Radiation induced isostaining: fact or fiction?

Two different experiments were set up to induce differential staining in M-1¹ cells or to induce isostaining in M-2 cells after -irradiation of human lymphocytes. Neither of these two unusual staining patterns previously described by Luchnik and Porjadkova (1977) were observed. Possible explanations for this disagreement are discussed.Abbreviations SCE Sister chromatid exchange - IS Isostained regions - S-1, S-2, S-3 and S-4 First, second, third and fourth DNA synthetic periods after addition of 5-bromodeoxyuridine - M-1, M-2, M-3, M-4 and M-5 First, second, third, fourth and fifth mitosis after addition of 5-bromodeoxyuridine - BrdU 5-bromodeoxyuridine - EMEM Eagle's Minimum Essential Medium     

Cytokine Plasma Levels: Reliable Predictors for Radiation Pneumonitis?

Background

Radiotherapy (RT) is the primary treatment modality for inoperable, locally advanced non-small-cell lung cancer (NSCLC), but even with highly conformal treatment planning, radiation pneumonitis (RP) remains the most serious, dose-limiting complication. Previous clinical reports proposed that cytokine plasma levels measured during RT allow to estimate the individual risk of patients to develop RP. The identification of such cytokine risk profiles would facilitate tailoring radiotherapy to maximize treatment efficacy and to minimize radiation toxicity. However, cytokines are produced not only in normal lung tissue after irradiation, but are also over-expressed in tumour cells of NSCLC specimens. This tumour-derived cytokine production may influence circulating plasma levels in NSCLC patients. The aim of the present study was to investigate the prognostic value of TNF-α, IL-1β, IL-6 and TGF-β1 plasma levels to predict radiation pneumonitis and to evaluate the impact of tumour-derived cytokine production on circulating plasma levels in patients irradiated for NSCLC.

Methodology/Principal Findings

In 52 NSCLC patients (stage I–III) cytokine plasma levels were investigated by ELISA before and weekly during RT, during follow-up (1/3/6/9 months after RT), and at the onset of RP. Tumour biopsies were immunohistochemically stained for IL-6 and TGF-β1, and immunoreactivity was quantified (grade 1–4). RP was evaluated according to LENT-SOMA scale. Tumour response was assessed according to RECIST criteria by chest-CT during follow-up. In our clinical study 21 out of 52 patients developed RP (grade I/II/III/IV: 11/3/6/1 patients). Unexpectedly, cytokine plasma levels measured before and during RT did not correlate with RP incidence. In most patients IL-6 and TGF-β1 plasma levels were already elevated before RT and correlated significantly with the IL-6 and TGF-β1 production in corresponding tumour biopsies. Moreover, IL-6 and TGF-β1 plasma levels measured during follow-up were significantly associated with the individual tumour responses of these patients.

Conclusions/Significance

The results of this study did not confirm that cytokine plasma levels, neither their absolute nor any relative values, may identify patients at risk for RP. In contrast, the clear correlations of IL-6 and TGF-β1 plasma levels with the cytokine production in corresponding tumour biopsies and with the individual tumour responses suggest that the tumour is the major source of circulating cytokines in patients receiving RT for advanced NSCLC.     

The Ordovician Radiation: A Follow-up to the Cambrian Explosion?

There was a major diversification known as the Ordovician Radiation,in the period immediately following the Cambrian. This eventis unique in taxonomic, ecologic and biogeographic aspects. While all of the phyla but one were established during the Cambrianexplosion, taxonomic increases during the Ordovician were manifestat lower taxonomic levels although ordinal level diversity doubled.Marine family diversity tripled and within clade diversity increasesoccurred at the genus and species levels. The Ordovician radiationestablished the Paleozoic Evolutionary Fauna; those taxa whichdominated the marine realm for the next 250 million years. Communitystructure dramatically increased in complexity. New communitieswere established and there were fundamental shifts in dominanceand abundance. Over the past ten years, there has been an effort to examinethis radiation at different scales. In comparison with the Cambrianexplosion which appears to be more globally mediated, localand regional studies of Ordovician faunas reveal sharp transitionswith timing and magnitudes that vary geographically. These transitionssuggest a more episodic and complex history than that revealedthrough synoptic global studies alone. Despite its apparent uniqueness, we cannot exclude the possibilitythat the Ordovician radiation was an extension of Cambrian diversitydynamics. That is, the Ordovician radiation may have been anevent independent of the Cambrian radiation and thus requiringa different set of explanations, or it may have been the inevitablefollow-up to the Cambrian radiation. Future studies should focuson resolving this issue.     

Radiation induced DNA DSBs: Contribution from stalled replication forks?

When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using γH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle. V79-4 cells and human HF19 fibroblast cells have been either irradiated with 0–20 Gy of γ radiation or, for comparison, treated with a low concentration of hydrogen peroxide, which is known to induce SSBs but not DSBs. Inhibition of the repair of oxidative DNA lesions by poly(ADP ribose) polymerase (PARP) inhibitor leads to an increase in radiation induced γH2AX and RAD51 foci which we propose is due to these lesions colliding with replication forks forming replication induced DSBs. It was confirmed that DSBs are not induced in G₁ phase cells by treatment with hydrogen peroxide but treatment does lead to DSB induction, specifically in S phase cells. We therefore suggest that radiation induced SSBs and non-DSB DNA damage contribute to the formation of replication induced DSBs, detected as RAD51 foci.     

Molecular Mechanisms of Radiation Induced DNA Damage: H-Abstraction and β-Cleavage

Quantum mechanical simulations of hydrogen abstraction by hydroxyl radical from methanol and ethanol yield barriers that agree very well with those measured experimentally. Analysis of the multi configurationally wave function indicates that the strength of the C-H bond is the electronic parameter that has a major contribution to the barrier for H-abstraction. Similar analysis applied to 2-deoxy-D-ribose shows that the strength of a C-H bond together with the steric accessibility of the hydrogen determine that H₄ is the most susceptible hydrogen for abstraction by a hydroxyl radical. Quantum mechanical simulations of β-cleavage show that a concerted mechanism in which a water molecule assists in the bond breaking process is more likely than a Sⁱ_n mechanism. However, the polar transition state suggests that the environment of the DNA and the surrounding water will have an important effect on the reaction.     

Quantification of γH2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX¹. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)². Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB^2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete γH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy². The loss of γH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary^4-8. The disappearence of γH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C^5,6. Further, removal of γH2AX by redistribution involving histone exchange with H2A.Z has been implicated^7,8. Importantly, the quantitative analysis of γH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of γH2AX foci in γ-irradiated adherent human keratinocytes⁹.Download video file.^{(225M, mp4)}     

Radiation damping compensation of selective pulses in water–protein exchange spectroscopy

The observation of nuclear Overhauser effects (NOEs) between bound water and biological macromolecules such as proteins and nucleic acids can be improved by inverting the water resonance selectively while compensating for radiation damping effects. The efficiency of inversion, the offset profiles, and the appearance of 2D NOE-NOESY spectra can be improved in comparison with earlier methods.     

Response of Tumor Spheroids to Radiation: Modeling and Parameter Estimation

We propose a spatially distributed continuous model for the spheroid response to radiation, in which the oxygen distribution is represented by means of a diffusion-consumption equation and the radiosensitivity parameters depend on the oxygen concentration. The induction of lethally damaged cells by a pulse of radiation, their death, and the degradation of dead cells are included. The compartments of lethally damaged cells and of dead cells are subdivided into different subcompartments to simulate the delays that occur in cell death and cell degradation, with a gain in model flexibility. It is shown that, for a single irradiation and under the hypothesis of a sufficiently small spheroid radius, the model can be reformulated as a linear stationary ordinary differential equation system. For this system, the parameter identifiability has been investigated, showing that the set of unknown parameters can be univocally identified by exploiting the response of the model to at least two different radiation doses. Experimental data from spheroids originated from different cell lines are used to identify the unknown parameters and to test the predictive capability of the model with satisfactory results.     

How Safe Is Safe Enough? Radiation Risk for a Human Mission to Mars

Astronauts on a mission to Mars would be exposed for up to 3 years to galactic cosmic rays (GCR) — made up of high-energy protons and high charge (Z) and energy (E) (HZE) nuclei. GCR exposure rate increases about three times as spacecraft venture out of Earth orbit into deep space where protection of the Earth''s magnetosphere and solid body are lost. NASA''s radiation standard limits astronaut exposures to a 3% risk of exposure induced death (REID) at the upper 95% confidence interval (CI) of the risk estimate. Fatal cancer risk has been considered the dominant risk for GCR, however recent epidemiological analysis of radiation risks for circulatory diseases allow for predictions of REID for circulatory diseases to be included with cancer risk predictions for space missions. Using NASA''s models of risks and uncertainties, we predicted that central estimates for radiation induced mortality and morbidity could exceed 5% and 10% with upper 95% CI near 10% and 20%, respectively for a Mars mission. Additional risks to the central nervous system (CNS) and qualitative differences in the biological effects of GCR compared to terrestrial radiation may significantly increase these estimates, and will require new knowledge to evaluate.     

Biochemical Changes in Rat Brain Exposed to Low Intensity 9.9 GHz Microwave Radiation

Present study concerns with various biochemical changes in the developing rat brain exposed to 9.9 GHz (square wave modulated, 1 kHz) at power density 0.125 mW/cm² (specific absorption rate 1.0 W/kg) for 2 h/day for 35 days. Thirty days old male wistar rats were used for this present study. Each group consists of eight animals. After the exposure, biochemical assays such as calcium ion efflux, calcium-dependent protein kinase (PKC), and ornithine decarboxylase (ODC) were performed on the brain tissue. Results of this study reveal that chronic exposure of rat to microwave radiation alter the activity of certain enzymes. There was a significant increase in calcium ion efflux and the activity of ODC. On the other hand, there is a significant decrease in PKC activity. Since these enzymes are related to growth, any alteration may lead to affect functioning of the brain and its development.     

Cardiovascular Changes in Atherosclerotic ApoE-Deficient Mice Exposed to Co60 (γ) Radiation

Background

There is evidence for a role of ionizing radiation in cardiovascular diseases. The goal of this work was to identify changes in oxidative and nitrative stress pathways and the status of the endothelinergic system during progression of atherosclerosis in ApoE-deficient mice after single and repeated exposure to ionizing radiation.

Methods and Results

B6.129P2-ApoE tmlUnc mice on a low-fat diet were acutely exposed (whole body) to Co60 (γ) (single dose 0, 0.5, and 2 Gy) at a dose rate of 36.32 cGy/min, or repeatedly (cumulative dose 0 and 2 Gy) at a dose-rate of 0.1 cGy/min for 5 d/wk, over a period of 4 weeks. Biological endpoints were investigated after 3–6 months of recovery post-radiation. The nitrative stress marker 3-nitrotyrosine and the vasoregulator peptides endothelin-1 and endothelin-3 in plasma were increased (p<0.05) in a dose-dependent manner 3–6 months after acute or chronic exposure to radiation. The oxidative stress marker 8-isoprostane was not affected by radiation, while plasma 8-hydroxydeoxyguanosine and L-3,4-dihydroxyphenylalanine decreased (p<0.05) after treatment. At 2Gy radiation dose, serum cholesterol was increased (p = 0.008) relative to controls. Percent lesion area increased (p = 0.005) with age of animal, but not with radiation treatment.

Conclusions

Our observations are consistent with persistent nitrative stress and activation of the endothelinergic system in ApoE−/− mice after low-level ionizing radiation exposures. These mechanisms are known factors in the progression of atherosclerosis and other cardiovascular diseases.     

Gastrointestinal Acute Radiation Syndrome in G?ttingen Minipigs (Sus Scrofa Domestica)

In the absence of supportive care, exposing Göttingen minipigs to γ-radiation doses of less than 2 Gy achieves lethality due to hematopoietic acute radiation syndrome. Doses of 2 to 5 Gy are associated with an accelerated hematopoietic syndrome, characterized by villus blunting and fusion, the beginning of sepsis, and a mild transient reduction in plasma citrulline concentration. We exposed male Göttingen minipigs (age, 5 mo; weight, 9 to 11 kg) to γ-radiation doses of 5 to 12 Gy (total body; ⁶⁰Co, 0.6 Gy/min) to test whether these animals exhibit classic gastrointestinal acute radiation syndrome (GI-ARS). After exposure, the minipigs were monitored for 10 d by using clinical signs, CBC counts, and parameters associated with the development of the gastrointestinal syndrome. Göttingen minipigs exposed to γ radiation of 5 to 12 Gy demonstrate a dose-dependent occurrence of all parameters classically associated with acute GI-ARS. These results suggest that Göttingen minipigs may be a suitable model for studying GI-ARS after total body irradiation, but the use of supportive care to extend survival beyond 10 d is recommended. This study is the first step toward determining the feasibility of using Göttingen minipigs in testing the efficacy of candidate drugs for the treatment of GI-ARS after total body irradiation.Abbreviations: GI-ARS, gastrointestinal acute radiation syndromeNo drugs have been approved for the use of preventing gastrointestinal acute radiation syndrome (GI-ARS) in irradiated patients. The effect of the acute exposure of the GI tract to radiation traditionally has been associated with the inhibition of mitotic activity in intestinal crypts and the interrupted migration of GI epithelial cells from the crypts to the tips of the villi, leading to denudation of the intestinal mucosal barrier. The monolayer of epithelial cells lining the GI mucosa performs many functions of vital importance, including electrolyte transport, secretion of digestive fluids, absorption of nutrients, excretion of toxins, and providing a barrier to the luminal environment and commensal bacteria. The loss of intestinal barrier integrity results in the loss of nutrients, water, and electrolytes; increased permeability to bacteria and antigens; sepsis; inflammation; and organ dysfunction.In humans, GI-ARS usually manifests at doses exceeding 5 to 6 Gy;¹⁰ death occurs within 2 wk in the absence of treatment. Radiation-induced GI damage is accompanied by bone marrow suppression; the sequelae of GI-ARS and hematopoietic ARS partially overlap but do not necessarily develop concomitantly.²³ Characteristic of GI-ARS is the early onset of symptoms (nausea, vomiting, watery diarrhea, cramps) within a few hours, and the overt illness is marked by vomiting and diarrhea. Bacterial translocation from the intestinal tract, loss of crypts, shortening of villi, decreased citrulline levels, onset of abdominal pain, vomiting, and diarrhea are accepted markers of the GI syndrome.²² Cardiovascular collapse, fluctuations in electrolyte concentrations, severe hemorrhage, and sepsis likely contribute to acute renal and multiorgan failure.²³Well-characterized animal models are required for efficacy testing of radiation countermeasures. So far, NHP are the only large animal model well-characterized in regard to the dose–survival relationship, symptoms, vital signs, and GI histology of GI-ARS.¹¹ In NHP, the LD_50/15 for GI-ARS in the presence of medical management including blood product transfusion is estimated to be 11.33 Gy.¹¹ Animals in the cited study¹⁰ were characterized by shorter survival time compared with hematopoietic ARS, diarrhea, dehydration, and dose-dependent loss of body weight, intestinal crypts, and villi. In addition to the total body irradiation model, a partial-body irradiation model was established in NHP, by using 5% sparing of the bone marrow, to evaluate the long-term effects of radiation on the GI system and to study concomitant subsyndromes and organ injuries, including bone marrow and lungs.¹⁰ Advantages of the partial body irradiation model are the ability to assess the development of various subsyndromes over time and the development of a polypharmacy approach targeting multiple organs. In dogs, acute GI-ARS was produced consistently by a single dose of 9.38 Gy to the abdomen.²⁰ In rodents, GI damage was induced by 8 to 15 Gy total body irradiation, depending upon the species and strain,^5,6,17,18 and was characterized by accelerated death, poor nutrient absorption, crypt depletion, intestinal epithelial cells denudation, infections, diarrhea, dehydration, and weight loss. In mice, both acute and delayed effects of the GI syndrome have been identified; study endpoints evaluating medical countermeasures against GI-ARS have been proposed.^2,3 Acute effects were assessed at several levels of bone-marrow shielding and supportive care and were demonstrated by the presence of diarrhea, apoptosis, villus blunting, crypt depletion, loss of mucosal integrity, and ulcerations in the GI tract. Delayed effects included higher-than-normal levels of apoptotic and mitotic crypt cells, premature reductions in gut function, and the presence of adenomas.^2,3Additional large animal models are urgently needed to expedite drug testing and marketing under FDA regulations. Minipigs may be a potential alternative model for studying hematopoietic ARS,¹⁵ but their high sensitivity to radiation prompts doubt regarding the feasibility of GI-ARS studies in this model. In the current study, we filled an important gap in development of minipigs as a model for ARS and addressed potential concerns regarding the feasibility of inducing classic GI-ARS in minipigs before they succumb to cardiovascular or respiratory complications. In previous publications, we documented hematopoietic ARS and accelerated hematopoietic ARS at higher radiation doses.^14,15 However, despite sporadic reports of specific types of radiation-induced injury to the GI tract of swine after total body irradiation, GI-ARS in that species has not been addressed comprehensively. To this end, we used a population of minipigs that was completely different from that in the previous studies and exposed the animals to much higher radiation doses than had been used previously. The subsequent results were markedly different from those of the previous reports, including loss of GI crypts and large decreases in circulating citrulline, both of which are important signs of GI-ARS. Our results demonstrate convincingly that GI-ARS occurs in swine and place this subsyndrome in the context of others studied comprehensively in the same strain. This achievement is an important milestone in the development of this much-needed large animal model of ARS, which promises to provide an alternative to NHP and dogs.We show here that exposure of minipigs to γ irradiation in the range 5 to 12 Gy resulted in the development of signs typical of GI-ARS, and we discuss the importance of choosing the appropriate endpoint criteria for the different subsyndromes of ARS. Most minipigs died within 10 d, shifting the question from the feasibility of inducing GI-ARS in minipigs to the levels of supportive care appropriate for drug efficacy testing in this model.     

Epigenetic DNA Methylation in Radiation Biology: On the Field or on the Sidelines?

DNA methylation has been studied with regard to chemotherapeutics for a number of years. The radiation field has just begun to look at this in the context of radiotherapy or radiation exposure. So far, the data suggest that radiation induces epigenetic reprogramming which indicates a purposeful response that influences the cell fate or alters the response to future exposure. Further studies may result in discovery of biomarkers for radiotherapy outcome or prediction of the degree of radiation resistance. Past and ongoing development of DNMT1 inhibitors that lead to DNA hypomethylation appear to sensitize many tumor types to radiation and may be an area with long term clinical implications. J. Cell. Biochem. 116: 212–217, 2015. © 2014 Wiley Periodicals, Inc.     

Effects of Long-Term Exposure to Low-Power 915 MHz Unmodulated Radiation on Phaseolus vulgaris L.

The morphophysiological response of Phaseolus vulgaris L. to low-power electromagnetic radiation was investigated in order to assess the potential harmful effects of long-term continuous exposure. The plants were grown in two separate electromagnetic field (EMF) shielded rooms, in a controlled, greenhouse-like environment. One batch was continuously irradiated during the growth period (from sowing to maturity) and the other one was used as a reference. An unmodulated signal at 915 MHz (the central frequency between the uplink and downlink of the GSM900 mobile communications band) was used, with a maximum power density of 10 mW/m² measured near the plants. The plants were analyzed using ultraviolet–visible, statistical, morphometric, and electron microscopy methods. Significant differences were observed regarding the height of the plants, number of inflorescences, and chlorophyll and carotenoid content, all closely connected with the ultrastructural changes observed in the leaves. The irradiated batch grew higher (19% increase in plant height, 20% increase in stem and leaves' dry mass), with 18% fewer inflorescences, and extremely long roots (34% increase in dry mass). The ultrastructure of the irradiated leaves showed irregular cells and a higher content of plastoglobules in the chloroplasts. All results indicate that the irradiated plants suffered significant morphological modifications during their long-term exposure to the specific EM radiation. Bioelectromagnetics. © 2020 Bioelectromagnetics Society     

Reduction of Solar UV Radiation Due to Urban High-Rise Buildings – A Coupled Modelling Study

Solar UV radiation has both adverse and beneficial effects to human health. Using models (a radiative transfer model coupled to a building shading model), together with satellite and surface measurements, we studied the un-obstructed and obstructed UV environments in a sub-tropical urban environment featured with relatively high pollution (aerosol) loadings and high-rise buildings. Seasonal patterns of the erythemal UV exposure rates were governed by solar zenith angles, seasonal variations of aerosol loadings and cloud effects. The radiative transfer modelling results agreed with measurements of erythemal UV exposure rates and spectral irradiances in UVA and UVB ranges. High-rise buildings and narrow road width (height to width, H/W, ratios up to 15) reduced the modelled total UV (UVA+UVB) radiation and leave 10% of the un-obstructed exposure rate at ground-level at noon. No more than 80% of the un-obstructed exposure rate was received in the open area surrounded by 20-storey buildings. Our modelled reduction of UVB radiation in the urban environment was consistent with similar measurements obtained for Australia. However, our results in more extreme environments (higher H/W ratios) were for the first time reported, with 18% of the un-obstructed exposure rate remained at the ground-level center of the street canyon.