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Preclinical mouse models that allow the evaluation of novel techniques in radiotherapy using charged particles like protons or heavier ions are discussed here. Focused beams of protons or carbon ions offer distinct physical characteristics that might contribute to an overall improved risk-benefit profile in radiotherapy. In the last decade, novel concepts in radiotherapy aimed to optimize the delivery of high radiation doses to the tumor while sparing the surrounding healthy tissue. 2D in vitro tumor cell cultures provide experimental models to determine the radiosensitivity of isolated cells to different radiation qualities. However, these cell culture models are not capable of mimicking the complexity and heterogeneity of 3-dimensionally growing clinical tumors with their individual tumor microenvironment. Therefore, small animal tumor models are urgently needed to study radiation responses after particle radiotherapy in vivo. Here we describe the recent developments and the accurate testing of novel techniques in particle radiotherapy using up-to-date preclinical animal models., (© 2018 S. Karger AG, Basel.)

Purpose: Proton minibeam radiation therapy is a novel approach to minimize normal tissue damage in the entrance channel by spatial fractionation while keeping tumor control through a homogeneous tumor dose using beam widening with an increasing track length. In the present study, the dose distributions for homogeneous broad beam and minibeam irradiation sessions were simulated. Also, in an animal study, acute normal tissue side effects of proton minibeam irradiation were compared with homogeneous irradiation in a tumor-free mouse ear model to account for the complex effects on the immune system and vasculature in an in vivo normal tissue model., Methods and Materials: At the ion microprobe SNAKE, 20-MeV protons were administered to the central part (7.2 × 7.2 mm(2)) of the ear of BALB/c mice, using either a homogeneous field with a dose of 60 Gy or 16 minibeams with a nominal 6000 Gy (4 × 4 minibeams, size 0.18 × 0.18 mm(2), with a distance of 1.8 mm). The same average dose was used over the irradiated area., Results: No ear swelling or other skin reactions were observed at any point after minibeam irradiation. In contrast, significant ear swelling (up to fourfold), erythema, and desquamation developed in homogeneously irradiated ears 3 to 4 weeks after irradiation. Hair loss and the disappearance of sebaceous glands were only detected in the homogeneously irradiated fields., Conclusions: These results show that proton minibeam radiation therapy results in reduced adverse effects compared with conventional homogeneous broad-beam irradiation and, therefore, might have the potential to decrease the incidence of side effects resulting from clinical proton and/or heavy ion therapy., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Glioblastoma (GBM) is the most common and most aggressive malignant primary brain tumor in adults. In spite of multimodal therapy concepts, consisting of surgery, radiotherapy and chemotherapy, the median survival, merely 15-18 months, is still poor. Mechanisms for resistance of GBM to radio(chemo)therapy are not fully understood yet and due to the genetic heterogeneity within the tumor including radiation-resistant tumor stem cells, there are several factors leading to therapy failure. Recent research revealed that, hypoxia during radiation and miRNAs may adversely affect the therapeutic response to radiotherapy. Further molecular alterations and prognostic markers like the DNA-repair protein O6-methylguanine-DNA methyltransferase (MGMT), anti-apoptotic molecular chaperones, and/or the activity of aldehyde dehydrogenase 1 (ALDH1) have also been identified to play a role in the sensitivity to cytostatic agents. Latest approaches in the field of radiotherapy to use particle irradiation or dose escalation strategies including modern molecular imaging, however, need further evaluation with regard to long-term outcome. In this review we focus on current information about the mechanisms and markers that mediate resistance to radio(chemo)therapy, and discuss the opportunities of Innovative Radiotherapy (iRT) concepts to improve treatment options for GBM patients.

Background: Breast cancer is the most common cancer in women worldwide and surgery, radiotherapy (RT) and chemotherapy (ChT) are frequently used to treat this cancer. Adjuvant RT has been shown to cause long-term changes in lymphocyte counts in the peripheral blood. Herein, the time course of changes in lymphocyte subpopulations upon RT was studied in patients with and without adjuvant ChT in order to explore its potential clinical impact., Materials and Methods: Total lymphocyte counts and the composition of lymphocyte subpopulations before RT (t0), after 30 Gy (t1), at the end of RT (t2), and 6 weeks (t3), 6 months (t4), and 1 year (t5) after RT were studied by flow cytometry., Results: Absolute lymphocyte counts were significantly lower in all breast cancer patients (n=40) before and also 1 year after RT compared to healthy controls. The percentage of CD3(+)/CD4(+) helper T cells and FoxP3(+) regulatory T cells increased significantly in patients without adjuvant ChT. Different NK cell subpopulations dropped during RT in patients with and without ChT, but recovered to initial levels 6months after RT (t4). During RT (t0-t2) the percentage of CD19(+) B cells significantly dropped in patients without ChT, but gradually increased in patients with adjuvant ChT. Both patient groups reached initial levels 6 months after RT (t4)., Conclusion: Different lymphocyte subpopulations respond differently to RT with and without adjuvant ChT. CD4(+) T cells increase during RT, whereas NK cells and B cells decrease in patients without ChT, but recover within 6 months after RT. Treg cells gradually increase in patients without ChT from t0 to t5, but not in patients with adjuvant ChT., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The copper-catalyzed asymmetric conjugate addition (ACA) of nucleophiles onto polyenic Michael acceptors represents an attractive and powerful methodology for the synthesis of relevant chiral molecules, as it enables in a straightforward manner the sequential generation of two or more stereogenic centers. In the last decade, various chiral copper-based catalysts were evaluated in combination with different nucleophiles and Michael acceptors, and have unambiguously demonstrated their usefulness in the control of the regio- and enantioselectivity of the addition. The aim of this review is to report recent breakthroughs achieved in this challenging field.

Despite enormous progress in radiation technologies (high precision image-guided irradiation, proton irradiation, heavy ion irradiation) and radiotherapeutic concepts (hypofractionated irradiation schemes), the clinical outcome of radiotherapy in locally advanced and metastasized tumors and in hypoxic tumors which are radiation-resistant remains unsatisfactory. Given their key influence on a number of biological and immunological parameters, this article considers the influence of irradiation-induced stress proteins on radiation-induced immunomodulation. Depending on its location, the major stress-inducible Heat shock protein 70 (Hsp70) has been found to fulfill multiple roles. On the one hand, increased intracellular Hsp70 levels have been found to play a key role in the recovery from stress such as radio(chemo)therapy, and on the other hand extracellular Hsp70 proteins are potent stimulators of the innate immune system and mediators of anti-tumor immunity. Furthermore, if loaded with tumor-derived peptides, members of the Heat Shock Protein 70 (HSP70) and 90 (HSP90) families can stimulate the adaptive immune system via antigen cross-presentation. An irradiation-induced enhancement of the selective expression of a membrane form of Hsp70 on the surface of tumor cells which can act as a recognition structure for activated NK cells might have significant clinical relevance, in that the outcome of irradiation therapy for advanced tumors could be improved by combining it with cell-based and other immunotherapies that target this membrane form of Hsp70., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

In conventional experiments on biological effects of radiation types of diverse quality, micrometer-scale double-strand break (DSB) clustering is inherently interlinked with clustering of energy deposition events on nanometer scale relevant for DSB induction. Due to this limitation, the role of the micrometer and nanometer scales in diverse biological endpoints cannot be fully separated. To address this issue, hybrid human-hamster AL cells have been irradiated with 45MeV (60keV/μm) lithium ions or 20MeV (2.6keV/μm) protons quasi-homogeneously distributed or focused to 0.5×1μm(2) spots on regular matrix patterns (point distances up to 10.6×10.6μm), with pre-defined particle numbers per spot to provide the same mean dose of 1.7Gy. The yields of dicentrics and their distribution among cells have been scored. In parallel, track-structure based simulations of DSB induction and chromosome aberration formation with PARTRAC have been performed. The results show that the sub-micrometer beam focusing does not enhance DSB yields, but significantly affects the DSB distribution within the nucleus and increases the chance to form DSB pairs in close proximity, which may lead to increased yields of chromosome aberrations. Indeed, the experiments show that focusing 20 lithium ions or 451 protons per spot on a 10.6μm grid induces two or three times more dicentrics, respectively, than a quasi-homogenous irradiation. The simulations reproduce the data in part, but in part suggest more complex behavior such as saturation or overkill not seen in the experiments. The direct experimental demonstration that sub-micrometer clustering of DSB plays a critical role in the induction of dicentrics improves the knowledge on the mechanisms by which these lethal lesions arise, and indicates how the assumptions of the biophysical model could be improved. It also provides a better understanding of the increased biological effectiveness of high-LET radiation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A silver-free methodology was developed for the synthesis of unprecedented N-heterocyclic carbene ruthenium indenylidene complexes bearing a bidentate picolinate ligand. The highly stable (SIPr)(picolinate)RuCl(indenylidene) complex 4a (SIPr = 1,3-bis(2-6-diisopropylphenyl)imidazolidin-2-ylidene) demonstrated excellent latent behaviour in ring closing metathesis (RCM) reaction and could be activated in the presence of a Brønsted acid. The versatility of the catalyst 4a was subsequently demonstrated in RCM, cross-metathesis (CM) and enyne metathesis reactions.

The risk of developing normal tissue injuries often limits the radiation dose that can be applied to the tumour in radiation therapy. Microbeam Radiation Therapy (MRT), a spatially fractionated photon radiotherapy is currently tested at the European Synchrotron Radiation Facility (ESRF) to improve normal tissue protection. MRT utilizes an array of microscopically thin and nearly parallel X-ray beams that are generated by a synchrotron. At the ion microprobe SNAKE in Munich focused proton microbeams ("proton microchannels") are studied to improve normal tissue protection. Here, we comparatively investigate microbeam/microchannel irradiations with sub-millimetre X-ray versus proton beams to minimize the risk of normal tissue damage in a human skin model, in vitro. Skin tissues were irradiated with a mean dose of 2 Gy over the irradiated area either with parallel synchrotron-generated X-ray beams at the ESRF or with 20 MeV protons at SNAKE using four different irradiation modes: homogeneous field, parallel lines and microchannel applications using two different channel sizes. Normal tissue viability as determined in an MTT test was significantly higher after proton or X-ray microchannel irradiation compared to a homogeneous field irradiation. In line with these findings genetic damage, as determined by the measurement of micronuclei in keratinocytes, was significantly reduced after proton or X-ray microchannel compared to a homogeneous field irradiation. Our data show that skin irradiation using either X-ray or proton microchannels maintain a higher cell viability and DNA integrity compared to a homogeneous irradiation, and thus might improve normal tissue protection after radiation therapy., (Copyright © 2015 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.)

The potential of proton microchannel radiotherapy to reduce radiation effects in the healthy tissue but to keep tumor control the same as in conventional proton therapy is further elucidated. The microchannels spread on their way to the tumor tissue resulting in different fractions of the healthy tissue covered with doses larger than the tumor dose, while the tumor gets homogeneously irradiated. The aim of this study was to evaluate the effect of increasing channel width on potential side effects in the normal tissue. A rectangular 180 × 180 µm(2) and two Gaussian-type dose distributions of σ = 260 µm and σ = 520 µm with an interchannel distance of 1.8 mm have been applied by 20-MeV protons to a 3D human skin model in order to simulate the widened channels and to compare the irradiation effects at different endpoints to those of a homogeneous proton irradiation. The number of protons applied was kept constant at all irradiation modes resulting in the same average dose of 2 Gy. All kinds of proton microchannel irradiation lead to higher cell viability and produce significantly less genetic damage than homogeneous proton irradiation, but the reduction is lower for the wider channel sizes. Our findings point toward the application of microchannel irradiation for clinical proton or heavy ion therapy to further reduce damage of normal tissues while maintaining tumor control via a homogeneous dose distribution inside the tumor.

Background: The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in the cytosol and integrated in the plasma membrane of tumor cells via lipid anchorage. Following stress such as non-lethal irradiation Hsp70 synthesis is up-regulated. Intracellular located Hsp70 is known to exert cytoprotective properties, however, less is known about membrane (m)Hsp70. Herein, we investigate the role of mHsp70 in the sensitivity towards irradiation in tumor sublines that differ in their cytosolic and/or mHsp70 levels., Methods: The isogenic human colon carcinoma sublines CX(+) with stable high and CX(-) with stable low expression of mHsp70 were generated by fluorescence activated cell sorting, the mouse mammary carcinoma sublines 4 T1 (4 T1 ctrl) and Hsp70 knock-down (4 T1 Hsp70 KD) were produced using the CRISPR/Cas9 system, and the Hsp70 down-regulation in human lung carcinoma sublines H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD was achieved by small interfering (si)RNA against Heat shock factor 1 (HSF-1). Cytosolic and mHsp70 was quantified by Western blot analysis/ELISA and flow cytometry; double strand breaks (DSBs) and apoptosis were measured by flow cytometry using antibodies against γH2AX and real-time PCR (RT-PCR) using primers and antibodies directed against apoptosis related genes; and radiation sensitivity was determined using clonogenic cell surviving assays., Results: CX(+)/CX(-) tumor cells exhibited similar cytosolic but differed significantly in their mHsp70 levels, 4 T1 ctrl/4 T1 Hsp70 KD cells showed significant differences in their cytosolic and mHsp70 levels and H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD lung carcinoma cell sublines had similar mHsp70 but significantly different cytosolic Hsp70 levels. γH2AX was significantly up-regulated in irradiated CX(-) and 4 T1 Hsp70 KD with low basal mHsp70 levels, but not in their mHsp70 high expressing counterparts, irrespectively of their cytosolic Hsp70 content. After irradiation γH2AX, Caspase 3/7 and Annexin V were up-regulated in the lung carcinoma sublines, but no significant differences were observed in H1339 ctrl/H1339 HSF-1 KD, and EPLC-272H ctrl/EPLC-272H HSF-1 KD that exhibit identical mHsp70 but different cytosolic Hsp70 levels. Clonogenic cell survival was significantly lower in CX(-) and 4 T1 Hsp70 KD cells with low mHsp70 expression, than in CX+ and 4 T1 ctrl cells, whereas no difference in clonogenic cell survival was observed in H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/ EPLC-272H HSF-1 KD sublines with identical mHsp70 but different cytosolic Hsp70 levels., Conclusion: In summary, our results indicate that mHsp70 has an impact on radiation resistance.

Provided that a selective accumulation of (10)B-containing compounds is introduced in tumor cells, following irradiation by thermal neutrons produces high-LET alpha-particles ((4)He) and recoiling lithium-7 ((7)Li) nuclei emitted during the capture of thermalized neutrons (0.025 eV) from (10)B. To estimate the biological effectiveness of this boron neutron capture [(10)B(n,α)(7)Li] reaction, the chromosome aberration assay and the flow cytometry apoptosis assay were applied. At the presence of the clinically used compounds BSH (sodium borocaptate) and BPA (p-boronophenylalanine), human lymphocytes were irradiated by sub-thermal neutrons. For analyzing chromosome aberrations, human lymphocytes were exposed to thermally equivalent neutron fluences of 1.82 × 10(11) cm(-2) or 7.30 × 10(11) cm(-2) (corresponding to thermal neutron doses of 0.062 and 0.248 Gy, respectively) in the presence of 0, 10, 20, and 30 ppm of BSH or BPA. Since the kerma coefficient of blood increased by 0.864 × 10(-12) Gy cm(2) per 10 ppm of (10)B, the kerma coefficients in blood increase from 0.34 × 10(-12) cm(2) (blood without BSH or BPA) up to 2.93 × 10(-12) Gy cm(2) in the presence of 30 ppm of (10)B. For the (10)B(n, α)(7)Li reaction, linear dose-response relations for dicentrics with coefficients α = 0.0546 ± 0.0081 Gy(-1) for BSH and α = 0.0654 ± 0.0075 Gy(-1) for BPA were obtained at 0.062 Gy as well as α = 0.0985 ± 0.0284 Gy(-1) for BSH and α = 0.1293 ± 0.0419 Gy(-1) for BPA at 0.248 Gy. At both doses, the corresponding (10)B(n, α)(7)Li reactions from BSH and BPA are not significantly different. A linear dose-response relation for dicentrics also was obtained for the induction of apoptosis by the (10)B(n, α)(7)Li reaction at 0.248 Gy. The linear coefficients α = 0.0249 ± 0.0119 Gy(-1) for BSH and α = 0.0334 ± 0.0064 Gy(-1) for BPA are not significantly different. Independently of the applied thermal neutron doses of 0.062 Gy or 0.248 Gy, the (10)B(n, α)(7)Li reaction from 30 ppm BSH or BPA induced an apparent RBE of about 2.2 for the production of dicentrics as compared to exposure to thermal neutrons alone. Since the apparent RBE value is defined as the product of the RBE of a thermal neutron dose alone times a boron localization factor which depends on the concentration of a (10)B-containing compound, this localization factor determines the biological effectiveness of the (10)B(n, α)(7)Li reaction.

Magnetic nanoparticles are highly desirable for biomedical research and treatment of cancer especially when combined with hyperthermia. The efficacy of nanoparticle-based therapies could be improved by generating radioactive nanoparticles with a convenient decay time and which simultaneously have the capability to be used for locally confined heating. The core-shell morphology of such novel nanoparticles presented in this work involves a polysilico-tungstate molecule of the polyoxometalate family as a precursor coating material, which transforms into an amorphous tungsten oxide coating upon annealing of the FePt core-shell nanoparticles. The content of tungsten atoms in the nanoparticle shell is neutron activated using cold neutrons at the Heinz Maier-Leibnitz (FRMII) neutron facility and thereby transformed into the radioisotope W-187. The sizeable natural abundance of 28% for the W-186 precursor isotope, a radiopharmaceutically advantageous gamma-beta ratio of γβ≈30% and a range of approximately 1mm in biological tissue for the 1.3MeV β-radiation are promising features of the nanoparticles' potential for cancer therapy. Moreover, a high temperature annealing treatment enhances the magnetic moment of nanoparticles in such a way that a magnetic heating effect of several degrees Celsius in liquid suspension - a prerequisite for hyperthermia treatment of cancer - was observed. A rise in temperature of approximately 3°C in aqueous suspension is shown for a moderate nanoparticle concentration of 0.5mg/ml after 15min in an 831kHz high-frequency alternating magnetic field of 250Gauss field strength (25mT). The biocompatibility based on a low cytotoxicity in the non-neutron-activated state in combination with the hydrophilic nature of the tungsten oxide shell makes the coated magnetic FePt nanoparticles ideal candidates for advanced radiopharmaceutical applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Tumor but not normal cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface (mHsp70) from where it can be actively released. Therefore, membrane (mHsp70) and soluble Hsp70 (sHsp70) were investigated as potential tumor biomarkers and for monitoring the outcome of radiation therapy., Methods: Biopsies and blood were collected from patients with squamous cell carcinoma of the head and neck (SCCHN) at different time points (before, during therapy and in the follow-up period). Hsp70 membrane expression was determined on single cell suspensions of tumor biopsies and reference tissues by flow cytometry, sHsp70 protein and antibody levels were determined in the serum of patients and healthy donors by ELISA and NK cell markers that are related to the presence of sHsp70 were analyzed in the patient's peripheral blood lymphocytes (PBL)., Results: Tumor biopsies exhibited significantly increased mHsp70 expression levels compared to the reference tissue. Soluble Hsp70 levels were significantly higher in SCCHN patients compared to healthy human volunteers and high mHsp70 expression levels on tumor cells were associated with high sHsp70 levels in the serum of patients. Following surgery and radiotherapy sHsp70 levels in patients dropped in patients without tumor relapse in the follow-up period. In contrast to sHsp70 protein, anti-Hsp70 antibody levels remained nearly unaltered in the serum of SCCHN patients before and after therapy. Furthermore, sHsp70 protein but not anti-Hsp70 antibody levels were found to be associated with the tumor volume in SCCHN patients before start of therapy. The expression densities of the activatory NK cell markers CD56, CD94, NKG2D, NKp30, Nkp44, and NKp46 differed in patients following therapeutic intervention. A significant increase in the density of NKG2D was observed in SCCHN patients in the follow-up period after surgery and radiotherapy., Conclusion: We suggest sHsp70 as a potential biomarker for detecting tumors and for monitoring the clinical outcome of radiotherapy in SCCHN patients.

Purpose: Tumor cells, in contrast to normal cells, frequently overexpress heat shock protein 70 (Hsp70) in the cytosol, present it on their cell surface, and actively release it. Therefore, soluble Hsp70 (sHsp70) was investigated as a potential tumor biomarker for monitoring the outcome of radiation therapy., Methods and Materials: Plasma from mice bearing membrane Hsp70 (mHsp70)-positive FaDu human squamous cell carcinoma of the head and neck and spontaneous pancreatic ductal adenocarcinoma (PDAC) was investigated. A cohort of mice with FaDu tumors (0.32 cm(3)) was irradiated with 30 Gy, and plasma was collected 24 hours after irradiation, after the tumors had shrunk to 50% of their starting volume and after complete remission. sHsp70 levels in the plasma were quantified by enzyme-linked immunosorbent assay., Results: sHsp70 levels were significantly higher in the blood of tumor-bearing mice than that of control animals. A correlation between increasing sHsp70 plasma levels and tumor volume in the range of 0.01 cm(3) to 0.66 cm(3) was observed. Radiation-induced regression of the tumors was associated with significantly decreased sHsp70 levels, which returned to the level of control animals after complete remission., Conclusion: We propose sHsp70 as an innovative biomarker for detecting tumors and for monitoring the clinical outcome of radiation therapy in cancer patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The new technology of laser-driven ion acceleration (LDA) has shown the potential for driving highly brilliant particle beams. Laser-driven ion acceleration differs from conventional proton sources by its ultra-high dose rate, whose radiobiological impact should be investigated thoroughly before adopting current clinical dose concepts. The growth of human FaDu tumors transplanted onto the hind leg of nude mice was measured sonographically. Tumors were irradiated with 20 Gy of 23 MeV protons at pulsed mode with single pulses of 1 ns duration or continuous mode (∼100 ms) in comparison to controls and to a dose-response curve for 6 MV photons. Tumor growth delay and the relative biological effectiveness (RBE) were calculated for all irradiation modes. The mean target dose reconstructed from Gafchromic films was 17.4 ± 0.8 Gy for the pulsed and 19.7 ± 1.1 Gy for the continuous irradiation mode. The mean tumor growth delay was 34 ± 6 days for pulsed, 35 ± 6 days for continuous protons, and 31 ± 7 days for photons 20 ± 1.2 Gy, resulting in RBEs of 1.22 ± 0.19 for pulsed and 1.10 ± 0.18 for continuous protons, respectively. In summary, protons were found to be significantly more effective in reducing the tumor volume than photons (P < 0.05). Together with the results of previous in vitro experiments, the in vivo data reveal no evidence for a substantially different radiobiology that is associated with the ultra-high dose rate of protons that might be generated from advanced laser technology in the future.

Male-mediated developmental toxicity has been of concern for many years. The public became aware of male-mediated developmental toxicity in the early 1990s when it was reported that men working at Sellafield might be causing leukemia in their children. Human and animal studies have contributed to our current understanding of male-mediated effects. Animal studies in the 1980s and 1990s suggested that genetic damage after radiation and chemical exposure might be transmitted to offspring. With the increasing understanding that there is histone retention and modification, protamine incorporation into the chromatin and DNA methylation in mature sperm and that spermatozoal RNA transcripts can play important roles in the epigenetic state of sperm, heritable studies began to be viewed differently. Recent reports using molecular approaches have demonstrated that DNA damage can be transmitted to babies from smoking fathers, and expanded simple tandem repeats minisatellite mutations were found in the germline of fathers who were exposed to radiation from the Chernobyl nuclear power plant disaster. In epidemiological studies, it is possible to clarify whether damage is transmitted to the sons after exposure of the fathers. Paternally transmitted damage to the offspring is now recognized as a complex issue with genetic as well as epigenetic components.

A series of N-heterocyclic carbene (NHC)/PR3 palladium(II) and palladium(0) complexes has been synthesized and fully characterized. X-ray crystallographic data have allowed comparison of ligand steric properties. The NHC ligand was found to vary its steric properties as a function of the phosphine co-ligand. These complexes display interesting catalytic properties in the Suzuki-Miyaura reaction performed in aqueous media. The pre-catalyst [PdCl2(IPr)(XPhos)] (IPr = N,N'-bis-(2,6-diisopropylphenyl)imidazol-2-ylidene; XPhos = 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl) was found to be the most efficient system, promoting the coupling of a wide range of aryl chlorides with boronic acids in aqueous media with a typical catalyst loading of 0.03 mol%.

The induction of chromosome aberrations in human lymphocytes irradiated in vitro with slow neutrons was examined to assess the maximum low-dose RBE (RBE(M)) relative to (60)Co γ-rays. For the blood irradiations, cold neutron beam available at the prompt gamma activation analysis facility at the Munich research reactor FRM II was used. The given flux of cold neutrons can be converted into a thermally equivalent one. Since blood was taken from the same donor whose blood had been used for previous irradiation experiments using widely varying neutron energies, the greatest possible accuracy was available for such an estimation of the RBE(M) avoiding the inter-individual variations or differences in methodology usually associated with inter-laboratory comparisons. The magnitude of the coefficient α of the linear dose-response relationship (α = 0.400 ± 0.018 Gy(-1)) and the derived RBE(M) of 36.4 ± 13.3 obtained for the production of dicentrics by thermal neutrons confirm our earlier observations of a strong decrease in α and RBE(M) with decreasing neutron energy lower than 0.385 MeV (RBE(M) = 94.4 ± 38.9). The magnitude of the presently estimated RBE(M) of thermal neutrons is-with some restrictions-not significantly different to previously reported RBE(M) values of two laboratories.

The application of a microchannel proton irradiation was compared to homogeneous irradiation in a three-dimensional human skin model. The goal is to minimize the risk of normal tissue damage by microchannel irradiation, while preserving local tumor control through a homogeneous irradiation of the tumor that is achieved because of beam widening with increasing track length. 20 MeV protons were administered to the skin models in 10- or 50-μm-wide irradiation channels on a quadratic raster with distances of 500 μm between each channel (center to center) applying an average dose of 2 Gy. For comparison, other samples were irradiated homogeneously at the same average dose. Normal tissue viability was significantly enhanced after microchannel proton irradiation compared to homogeneous irradiation. Levels of inflammatory parameters, such as Interleukin-6, TGF-Beta, and Pro-MMP1, were significantly lower in the supernatant of the human skin tissue after microchannel irradiation than after homogeneous irradiation. The genetic damage as determined by the measurement of micronuclei in keratinocytes also differed significantly. This difference was quantified via dose modification factors (DMF) describing the effect of each irradiation mode relative to homogeneous X-ray irradiation, so that the DMF of 1.21 ± 0.20 after homogeneous proton irradiation was reduced to 0.23 ± 0.11 and 0.40 ± 0.12 after microchannel irradiation using 10- and 50-μm-wide channels, respectively. Our data indicate that proton microchannel irradiation maintains cell viability while significantly reducing inflammatory responses and genetic damage compared to homogeneous irradiation, and thus might improve protection of normal tissue after irradiation.

Background: Older men tend to have poorer semen quality and are generally at higher risks for infertility and abnormal reproductive outcomes., Methods: We employed proton-induced X-ray emission (PIXE, 3 MeV proton beam) to investigate the concentrations of zinc, copper, calcium, sulfur, chlorine, potassium, titanium, iron and nickel in washed sperm and seminal plasma from non-smoking groups of 10 older men (65-80 years old) and 10 younger men (22-28 years old) who were concurrently assayed for sperm function and genomicly defective sperm., Results: The older group showed elevated zinc, copper and calcium in sperm and elevated sulfur in seminal plasma compared with the younger men. The older group also showed reduced motility as well as increased sperm DNA fragmentation, achondroplasia mutations, DNA strand breaks and chromosomal aberrations. Sperm calcium and copper were positively associated with sperm DNA fragmentation (P < 0.03). Seminal sulfur was positively associated with sperm DNA fragmentation and chromosomal aberrations (P < 0.04), and negatively associated with sperm motility (P < 0.05). Sperm calcium was negatively associated with sperm motility, independent of male age (P = 0.01)., Conclusions: We identified major differences in elemental concentrations between sperm and seminal plasma and that higher sperm copper, sulfur and calcium are quantitatively associated with poorer semen quality and increased frequencies of genomic sperm defects.

A series of [Pd(NHC)(PR(3))] complexes quantitatively form [Pd(η(2)-O(2))(NHC)(PR(3))] products when submitted to an dioxygen atmosphere. Synthetic details and structural features of these Pd-peroxo complexes are described. The palladium compounds were found to be efficient catalysts in the oxidation of alcohols into corresponding carbonyl compounds using O(2) or air as oxidant. Reactions proceed at low catalyst loadings with a wide range of alcohols.

This study shows that enhanced radiobiological effectiveness (RBE) values can be generated focusing low linear energy transfer (LET) radiation and thus changing the microdose distribution. 20 MeV protons (LET = 2.65 keV µm(-1)) are focused to submicrometer diameter at the ion microprobe superconducting nanoprobe for applied nuclear (Kern) physics experiments of the Munich tandem accelerator. The RBE values, as determined by measuring micronuclei (RBE(MN) = 1.48 ± 0.07) and dicentrics (RBE(D) = 1.92 ± 0.15), in human-hamster hybrid (A(L)) cells are significantly higher when 117 protons were focused to a submicrometer irradiation field within a 5.4 × 5.4 µm(2) matrix compared to quasi homogeneous in a 1 × 1 µm(2) matrix applied protons (RBE(MN) = 1.28 ± 0.07; RBE(D) = 1.41 ± 0.14) at the same average dose of 1.7 Gy. The RBE values are normalized to standard 70 kV (dicentrics) or 200 kV (micronuclei) x-ray irradiation. The 117 protons applied per point deposit the same amount of energy like a (12)C ion with 55 MeV total energy (4.48 MeV u(-1)). The enhancements are about half of that obtained for (12)C ions (RBE(MN) = 2.20 ± 0.06 and RBE(D) = 3.21 ± 0.10) and they are attributed to intertrack interactions of the induced damages. The measured RBE values show differences from predictions of the local effect model (LEM III) that is used to calculate RBE values for irradiation plans to treat tumors with high LET particles.

The relevance of preconceptional and prenatal toxicant exposures for genomic stability in offspring is difficult to analyze in human populations, because gestational exposures usually cannot be separated from preconceptional exposures. To analyze the roles of exposures during gestation and conception on genomic stability in the offspring, stability was assessed via the Comet assay and highly sensitive, semiautomated confocal laser scans of γH2AX foci in cord, maternal, and paternal blood as well as spermatozoa from 39 families in Crete, Greece, and the United Kingdom. With use of multivariate linear regression analysis with backward selection, preconceptional paternal smoking (% tail DNA: P>0.032; γH2AX foci: P>0.018) and gestational maternal (% tail DNA: P>0.033) smoking were found to statistically significantly predict DNA damage in the cord blood of F1 offspring. Maternal passive smoke exposure was not identified as a predictor of DNA damage in cord blood, indicating that the effect of paternal smoking may be transmitted via the spermatozoal genome. Taken together, these studies reveal a role for cigarette smoke in the induction of DNA alterations in human F1 offspring via exposures of the fetus in utero or the paternal germline. Moreover, the identification of transgenerational DNA alterations in the unexposed F1 offspring of smoking-exposed fathers supports the claim that cigarette smoke is a human germ cell mutagen.

The use of particle ion beams in cancer radiotherapy has a long history. Today, beams of protons or heavy ions, predominantly carbon ions, can be accelerated to precisely calculated energies which can be accurately targeted to tumors. This particle therapy works by damaging the DNA of tissue cells, ultimately causing their death. Among the different types of DNA lesions, the formation of DNA double strand breaks is considered to be the most relevant of deleterious damages of ionizing radiation in cells. It is well-known that the extremely large localized energy deposition can lead to complex types of DNA double strand breaks. These effects can lead to cell death, mutations, genomic instability, or carcinogenesis. Complex double strand breaks can increase the probability of mis-rejoining by NHEJ. As a consequence differences in the repair kinetics following high and low LET irradiation qualities are attributed mainly to quantitative differences in their contributions of the fast and slow repair component. In general, there is a higher contribution of the slow component of DNA double strand repair after exposure to high LET radiation, which is thought to reflect the increased amount of complex DNA double strand breaks. These can be accurately measured by the γ-H2AX assay, because the number of phosphorylated H2AX foci correlates well with the number of double strand breaks induced by low or / and high LET radiation.

Ionizing irradiation is an important clinical approach to treat solid tumors. Modern radiation technologies aim to selectively kill tumor cells and protect the surrounding normal tissue. The standard paradigm for radiation effects in cellular systems involves damage of the DNA including DNA double-strand breaks, which are considered as most effective in destroying tumor cells. Due to their enhanced physical and radiobiological properties, high-linear energy transfer radiation qualities are of special interest in tumor therapy. Future radiation therapy strategies aim to utilize carbon ions to effectively treat highly aggressive tumors. More recently, evidence is emerging for non-DNA targeted effects of radiation, including mutations, chromosomal aberrations, and changes in gene expression, which can occur in cells that were not directly exposed to radiation. Radiation oncologists are only gradually beginning to appreciate the clinical relevance of radiation-induced bystander effects, genomic instability, and abscopal effects. Since these effects are sensed by the immune system, a combination of immunotherapy and irradiation presents a new therapeutic opportunity in the future.

Benzene is a primary industrial chemical and a ubiquitous environmental pollutant that causes human leukemia and maybe other malignancies. Occupational exposure to benzene has been associated with increased chromosomal aneuploidies in blood lymphocytes and, in separate studies, in sperm. However, aneuploidy detection in somatic and germ cells within the same benzene-exposed individuals has never been reported. To compare aneuploidies in blood lymphocytes and sperm within the same individuals exposed to benzene, a cross-sectional study was conducted in 33 benzene-exposed male workers and 33 unexposed workers from Chinese factories. Air benzene concentrations in the exposed workers ranged from below the detection limit to 24 ppm (median, 2.9 ppm) and were undetectable in the unexposed subjects. Aneuploidies of chromosomes 21, X, and Y in blood lymphocytes were examined by multicolor fluorescence in situ hybridization and were compared to the previously reported aneuploidies in sperm. The results showed that benzene exposure was positively associated with the gain of chromosome 21 but not sex chromosomes in blood lymphocytes. This was in contrast to analysis of sperm, where the gain of sex chromosomes, but not chromosome 21, was significantly increased in the exposed workers. Furthermore, a significant correlation in the gain of sex chromosomes between blood lymphocytes and sperm was observed among the unexposed subjects, but not among the exposed workers. The findings suggest that benzene exposure induces aneuploidies in both blood cells and sperm within the same individuals, but selectively affects chromosome 21 in blood lymphocytes and the sex chromosomes in sperm., (Copyright © 2012 Wiley Periodicals, Inc.)

In particle tumor therapy including beam scanning at accelerators, the dose per voxel is delivered within about 100 ms. In contrast, the new technology of laser plasma acceleration will produce ultimately shorter particle packages that deliver the dose within a nanosecond. Here, possible differences for relative biological effectiveness in creating DNA double-strand breaks in pulsed or continuous irradiation mode are studied. HeLa cells were irradiated with 1 or 5 Gy of 20-MeV protons at the Munich tandem accelerator, either at continuous mode (100 ms), or applying a single pulse of 1-ns duration. Cells were fixed 1 h after 1-Gy irradiation and 24 h after 5-Gy irradiation, respectively. A dose-effect curve based on five doses of X-rays was taken as reference. The total number of phosphorylated histone H2AX (gamma-H2AX) foci per cell was determined using a custom-made software macro for gamma-H2AX foci counting. For 1 h after 1-Gy 20-MeV proton exposures, values for the relative biological effectiveness (RBE) of 0.97 ± 0.19 for pulsed and 1.13 ± 0.21 for continuous irradiations were obtained in the first experiment 1.13 ± 0.09 and 1.16 ± 0.09 in the second experiment. After 5 Gy and 24 h, RBE values of 0.99 ± 0.29 and 0.91 ± 0.23 were calculated, respectively. Based on the gamma-H2AX foci numbers obtained, no significant differences in RBE between pulsed and continuous proton irradiation in HeLa cells were detected. These results are well in line with our data on micronucleus induction in HeLa cells.

Background: Benzene is an industrial chemical that causes blood disorders, including acute myeloid leukemia. We previously reported that occupational exposures near the U.S. Occupational Safety and Health Administration permissible exposure limit (8 hr) of 1 ppm was associated with sperm aneuploidy., Objective: We investigated whether occupational exposures near 1 ppm increase the incidence of sperm carrying structural chromosomal aberrations., Methods: We applied a sperm fluorescence in situ hybridization assay to measure frequencies of sperm carrying partial chromosomal duplications or deletions of 1cen or 1p36.3 or breaks within 1cen-1q12 among 30 benzene-exposed and 11 unexposed workers in Tianjin, China, as part of the China Benzene and Sperm Study (C-BASS). Exposed workers were categorized into low-, moderate-, and high-exposure groups based on urinary benzene (medians: 2.9, 11.0, and 110.6 µg/L, respectively). Median air benzene concentrations in the three exposure groups were 1.2, 3.7, and 8.4 ppm, respectively., Results: Adjusted incidence rate ratios (IRRs) and 95% confidence intervals (CIs) for all structural aberrations combined were 1.42 (95% CI: 1.10, 1.83), 1.44 (95% CI: 1.12, 1.85), and 1.75 (95% CI: 1.36, 2.24) and for deletion of 1p36.3 alone were 4.31 (95% CI: 1.18, 15.78), 6.02 (95% CI: 1.69, 21.39), and 7.88 (95% CI: 2.21, 28.05) for men with low, moderate, and high exposure, respectively, compared with unexposed men. Chromosome breaks were significantly increased in the high-exposure group [IRR 1.49 (95% CI: 1.10, 2.02)]., Conclusions: Occupational exposures to benzene were associated with increased incidence of chromosomally defective sperm, raising concerns for worker infertility and spontaneous abortions as well as mental retardation and inherited defects in their children. Our sperm findings point to benzene as a possible risk factor for de novo 1p36 deletion syndrome. Because chromosomal aberrations in sperm can arise from defective stem cells/spermatogonia, our findings raise concerns that occupational exposure to benzene may have persistent reproductive effects in formerly exposed workers.

Together with surgery and chemotherapy, ionizing irradiation is one of the key therapeutic approaches to treat cancer. More than 50 percent of all cancer patients will receive radiotherapeutic intervention at some stage of their disease. The more precise instrumentation for delivery of radiotherapy and the emphasis on hypofractionation technologies have drastically improved loco-regional tumor control within the last decades. However, the appearance of distant metastases often requires additional systemic treatment modalities such as chemotherapy. High dose chemotherapy is generally considered as immunosuppressive and can cause severe adverse effects. Therefore, we want to elucidate the effects of ionizing irradiation on the immune system and provide immunological treatment strategies which are induced by the host's stress response. Similar to other stressors, ionizing irradiation is known to enhance the synthesis of a variety of immune-stimulatory and -modulating molecules such as heat shock proteins (HSP), high mobility group box 1 (HMGB1) and survivin. Herein, we focus on HSP that exhibit an unusual cell membrane localization and release mechanism in tumor cells. These tumor-specific characteristics render HSP as ideal targets for therapeutic interventions. Depending on their intra/membrane and extracellular localization HSP have the ability to protect tumor cells from stress-induced lethal damage by interfering with antiapoptotic pathways or to elicit anti-cancer immunity.

Background: Laser acceleration of protons and heavy ions may in the future be used in radiation therapy. Laser-driven particle beams are pulsed and ultra high dose rates of >10⁹ Gy s⁻¹ may be achieved. Here we compare the radiobiological effects of pulsed and continuous proton beams., Methods: The ion microbeam SNAKE at the Munich tandem accelerator was used to directly compare a pulsed and a continuous 20 MeV proton beam, which delivered a dose of 3 Gy to a HeLa cell monolayer within < 1 ns or 100 ms, respectively. Investigated endpoints were G2 phase cell cycle arrest, apoptosis, and colony formation., Results: At 10 h after pulsed irradiation, the fraction of G2 cells was significantly lower than after irradiation with the continuous beam, while all other endpoints including colony formation were not significantly different. We determined the relative biological effectiveness (RBE) for pulsed and continuous proton beams relative to x-irradiation as 0.91 ± 0.26 and 0.86 ± 0.33 (mean and SD), respectively., Conclusions: At the dose rates investigated here, which are expected to correspond to those in radiation therapy using laser-driven particles, the RBE of the pulsed and the (conventional) continuous irradiation mode do not differ significantly.

There is rapidly increasing information on the issue of three-dimensional nuclear architecture, according to which chromosomes are organized in localized territories and chromosome arms in exclusive domains within a given territory. The aim of the present study was to investigate the impact of different cell exposure conditions on cytogenetic damage induced by high-LET radiation. To this end the yield ratio of dicentrics to centric rings (F value) induced by (241)Am α particles was analyzed in monolayer cultures of human lymphocytes that were either settled or attached to foils, simulating a rounded or spread out cellular geometry, respectively. Monolayers were exposed in special irradiation chambers to 0.1 and 1.0 Gy and subsequently analyzed for chromosome aberrations. Independent of these different dose levels, significantly different F values of 10.07 ± 1.73 and 4.27 ± 0.44 have been determined in attached and settled lymphocytes, respectively. Since the diameter of nuclei vertically traversed by α particles in attached cells is about one-half that in settled cells, these F values support the postulate that proximity effects regarding the chromatin geometry in flattened or spherical human lymphocytes influence the formation of high-LET radiation-induced dicentrics and centric rings. A comparison with our earlier data sets obtained for both in vitro and in vivo exposure of human lymphocytes to α particles or (137)Cs γ rays supports the notion that the F value depends on the radiation quality when investigations are confined to spherical human lymphocytes. Thus the F value should not be ruled out as a practical chromosomal "fingerprint" for past exposure to high-LET radiation.

A technical set-up for irradiation of subcutaneous tumours in mice with nanosecond-pulsed proton beams or continuous proton beams is described and was successfully used in a first experiment to explore future potential of laser-driven particle beams, which are pulsed due to the acceleration process, for radiation therapy. The chosen concept uses a microbeam approach. By focusing the beam to approximately 100 × 100 μm(2), the necessary fluence of 10(9) protons per cm(2) to deliver a dose of 20 Gy with one-nanosecond shot in the Bragg peak of 23 MeV protons is achieved. Electrical and mechanical beam scanning combines rapid dose delivery with large scan ranges. Aluminium sheets one millimetre in front of the target are used as beam energy degrader, necessary for adjusting the depth-dose profile. The required procedures for treatment planning and dose verification are presented. In a first experiment, 24 tumours in mice were successfully irradiated with 23 MeV protons and a single dose of 20 Gy in pulsed or continuous mode with dose differences between both modes of 10%. So far, no significant difference in tumour growth delay was observed.

The use of phosphites in second generation, ruthenium-based olefin metathesis pre-catalysts leads to an improvement in catalyst stability and activity at low catalyst loadings., (This journal is © The Royal Society of Chemistry 2011)

Laser accelerated radiotherapy is a potential cancer treatment with proton and carbon-ion beams that is currently under development. Ultra-fast high-energy laser pulses will accelerate ion beams that deliver their dose to a patient in a "pulsed mode" that is expected to differ from conventional irradiation by increasing the dose delivery rate to a tissue voxel by approximately 8 orders of magnitude. In two independently performed experiments at the ion microprobe SNAKE of the 14 MV Munich tandem accelerator, A(L) cells were exposed either to protons with 1-ns pulse durations or to protons applied over 150 ms in continuous irradiation mode. A slightly but consistently lower aberration yield was observed for the pulsed compared to the continuous mode of proton irradiation. This difference was not statistically significant when each aberration type was analyzed separately (P values between 0.61 and 0.85 in experiment I and P values between 0.32 and 0.64 in experiment II). However, excluding the total aberrations, which were not analyzed as independent radiation-induced effects, the mean ratio of the yields of dicentrics, centric rings and excess acentrics scored together showed (with 95% CI) a significant difference of 0.90 (0.81; 0.98) between the pulsed and the continuous irradiation modes. A similar tendency was also determined for the corresponding RBE values relative to 70 kV X rays. Since the different findings for the comparisons of individual chromosome aberration types and combined comparisons could be explained by different sample sizes with the consequence that the individual comparisons had less statistical power to identify a difference, it can be concluded that 20 MeV protons may be slightly less effective in the pulsed mode.

The synthesis, characterisation and catalytic behaviour of ruthenium indenylidene complexes bearing an N-heterocyclic carbene and triisopropylphosphite are described.

Purpose: In order to obtain more insight into heavy ion tumour therapy, some features of the underlying molecular mechanisms controlling the cellular response to high linear energy transfer (LET) radiation are currently analysed., Materials and Methods: We analysed the decay of the integrated fluorescence intensity of gamma-H2AX (phosphorylated histone H2AX) which is thought to reflect the repair kinetics of radiation-induced DNA double-strand breaks (DSB) using Laser-Scanning-Cytometry. Asynchronous human HeLa cells were irradiated with a single dose of either 1.89 Gy of 55 MeV carbon ions or 5 Gy of 70 kV X-rays., Results: Measurements of the gamma-H2AX-intensities from 15-60 min resulted in a 16 % decrease for carbon ions and in a 43 % decrease for X-rays. After 21 h, the decrease was 77 % for carbon ions and 85 % for X-rays. The corresponding time-effect relationship was fitted by a bi-exponential function showing a fast and a slow component with identical half-life values for both radiation qualities being 24 +/- 4 min and 13.9 +/- 0.7 h, respectively. Apparent differences in the kinetics following high and low LET irradiation could completely be attributed to quantitative differences in their contributions, with the slow component being responsible for 47 % of the repair after exposure to X-rays as compared to 80 % after carbon ion irradiation., Conclusion: gamma-H2AX loss kinetics follows a bi-exponential decline with two definite decay times independent of LET. The higher contribution of the slow component determined for carbon ion exposure is thought to reflect the increased amount of complex DSB induced by high LET radiation.

Background: Benzene is a common industrial chemical known to induce leukemia and other blood disorders, as well as aneuploidy, in both human blood cells and sperm at exposures > 10 ppm. Recent reports have identified health effects at exposure levels < 1 ppm, the permissible exposure limit (PEL; 8 hr) set by the U.S. Occupational Safety and Health Administration., Objective: We investigated whether occupational exposures to benzene near 1 ppm induce aneuploidy in sperm., Methods: We used multicolor fluorescence in situ hybridization to measure the incidence of sperm with numerical abnormalities of chromosomes X, Y, and 21 among 33 benzene-exposed men and 33 unexposed men from Chinese factories. Individual exposures were assessed using personal air monitoring and urinary concentrations of benzene and trans,trans-muconic acid (E,E-MA). Air benzene concentrations were not detectable in unexposed men; in exposed men, concentrations ranged from below the detection limit to 24 ppm (median, 2.9 ppm), with 27% of exposed men (n = 9) having concentrations of

Background and Purpose: Laser accelerated radiotherapy is a prospect for cancer treatment with proton and/or carbon ion beams that is currently under fast development. In principal, ultra fast, high-energy laser pulses will lead to a "pulsed" delivery of the induced ion beam with pulse durations of 1ns and below, whereas conventional proton beams deriving from a cyclotron or synchrotron apply the dose within 100 ms ("continuous")., Materials and Methods: A simulation of both irradiation modes could be established at the Munich tandem accelerator with a 20MeV proton beam, and a wide-field fast scanning system was implemented that allowed for application of up to 5 Gy per tissue voxel in a single pulse. The relative biological effectiveness (RBE) of pulsed and continuous modes of irradiation with 20 MeV protons relative to the reference radiation 70 kV X-rays was examined in a human tissue model (3D human reconstructed skin, EpiDermFT) which preserves the three-dimensional geometric arrangement and communication of cells present in tissues in vivo. Using the induction of micronuclei (MN) in keratinocytes as the biological endpoint, the RBE was calculated as the ratio between the dose of 70 kV X-rays and 3 Gy of 20 MeV protons (pulsed or continuous) which produced equal response., Results: For pulsed and continuous 20 MV proton exposures of the human skin model, RBE values of 1.08+/-0.20 and 1.22+/-0.15 versus 70 kV X-rays were obtained in a first experiment and 1.00+/-0.14 and 1.13+/-0.14 in a second experiment during distinct beam access times, respectively. The approximately 10% difference in RBE between the respective irradiation modes in both experiments was associated with large uncertainties which were not statistically significant (p approximately 0.5)., Conclusion: These findings represent an important step on the way towards application of laser-accelerated protons for clinical radiotherapy. Further clinically relevant endpoints in normal and tumor tissue have to be evaluated., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

To obtain greater insight into the future potential of tumor radiotherapy using proton beams generated from high-intensity lasers, it is important to characterize the ionization quality of the new beams by measuring the relative biological effectiveness (RBE) under conditions where the full dose at one irradiation site will be deposited by a few proton pulses less than 1 ns in duration. HeLa cells attached to a Mylar foil were irradiated with 70 kV X rays to obtain a reference dose-response curve or with 3 Gy of 20 MeV protons at the Munich tandem accelerator (Garching), either using a continuous mode where a cell sample was irradiated within a 100-ms time span or using a pulsed mode where radiation was given in a single proton pulse of about 1 ns. After irradiation cytochalasin B was added; 24 h later cells were fixed and stained with acridine orange and micronuclei were counted. The X-ray dose-response curve for the production of micronuclei in HeLa cells followed a linear-quadratic model. The corresponding RBE values for 20 MeV protons in pulsed and continuous irradiation modes were 1.07 +/- 0.08 and 1.06 +/- 0.10 in the first proton experiment and 1.09 +/- 0.08 and 1.05 +/- 0.11 in the second, respectively. There was no evidence for a difference in the RBE for pulsed and continuous irradiation of HeLa cells with 20 MeV protons.

The aim of this study was to investigate and quantify two biomarkers for radiation exposure (dicentrics and gamma-H2AX foci) in human lymphocytes after CT scans in the presence of an iodinated contrast agent. Blood samples from a healthy donor were exposed to CT scans in the absence or presence of iotrolan 300 at iodine concentrations of 5 or 50 mg ml(-1) blood. The samples were exposed to 0.025, 0.05, 0.1 and 1 Gy in a tissue equivalent body phantom. Chromosome aberration scoring and automated microscopic analysis of gamma-H2AX foci were performed in parts of the same samples. The theoretical physical dose enhancement factor (DEF) was calculated on the basis of the mass energy-absorption coefficients of iodine and blood and the photon energy spectrum of the CT tube. No significant differences in the yields of dicentrics and gamma-H2AX foci were observed in the absence or presence of 5 mg iodine ml(-1) blood up to 0.1 Gy, whereas at 1 Gy the yields were elevated for both biomarkers. At an iodine concentration of 50 mg ml(-1) serving as a positive control, a biological DEF of 9.5 +/- 1.4 and 2.3 +/- 0.5 was determined for dicentrics and gamma-H2AX foci, respectively. A physical DEF of 1.56 and 6.30 was calculated for 5 and 50 mg iodine ml(-1), respectively. Thus, it can be concluded that in the diagnostic dose range (radiation and contrast dose), no relevant biological dose-enhancing effect could be detected, whereas a clear biological dose-enhancing effect could be found for a contrast dose well outside the diagnostic CT range for the complete radiation dose range with both methods.

Cigarette smoking in men has been associated with increased chromosomal abnormalities in sperm and with increased risks for spontaneous abortions, birth defects and neonatal death. Little is known, however, about the reproductive consequences of paternal exposure to second-hand smoke. We used a mouse model to investigate the effects of paternal exposure to sidestream (SS) smoke, the main constituent of second-hand smoke, on the genetic integrity and function of sperm, and to determine whether male germ cells were equally sensitive to mainstream (MS) and SS smoke. A series of sperm DNA quality and reproductive endpoints were investigated after exposing male mice for two weeks to MS or SS smoke. Our results indicated that: (i) only SS smoke significantly affected sperm motility; (ii) only MS smoke induced DNA strand breaks in sperm; (iii) both MS and SS smoke increased sperm chromatin structure abnormalities; and (iv) MS smoke affected both fertilization and the rate of early embryonic development, while SS smoke affected fertilization only. These results show that MS and SS smoke have differential effects on the genetic integrity and function of sperm and provide further evidence that male exposure to second-hand smoke, as well as direct cigarette smoke, may diminish a couple's chance for a successful pregnancy and the birth of a healthy baby.

Background: The trend for men to have children at older age raises concerns that advancing age may increase the production of genetically defective sperm, increasing the risks of transmitting germ-line mutations., Methods: We investigated the associations between male age and sperm DNA damage and the influence of several lifestyle factors in a healthy non-clinical group of 80 non-smokers (mean age: 46.4 years, range: 22-80 years) with no known fertility problems using the sperm Comet analyses., Results: The average percentage of DNA that migrated out of the sperm nucleus under alkaline electrophoresis increased with age (0.18% per year, P = 0.006), but there was no age association for damage measured under neutral conditions (P = 0.7). Men who consumed >3 cups coffee per day had approximately 20% higher percentage tail DNA under neutral but not alkaline conditions compared with men who consumed no caffeine (P = 0.005)., Conclusions: Our findings indicate that (i) older men have increased sperm DNA damage associated with alkali-labile sites or single-strand DNA breaks and (ii) independent of age, men with substantial daily caffeine consumption have increased sperm DNA damage associated with double-strand DNA breaks. DNA damage in sperm can be converted to chromosomal aberrations and gene mutations after fertilization, increasing the risks of developmental defects and genetic diseases among offspring.

Background: Semen quality is associated with fertility status, but there is little quantitative information on risk factors that affect semen quality, especially in non-clinical populations. Advancing male age has been associated with a decline in semen quality, with the largest effect being on sperm motility. However, there is little quantitative data on the specific components of sperm motion that are affected by male age., Methods: We performed linear regression analyses of 14 aspects of semen quality measured by computer-assisted semen analysis (CASA) in a non-clinical cohort of 90 non-smoking men, aged 22-80 years, who had no history of infertility or reproductive problems., Results: We found age-associated declines in CASA-determined motility (% motile, 0.8% per year; % progressively motile, 0.9% per year; % rapidly motile, 0.4% per year, P

The father, like the mother, can transmit genetic defects to his offspring that are detrimental for normal development and a healthy life. Epidemiological studies have identified associations between several paternal exposures and abnormal reproductive outcomes, but these types of studies are inherently complex and expensive, and the risk factors for the paternal contribution to abnormal reproductive outcomes remain poorly understood. Several sensitive methods have been developed for detecting mutations and chromosomal damage directly in sperm. These assays are potential bioindicators for paternal risk factors for infertility, spontaneous abortions, aneuploidy syndromes, and genetic diseases in children. Among these methods, fluorescence in situ hybridization (FISH) has been adapted for the detection of numerical and structural chromosomal abnormalities in the sperm of an expanding number of species, including humans and rodents. Sperm FISH has identified several potential paternal risk factors such as age, drugs, lifestyles, and various environmental/occupational exposures. Here, we summarize the status of the development and usage of these sperm-FISH assays and suggest strategies for prioritizing chemical agents for epidemiological investigations to assess paternal risk for abnormal reproductive outcome.

In recent years, two techniques for detecting genetic damage in the whole genome have gained importance: the alkaline comet assay, to detect DNA damage such as strand breaks and alkali-labile sites, and a multicolour FISH method, spectral karyotyping (SKY), to identify chromosomal aberrations simultaneously in all metaphase chromosomes. In the present study, the induction of DNA damage in human sperm and lymphocytes in vitro has been studied employing an anticancer drug, doxorubicin (DX). An increase in DNA damage was observed with the comet assay as the median per cent head DNA of sperm significantly decreased from 82.07 and 85.14% in the untreated control groups to 63.48 and 72.52% at doses of 0.8 micro M DX. At 1.6 micro M the percentage declined to 60.96% (the corresponding tail moment increased from 4.42 to 12.19). In stimulated lymphocytes, a significant increase was observed in tail moment, from 0.72 and 0.53 in controls to 15.17 and 12.10 at 0.2 micro M DX, continuing at the same level to a final concentration of 1.6 micro M. Structural aberrations found in the parallel SKY study in stimulated lymphocytes at 0.2 micro M DX consisted of 14% chromatid-type and 2% chromosome-type aberrations; none were found in controls. The SKY results correlate very well with the findings of the comet assay in lymphocytes where DNA damage was observed at similar doses. This study is the first reporting use of the comet assay and SKY analysis in parallel after chemical treatment. The potential of the two techniques together is evident, as they represent a set of assays feasible for evaluating damage in human somatic and germ cells after chemical treatment (i) by direct observation of two different end-points, detecting general DNA damage and chromosomal aberrations and (ii) by extrapolation from lymphocytes to sperm, which provides a 'parallelogram' approach in human cells.

Background: Modern reproductive technologies are enabling the treatment of infertile men with severe disturbances of spermatogenesis. The possibility of elevated frequencies of genetically and chromosomally defective sperm has become an issue of concern with the increased usage of ICSI, which can enable men with severely impaired sperm production to father children. Several papers have been published reporting aneuploidy in oligozoospermic patients, but relatively little is known about chromosome structural aberrations in the sperm of these patients., Methods: We examined sperm from infertile, oligozoospermic individuals for structural and numerical chromosomal abnormalities using a multicolour ACM fluorescence in situ hybridization (FISH) assay that utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical chromosomal abnormalities plus two categories of structural aberrations: duplications and deletions of 1pter and 1cen, and chromosomal breaks within the 1cen-1q12 region., Results: There was a significant increase in the average frequencies of sperm with duplications and deletions in the infertility patients compared with the healthy concurrent controls. There was also a significantly elevated level of breaks within the 1cen-1q12 region. There was no evidence for an increase in chromosome 1 disomy, or in diploidy., Conclusions: Our data reveal that oligozoospermia is associated with chromosomal structural abnormalities, suggesting that oligozoospermic men carry a higher burden of transmissible, chromosome damage. The findings raise the possibility of elevated levels of transmissible chromosomal defects following ICSI treatment.

Reactive oxygen species (ROS) are produced by a wide variety of exogenous chemicals and metabolic processes and cause a broad spectrum of damage to biological systems. As a consequence, ROS react with DNA, among many other biological targets, disrupting its structure and functionality. Estrogen-like compounds mediate DNA damage by ROS generation, implying that their effects can be modulated by antioxidants such as catalase, superoxide dismutase, and vitamin C. We examined DNA damage in human lymphocytes and sperm after treatment with four estrogen-like compounds (beta-estradiol, diethylstilbestrol, daidzein, and genistein) and its modulation by flavonoids (quercetin and kaempferol) using the Comet assay. The results indicated that quercetin and kaempferol reduced the DNA damage produced in sperm and lymphocytes by the four estrogenic compounds. The flavonoids also reduced the DNA damage induced by hydrogen peroxide, which was used as a positive control. Our results demonstrate that the antioxidant properties of flavonoids can protect the integrity of human sperm and lymphocyte DNA from ROS induced by estrogenic compounds.

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.

Background: The possibility that oligozoospermic men may have elevated levels of genetic damage in their sperm is of particular concern as they could transmit defects to their offspring., Methods: Sperm samples were obtained from 12 infertile, oligozoospermic patients and 12 healthy normozoospermic volunteers. Fluorescence in-situ hybridization (FISH) was used to determine aneuploidy rates in sperm and inverse restriction site mutation (iRSM) assay to determine gene mutations; defective chromatin packaging was quantified by sperm chromatin structure assay (SCSA) and DNA strand breaks by the Comet assay., Results: FISH analysis showed a significant increase in gonosomal X,Y,18 (P < 0.01) disomy and diploid sperm with X,Y,18,18 (P < 0.05) in the infertility patients compared with the controls. A significant increase (P < 0.01) in disturbed sperm chromatin was found in the infertility patients compared with the control group using the SCSA assay. In the Comet assay, a significant increase (P < 0.01) in the tail moment was found in the infertility patients compared with the control group, indicating significantly high levels of DNA strand breaks. There was no significant increase in point mutations detected by iRSM assay., Conclusions: The data indicate that infertile oligozoospermic men have an elevated level of XY aneuploidy and XY diploidy in the germ-line, as well as elevated levels of sperm chromatin disturbances and sperm DNA strand breaks. These data demonstrate that oligozoospermic infertility patients show several different types of genetic damage in their sperm. Thus, such men appear to have defects at a variety of levels of spermatogenesis and their infertility may not just be a result of the oligozoospermia.