Contribution of Genetic Background to the Radiation Risk for Cancer and Non-Cancer Diseases in Ptch1+/- Mice.

Experimental mouse studies are important to gain a comprehensive, quantitative and mechanistic understanding of the biological factors that modify individual risk of radiation-induced health effects, including age at exposure, dose, dose rate, organ/tissue specificity and genetic factors. In this study, neonatal Ptch1+/- mice bred on CD1 and C57Bl/6 background received whole-body irradiation at postnatal day 2. This time point represents a critical phase in the development of the eye lens, cerebellum and dentate gyrus (DG), when they are also particularly susceptible to radiation effects. Irradiation was performed with γ rays (60Co) at doses of 0.5, 1 and 2 Gy, delivered at 0.3 Gy/min or 0.063 Gy/min. Wild-type and mutant mice were monitored for survival, lens opacity, medulloblastoma (MB) and neurogenesis defects. We identified an inverse genetic background-driven relationship between the radiosensitivity to induction of lens opacity and MB and that to neurogenesis deficit in Ptch1+/- mutants. In fact, high incidence of radiation-induced cataract and MB were observed in Ptch1+/-/CD1 mutants that instead showed no consequence of radiation exposure on neurogenesis. On the contrary, no induction of radiogenic cataract and MB was reported in Ptch1+/-/C57Bl/6 mice that were instead susceptible to induction of neurogenesis defects. Compared to Ptch1+/-/CD1, the cerebellum of Ptch1+/-/C57Bl/6 mice showed increased radiosensitivity to apoptosis, suggesting that differences in processing radiation-induced DNA damage may underlie the opposite strain-related radiosensitivity to cancer and non-cancer pathologies. Altogether, our results showed lack of dose-rate-related effects and marked influence of genetic background on the radiosensitivity of Ptch1+/-mice, supporting a major contribution of individual sensitivity to radiation risk in the population.

miRNA-Signature of Irradiated Ptch1+/- Mouse Lens is Dependent on Genetic Background.

One harmful long-term effect of ionizing radiation is cataract development. Recent studies have been focused on elucidating the mechanistic pathways involved in this pathogenesis. Since accumulating evidence has established a role of microRNAs in ocular diseases, including cataract, the goal of this work was to determine the microRNA signature of the mouse lens, at short time periods postirradiation, to understand the mechanisms related to radio-induced cataractogenesis. To evaluate the differences in the microRNA profiles, 10-week-old Patched1 heterozygous (Ptch1+/-) mice, bred onto two different genetic backgrounds (CD1 and C57Bl/6J), received whole-body 2 Gy γ-ray irradiation, and 24 h later lenses were collected. Next-generation sequencing and bioinformatics analysis revealed that genetic background markedly influenced the list of the deregulated microRNAs and the mainly predicted perturbed biological functions of 2 Gy irradiated Ptch1+/- mouse lenses. We identified a subset of microRNAs with a contra-regulated expression between strains, with a key role in regulating Toll-like receptor (TLR)-signaling pathways. Furthermore, a detailed analysis of miRNome data showed a completely different DNA damage response in mouse lenses 24 h postirradiation, mainly mediated by a marked upregulation of p53 signaling in Ptch1+/-/C57Bl/6J lenses that was not detected on a CD1 background. We propose a strict interplay between p53 and TLR signaling in Ptch1+/-/C57Bl/6J lenses shortly after irradiation that could explain both the resistance of this strain to developing lens opacities and the susceptibility of CD1 background to radiation-induced cataractogenesis through activation of epithelial-mesenchymal transition.

Radiation-Induced Lens Opacity and Cataractogenesis: A Lifetime Study Using Mice of Varying Genetic Backgrounds.

Recent epidemiological findings and reanalysis of historical data suggest lens opacities resulting from ionizing radiation exposures are likely induced at lower doses than previously thought. These observations have led to ICRP recommendations for a reduction in the occupational dose limits for the eye lens, as well as subsequent implementation in EU member states. The EU CONCERT LDLensRad project was initiated to further understand the effects of ionizing radiation on the lens and identify the mechanism(s) involved in radiation-induced cataract, as well as the impact of dose and dose-rate. Here, we present the results of a long-term study of changes to lens opacity in male and female adult mice from a variety of different genetic (radiosensitive or radioresistant) backgrounds, including mutant strains Ercc2 and Ptch1, which were assumed to be susceptible to radiation-induced lens opacities. Mice received 0.5, 1 and 2 Gy 60Co gamma-ray irradiation at dose rates of 0.063 and 0.3 Gy min-1. Scheimpflug imaging was used to quantify lens opacification as an early indicator of cataract, with monthly observations taken postirradiation for an 18-month period in all strains apart from 129S2, which were observed for 12 months. Opacification of the lens was found to increase with time postirradiation (with age) for most mouse models, with ionizing radiation exposure increasing opacities further. Sex, dose, dose rate and genetic background were all found to be significant contributors to opacification; however, significant interactions were identified, which meant that the impact of these factors was strain dependent. Mean lens density increased with higher dose and dose rate in the presence of Ercc2 and Ptch1 mutations. This project was the first to focus on low (<1 Gy) dose, multiple dose rate, sex and strain effects in lens opacification, and clearly demonstrates the importance of these experimental factors in radiobiological investigations on the lens. The results provide insight into the effects of ionizing radiation on the lens as well as the need for further work in this area to underpin appropriate radiation protection legislation and guidance.

A COMPARISON BETWEEN X-RAY AND CARBON ION IRRADIATION IN HUMAN NEURAL STEM CELLS.

Glioblastoma multiforme (GBM) is characterized by a poor prognosis and a median survival of ~12-18 months. GBM is usually managed by neurosurgery followed by both chemotherapy and radiotherapy. Since GBM develops resistance to conventional therapies, treatment with C-ions is promising to completely eradicate the tumoural mass. During cranial irradiation, exposure of healthy tissues is inevitable. Because of the presence of neural stem cells, a deep investigation on the effects of C-ion irradiation with respect to X-ray induced damage is mandatory to allow a better definition of treatments. In this work, the comparison of X-rays and C-ion irradiation-induced effects on human neural stem cell, focusing on multiple endpoints, such as cell viability, cytokine secretion and spheroid formation is presented. Results show different temporal and dose responses of human neural stem cells to the different radiation qualities, suggesting different underpinning mechanisms of radiation-induced damages.

WHAT ROLES FOR TRACK-STRUCTURE AND MICRODOSIMETRY IN THE ERA OF -omics AND SYSTEMS BIOLOGY?

Ionizing radiation is a peculiar perturbation when it comes to damage to biological systems: it proceeds through discrete energy depositions, over a short temporal scale and a spatial scale critical for subcellular targets as DNA, whose damage complexity determines the outcome of the exposure. This lies at the basis of the success of track structure (and nanodosimetry) and microdosimetry in radiation biology. However, such reductionist approaches cannot account for the complex network of interactions regulating the overall response of the system to radiation, particularly when effects are manifest at the supracellular level and involve long times. Systems radiation biology is increasingly gaining ground, but the gap between reductionist and holistic approaches is becoming larger. This paper presents considerations on what roles track structure and microdosimetry can have in the attempt to fill this gap, and on how they can be further exploited to interpret radiobiological data and inform systemic approaches.

BIOINFORMATIC ANALYSIS OF DOSE- AND TIME-DEPENDENT miRNome RESPONSES.

The advent of new 'omics' techniques determined a massive boost in the measurement of the whole spectra of molecules within cells, favoring promising new radiobiological studies at low doses. The main aim of this work was to assess the radiation-induced perturbations of miRNA profiles and their temporal dynamics. Human Umbilical Vein Endothelial Cells were irradiated with low doses of γ-rays. At different time points post-irradiation, cells were harvested and miRNAs isolated. A full mapping of the miRNA sequences via Next-Generation-Sequencing analysis was performed followed by bioinformatic analyses. Pathway enrichment analyses on the differentially expressed miRNAs focused both on the averaged effects of different doses over the 24-h experiment and on the altered temporal dynamics of the miRNA profiles. These complementary analyses provided a picture of the dose- and time-dependent miRNAs responses, allowing to better explore the candidate biomarkers linked to radiation exposures and their corresponding pathways and functions.

AT THE PHYSICS-BIOLOGY INTERFACE: THE NEUTRON AFFAIR.

We present predictions of neutron relative biological effectiveness (RBE) for cell irradiations with neutron beams at PTB-Braunschweig. A neutron RBE model is adopted to evaluate initial DNA damage induction given the neutron-induced charged particle field. RBE values are predicted for cell exposures to quasi-monoenergetic beams (0.56 MeV, 1.2 MeV) and to a broad energy distribution neutron field with dose-averaged energy of 5.75 MeV. Results are compared to what obtained with our RBE predictions for neutrons at similar energies, when a 30-cm sphere is irradiated in an isotropic neutron field. RBE values for experimental conditions are higher for the lowest neutron energies, because, as expected, target geometry determines the weight of the low-effectiveness photon component of the neutron dose. These results highlight the importance of characterizing neutron fields in terms of physical interactions, to fully understand neutron-induced biological effects, contributing to risk estimation and to the improvement of radiation protection standards.

The origin of neutron biological effectiveness as a function of energy.

The understanding of the impact of radiation quality in early and late responses of biological targets to ionizing radiation exposure necessarily grounds on the results of mechanistic studies starting from physical interactions. This is particularly true when, already at the physical stage, the radiation field is mixed, as it is the case for neutron exposure. Neutron Relative Biological Effectiveness (RBE) is energy dependent, maximal for energies ~1 MeV, varying significantly among different experiments. The aim of this work is to shed light on neutron biological effectiveness as a function of field characteristics, with a comprehensive modeling approach: this brings together transport calculations of neutrons through matter (with the code PHITS) and the predictive power of the biophysical track structure code PARTRAC in terms of DNA damage evaluation. Two different energy dependent neutron RBE models are proposed: the first is phenomenological and based only on the characterization of linear energy transfer on a microscopic scale; the second is purely ab-initio and based on the induction of complex DNA damage. Results for the two models are compared and found in good qualitative agreement with current standards for radiation protection factors, which are agreed upon on the basis of RBE data.

Energy dependence of the complexity of DNA damage induced by carbon ions.

To assess the complexity of DNA damage induced by carbon ions as a function of their energy and LET, 2-Gy irradiations by 100 keV u(-1)-400 MeV u(-1) carbon ions were investigated using the PARTRAC code. The total number of fragments and the yield of fragments of <30 bp were calculated. The authors found a particularly important contribution of DNA fragmentation in the range of <1 kbp for specific energies of <6 MeV u(-1). They also considered the effect of different specific energies with the same LET, i.e. before and after the Bragg peak. As a first step towards a full characterisation of secondary particle production from carbon ions interacting with tissue, a comparison between DNA-damage induction by primary carbon ions and alpha particles resulting from carbon break-up is presented, for specific energies of >1 MeV u(-1).

Reaction mechanism interplay in determining the biological effectiveness of neutrons as a function of energy.

Neutron relative biological effectiveness (RBE) is found to be energy dependent, being maximal for energies ∼1 MeV. This is reflected in the choice of radiation weighting factors wR for radiation protection purposes. In order to trace back the physical origin of this behaviour, a detailed study of energy deposition processes with their full dependences is necessary. In this work, the Monte Carlo transport code PHITS was used to characterise main secondary products responsible for energy deposition in a 'human-sized' soft tissue spherical phantom, irradiated by monoenergetic neutrons with energies around the maximal RBE/wR. Thereafter, results on the microdosimetric characterisation of secondary protons were used as an input to track structure calculations performed with PARTRAC, thus evaluating the corresponding DNA damage induction. Within the proposed simplified approach, evidence is suggested for a relevant role of secondary protons in inducing the maximal biological effectiveness for 1 MeV neutrons.

Mechanisms of the induction of apoptosis mediated by radiation-induced cytokine release.

The aim of the present work was to investigate the mechanisms of radiation-induced bystander signalling leading to apoptosis in non-irradiated co-cultured cells. Cultured non-transformed cells were irradiated, and the effect on the apoptosis rate on co-cultured non-irradiated malignant cells was determined. For this, two different levels of the investigation are presented, i.e. release of signalling proteins and transcriptomic profiling of the irradiated and non-irradiated co-cultured cells. Concerning the signalling proteins, in this study, the attention was focussed on the release of the active and latent forms of the transforming growth factor-ß1 protein. Moreover, global gene expression profiles of non-transformed and transformed cells in untreated co-cultures were compared with those of 0.5-Gy-irradiated non-transformed cells co-cultured with the transformed cells. The results show an effect of radiation on the release of signalling proteins in the medium, although no significant differences in release rates were detectable when varying the doses in the range from 0.25 to 1 Gy. Moreover, gene expression results suggest an effect of radiation on both cell populations, pointing out specific signalling pathways that might be involved in the enhanced induction of apoptosis.

Investigation of radiation-induced multilayered signalling response of the inflammatory pathway.

Ionising radiation exposure of cells might induce the perturbation of cell functions and, in particular, the activation or inhibition of several important pathways. This perturbation can cause the deregulation of both intra- and extra-cellular signalling cascades (such as the inflammatory pathway) and alter not only the behaviour of directly exposed cells but also the neighbouring non-irradiated ones, through the so-called bystander effect. The aim of the present work was to investigate the complex nonlinear interactions between the inflammatory pathway and other strictly interlaced signalling pathways, such as Erk1/2 and Akt/PKB, focusing on the radiation-induced perturbation of such pathways in the dose range of 0-2 Gy. The results show how radiation affects these interconnected pathways and how confounding factors, such as the change of culture medium, can hide radiation-induced perturbations.

Radiosensitivity in lymphoblastoid cell lines derived from Shwachman-Diamond syndrome patients.

Shwachman-Diamond syndrome is an autosomal-recessive disorder characterised by bone marrow failure and a cumulative risk of progression to acute myeloid leukaemia. The Shwachman-Bodian-Diamond syndrome (SBDS) gene, the only gene known to be causative of the pathology, is involved in ribosomal biogenesis, stress responses and DNA repair, and the lack of SBDS sensitises cells to many stressors and leads to mitotic spindle destabilisation. The effect of ionising radiation on SBDS-deficient cells was investigated using immortalised lymphocytes from SDS patients in comparison with positive and negative controls in order to test whether, in response to ionising radiation exposure, any impairment in the DNA repair machinery could be observed. After irradiating cells with different doses of X-rays or gamma-rays, DNA repair kinetics and the residual damages using the alkaline COMET assay and the γ-H2AX assay were assessed, respectively. In this work, preliminary data about the comparison between ionising radiation effects in different patients-derived cells and healthy control cells are presented.

In vitro γ-ray-induced inflammatory response is dominated by culturing conditions rather than radiation exposures.

The inflammatory pathway has a pivotal role in regulating the fate and functions of cells after a wide range of stimuli, including ionizing radiation. However, the molecular mechanisms governing such responses have not been completely elucidated yet. In particular, the complex activation dynamics of the Nuclear transcription Factor kB (NF-kB), the key molecule governing the inflammatory pathway, still lacks a complete characterization. In this work we focused on the activation dynamics of the NF-kB (subunit p65) pathway following different stimuli. Quantitative measurements of NF-kB were performed and results interpreted within a systems theory approach, based on the negative feedback loop feature of this pathway. Time-series data of nuclear NF-kB concentration showed no evidence of γ-ray induced activation of the pathway for doses up to 5 Gy but highlighted important transient effects of common environmental stress (e.g. CO2, temperature) and laboratory procedures, e.g. replacing the culture medium, which dominate the in vitro inflammatory response.

Carbapenemases of Chryseobacterium (Flavobacterium) meningosepticum: distribution of blaB and characterization of a novel metallo-beta-lactamase gene, blaB3, in the type strain, NCTC 10016.

Genes encoding carbapenemases in 15 reference strains of Chryseobacterium (Flavobacterium) meningosepticum from the United Kingdom National Collection of Type Cultures and in one recent clinical isolate were investigated. All the strains hydrolyzed imipenem, but their levels of resistance to carbapenems varied, with imipenem and meropenem MICs ranging from 2 to >32 microg/ml. The blaB gene, which encodes a molecular-class B carbapenemase, was detected in only six reference strains and in clinical isolate 97/P/5448. The gene from 97/P/5448 had 98% nucleotide identity with the published sequence of blaB (from strain NCTC 10585) and was designated blaB2. A distinct carbapenemase gene, designated blaB3, was cloned from the type strain of C. meningosepticum, NCTC 10016. blaB3 had an open reading frame of 750 bp with 82% nucleotide identity to blaB and blaB2 and encoded a beta-lactamase of 249 amino acids, including the putative signal peptide. This beta-lactamase showed 87.6 and 86.7% amino acid homology with BlaB and BlaB2, respectively. blaB3 was detected in one other reference strain besides NCTC 10016, but the genetic basis of the carbapenemase activity detected in the other seven reference strains was not defined. Thus, neither blaB nor blaB3 was ubiquitous in the strains of C. meningosepticum studied, indicating that the reference strains may represent more than one bacterial species, each with its own intrinsic metallo-beta-lactamase. Further taxonomic studies of C. meningosepticum are necessary to resolve this topic. Chryseobacterium spp. are environmental organisms and occasional opportunist pathogens. They apparently represent a reservoir of diverse metallo-beta-lactamases, which potentially spread to gram-negative bacteria of greater clinical significance.

Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece.

Resistance to imipenem and meropenem was observed in 211 (16.5%) isolates of Pseudomonas aeruginosa recovered in a Greek university hospital during 1996 to 1998. In six isolates selected from throughout this period, high-level resistance to both carbapenems (MICs >/= 128 microg/ml) was associated with production of the class B beta-lactamase VIM-1. bla(VIM)-bearing isolates belonged to serotype O:12 and were indistinguishable by pulsed-field gel electrophoresis.

Antimicrobial resistance amongst Klebsiella spp. collected from intensive care units in Southern and Western Europe in 1997-1998.

A 1994 survey of 35 intensive care units (ICUs) in Western and Southern Europe found extended-spectrum beta-lactamases (ESBLs) in 220/966 (23%) klebsiellae. A follow-up survey from May 1997 to October 1998 collected klebsiellae from 24 ICUs, including 23 that participated in 1994. Twenty-one ICUs sent 433 eligible isolates, of which 110 (25%) had ESBLs. The prevalence of ESBLs had not changed significantly from 1994 but the proportion of ESBL-producers resistant to piperacillin/tazobactam had risen from 31% to 63% (P < 0.001), and most of this resistance was high level (MICs >/= 128 + 4 mg/L). The proportion of Klebsiella oxytoca isolates hyperproducing K1 beta-lactamase rose from 8% in 1994 to 21% in 1997-1998 (P < 0. 001). Most klebsiellae (99%) were very susceptible to meropenem (mode MIC 0.03 mg/L) but three had decreased susceptibility (MICs 2-4 mg/L). These could not hydrolyse carbapenems. Aminoglycoside resistance was not significantly changed in prevalence from 1994; ciprofloxacin resistance occurred in 31% of ESBL-producers in both years, but had increased among non-producers (2% in 1994 versus 7% in 1997-1998, P < 0.001).

Effect of conalbumin on the activity of Syn 2190, a 1,5 dihydroxy-4-pyridon monobactam inhibitor of AmpC beta-lactamases.

Syn 2190, a 1,5 dihydroxy-4-pyridon monobactam inhibitor of AmpC enzymes, was tested against beta-lactamase-producing bacteria with piperacillin, piperacillin-tazobactam and ceftazidime as partner drugs. In the presence of conalbumin as an iron chelator, Syn 2190 potentiated these drugs against most AmpC producers, although Klebsiella spp. with plasmidic AmpC enzymes were an exception. Potentiation was much weaker without conalbumin, suggesting that Syn 2190 exploits a ferric uptake pathway, as do catecholic cephalosporins. Syn 2190 had little ability to potentiate partner drugs against strains with other beta-lactamase types but, with conalbumin, increased the activity of piperacillin-tazobactam against Escherichia coli transconjugants producing various class A or D enzymes.