmental low-dose gamma radiation. Epidemiological data indicate that such low radiation doses may negatively affect liver function and result in the development of liver disease. However, the biological mechanisms behind these adverse effects are unknown. The aim of this study was to investigate radiation-induced damage in the liver after low radiation doses. Neonatal male NMRI mice were exposed to total body irradiation on postnatal day 10 using acute single doses ranging from 0.02 to 1.0 Gy. Early (1 day) and late (7 months) changes in the liver proteome were tracked using isotope-coded protein label technology and quantitative mass spectrometry. Our data indicate that low and moderate radiation doses induce an immediate inhibition of the glycolysis pathway and pyruvate dehydrogenase availability in the liver. Furthermore, they lead to significant long-term alterations in lipid metabolism and increased liver inflammation accompanying inactivation of the transcription factor peroxisome proliferator-activated receptor alpha. This study contributes to the understanding of the potential risk of liver damage in populations environmentally exposed to ionizing radiation

Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction

Epidemiological data from radiotherapy patients show the damaging effect of ionizing radiation on heart and vasculature. The endothelium is the main target of radiation damage and contributes essentially to the development of cardiac injury. However, the molecular mechanisms behind the radiation-induced endothelial dysfunction are not fully understood. In the present study, 10-week-old C57Bl/6 mice received local X-ray heart doses of 8 or 16 Gy and were sacrificed after 16 weeks; the controls were sham-irradiated. The cardiac microvascular endothelial cells were isolated from the heart tissue using streptavidin-CD31-coated microbeads. The cells were lysed and proteins were labeled with duplex isotope-coded protein label methodology for quantification. All samples were analyzed by LC–ESI–MS/MS and Proteome Discoverer software. The proteomics data were further studied by bioinformatics tools and validated by targeted transcriptomics, immunoblotting, immunohistochemistry, and serum profiling. Radiation-induced endothelial dysfunction was characterized by impaired energy metabolism and perturbation of the insulin/IGF-PI3K-Akt signaling pathway. The data also strongly suggested premature endothelial senescence, increased oxidative stress, decreased NO availability, and enhanced inflammation as main causes of radiation-induced long-term vascular dysfunction. Detailed data on molecular mechanisms of radiation-induced vascular injury as compiled here are essential in developing radiotherapy strategies that minimize cardiovascular complications

Differential Impact of Single-Dose Fe Ion and X-Ray Irradiation on Endothelial Cell Transcriptomic and Proteomic Responses.

Background and Purpose: Radiotherapy is an essential tool for cancer treatment. In order to spare normal tissues and to reduce the risk of normal tissue complications, particle therapy is a method of choice. Although a large part of healthy tissues can be spared due to improved depth dose characteristics, little is known about the biological and molecular mechanisms altered after particle irradiation in healthy tissues. Elucidation of these effects is also required in the context of long term space flights, as particle radiation is the main contributor to the radiation effects observed in space. Endothelial cells (EC), forming the inner layer of all vascular structures, are especially sensitive to irradiation and, if damaged, contribute to radiation-induced cardiovascular disease. Materials and Methods: Transcriptomics, proteomics and cytokine analyses were used to compare the response of ECs irradiated or not with a single 2 Gy dose of X-rays or Fe ions measured one and 7 days post-irradiation. To support the observed inflammatory effects, monocyte adhesion on ECs was also assessed. Results: Experimental data indicate time- and radiation quality-dependent changes of the EC response to irradiation. The irradiation impact was more pronounced and longer lasting for Fe ions than for X-rays. Both radiation qualities decreased the expression of genes involved in cell-cell adhesion and enhanced the expression of proteins involved in caveolar mediated endocytosis signaling. Endothelial inflammation and adhesiveness were increased with X-rays, but decreased after Fe ion exposure. Conclusions: Fe ions induce pro-atherosclerotic processes in ECs that are different in nature and kinetics than those induced by X-rays, highlighting radiation quality-dependent differences which can be linked to the induction and progression of cardiovascular diseases (CVD). Our findings give a better understanding of the underlying processes triggered by particle irradiation in ECs, a crucial aspect for the development of protective measures for cancer patients undergoing particle therapy and for astronauts in space