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Purpose: To establish stable in vitro growth of keratinocytes from very small biopsy specimens and successfully apply new test systems to determine their radiosensitivity. Materials and Methods: Oral mucosa biopsies (diameter: 1.7 mm) from 15 subjects were immobilized with custom-made cups onto culture plates. Outgrowing cells were tested for cytokeratin 5/14 and Ki67, expanded, radiated at different doses, and seeded onto circumscribed areas before being allowed to spread centrifugally. In this newly developed spreading assay, cell-covered areas were measured by image analysis. For statistical analysis, a linear mixed regression model was used; additionally, results were correlated to the radiation dose applied. Colony forming efficiency (CFE) was used to validate the results. DNA damage repair was analysed by gammaH2AX and 53BP1 foci quantification using immunofluorescence microscopy 24 h and 96 h after irradiation. Results: Stable keratinocyte growth continued for up to 7 weeks in 14 biopsies. Cells spread reliably from an initial 16.6 mm2 up to a median of 119.2 mm2 (range: 54.4–290). Radiated cells spread to only 100.7 mm2 (2 Gy; range: 55.3–266.7); 73.2 mm2 (4 Gy; 15–240.4); 47 mm2 (6 Gy; 2–111.9), and 22.7 mm2 (8 Gy; 0–80). Similarly, CFE decreased from 0.223 (0 Gy) to 0.0028 (8 Gy). Using an individual donor as a random factor, cell spread correlated with CFE, where radiation dose was the main driver (decrease by 0.50, adjusted for area). Upon irradiation with 6 Gy, radiation-induced DNA damage was increased after 24 h in all samples, and even after 96 h in 5 out of 7 samples, as detected by a higher number of gammaH2AX/53BP1 foci in irradiated cells (mean 3.7 for 24 h; mean 0.6 for 96 h). Conclusion: In vitro propagation of keratinocytes derived from a small biopsy is feasible. Radiation impairs cellular migration and proliferation, and the newly described spreading assay allows ranking for cellular radioresistance. The keratinocyte model also supports classical functional assays such as clonogenic survival and DNA double strand repair. The clinical relevance awaits upcoming investigations.

Recent findings related to childhood leukaemia incidence near nuclear installations have raised questions which can be answered neither by current knowledge on radiation risk nor by other established risk factors. In 2012, a workshop was organised on this topic with two objectives: (a) review of results and discussion of methodological limitations of studies near nuclear installations; (b) identification of directions for future research into the causes and pathogenesis of childhood leukaemia. The workshop gathered 42 participants from different disciplines, extending widely outside of the radiation protection field. Regarding the proximity of nuclear installations, the need for continuous surveillance of childhood leukaemia incidence was highlighted, including a better characterisation of the local population. The creation of collaborative working groups was recommended for consistency in methodologies and the possibility of combining data for future analyses. Regarding the causes of childhood leukaemia, major fields of research were discussed (environmental risk factors, genetics, infections, immunity, stem cells, experimental research). The need for multidisciplinary collaboration in developing research activities was underlined, including the prevalence of potential predisposition markers and investigating further the infectious aetiology hypothesis. Animal studies and genetic/epigenetic approaches appear of great interest. Routes for future research were pointed out. [ABSTRACT FROM AUTHOR]

Background: Sensitivity to ionizing radiation differs between individuals, but there is a limited understanding of the biological mechanisms that account for these variations. One example of such mechanisms are the mutations in the ATM (mutated ataxia telangiectasia) gene, that cause the rare recessively inherited disease Ataxia telangiectasia (AT). Hallmark features include chromosomal instability and increased sensitivity to ionizing radiation (IR)., Objectives: To deepen the molecular understanding of radiosensitivity and to identify potential new markers to predict it, human ATM-mutated and proficient cells were compared on a proteomic level., Design: In this study, we analyzed 3 cell lines from AT patients, with varying radiosensitivity, and 2 cell lines from healthy volunteers, 24 hours and 72 hours post-10 Gy irradiation., Methods: We used label-free mass spectrometry to identify differences in signaling pathways after irradiation in normal and radiosensitive individuals. Cell viability was initially determined by water soluble tetrazolium (WST) assay and DNA damage response was analyzed with 53BP1 repair foci formation along with KRAB-associated protein 1 (KAP1) phosphorylation., Results: Proteomic analysis identified 4028 proteins, which were used in subsequent in silico pathway enrichment analysis to predict affected biological pathways post-IR. In AT cells, networks were heterogeneous at both time points with no common pathway identified. Mitotic cell cycle progress was the most prominent pathway altered after IR in cells from healthy donors. In particular, components of the chromosome passenger complex (INCENP and CDCA8) were significantly downregulated after 72 hours. This could also be verified at the mRNA level., Conclusion: Altogether, the most striking result was that proteins forming the chromosome passenger complex were downregulated after radiation exposure in healthy normosensitive control cells, but not in radiosensitive ATM-deficient cells. Thus, mitosis-associated proteins form an interesting compound to gain insights into the development and prediction of radiosensitivity., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

Objective: Head and neck cancer (HNC) accounts for almost 890,000 new cases per year. Radiotherapy (RT) is used to treat the majority of these patients. A common side-effect of RT is the onset of oral mucositis, which decreases the quality of life and represents the major dose-limiting factor in RT. To understand the origin of oral mucositis, the biological mechanisms post-ionizing radiation (IR) need to be clarified. Such knowledge is valuable to develop new treatment targets for oral mucositis and markers for the early identification of "at-risk" patients., Methods: Primary keratinocytes from healthy volunteers were biopsied, irradiated in vitro (0 and 6 Gy), and subjected to mass spectrometry-based analyses 96 h after irradiation. Web-based tools were used to predict triggered biological pathways. The results were validated in the OKF6 cell culture model. Immunoblotting and mRNA validation was performed and cytokines present in cell culture media post-IR were quantified., Results: Mass spectrometry-based proteomics identified 5879 proteins in primary keratinocytes and 4597 proteins in OKF6 cells. Amongst them, 212 proteins in primary keratinocytes and 169 proteins in OKF6 cells were differentially abundant 96 h after 6 Gy irradiation compared to sham-irradiated controls. In silico pathway enrichment analysis predicted interferon (IFN) response and DNA strand elongation pathways as mostly affected pathways in both cell systems. Immunoblot validations showed a decrease in minichromosome maintenance (MCM) complex proteins 2-7 and an increase in IFN-associated proteins STAT1 and ISG15. In line with affected IFN signalling, mRNA levels of IFNβ and interleukin 6 (IL-6) increased significantly following irradiation and also levels of secreted IL-1β, IL-6, IP-10, and ISG15 were elevated., Conclusion: This study has investigated biological mechanisms in keratinocytes post- in vitro ionizing radiation. A common radiation signature in keratinocytes was identified. The role of IFN response in keratinocytes along with increased levels of pro-inflammatory cytokines and proteins could hint towards a possible mechanism for oral mucositis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Subedi, Huber, Sterr, Dietz, Strasser, Kaestle, Hauck, Duchrow, Aldrian, Monroy Ordonez, Luka, Thomsen, Henke, Gomolka, Rößler, Azimzadeh, Moertl and Hornhardt.)

Purpose: To establish stable in vitro growth of keratinocytes from very small biopsy specimens and successfully apply new test systems to determine their radiosensitivity., Materials and Methods: Oral mucosa biopsies (diameter: 1.7 mm) from 15 subjects were immobilized with custom-made cups onto culture plates. Outgrowing cells were tested for cytokeratin 5/14 and Ki67, expanded, radiated at different doses, and seeded onto circumscribed areas before being allowed to spread centrifugally. In this newly developed spreading assay, cell-covered areas were measured by image analysis. For statistical analysis, a linear mixed regression model was used; additionally, results were correlated to the radiation dose applied. Colony forming efficiency (CFE) was used to validate the results. DNA damage repair was analysed by gammaH2AX and 53BP1 foci quantification using immunofluorescence microscopy 24 h and 96 h after irradiation., Results: Stable keratinocyte growth continued for up to 7 weeks in 14 biopsies. Cells spread reliably from an initial 16.6 mm 2 up to a median of 119.2 mm 2 (range: 54.4-290). Radiated cells spread to only 100.7 mm 2 (2 Gy; range: 55.3-266.7); 73.2 mm 2 (4 Gy; 15-240.4); 47 mm 2 (6 Gy; 2-111.9), and 22.7 mm 2 (8 Gy; 0-80). Similarly, CFE decreased from 0.223 (0 Gy) to 0.0028 (8 Gy). Using an individual donor as a random factor, cell spread correlated with CFE, where radiation dose was the main driver (decrease by 0.50, adjusted for area). Upon irradiation with 6 Gy, radiation-induced DNA damage was increased after 24 h in all samples, and even after 96 h in 5 out of 7 samples, as detected by a higher number of gammaH2AX/53BP1 foci in irradiated cells (mean 3.7 for 24 h; mean 0.6 for 96 h)., Conclusion: In vitro propagation of keratinocytes derived from a small biopsy is feasible. Radiation impairs cellular migration and proliferation, and the newly described spreading assay allows ranking for cellular radioresistance. The keratinocyte model also supports classical functional assays such as clonogenic survival and DNA double strand repair. The clinical relevance awaits upcoming investigations., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Childhood leukemia (CL) is undoubtedly caused by a multifactorial process with genetic as well as environmental factors playing a role. But in spite of several efforts in a variety of scientific fields, the causes of the disease and the interplay of possible risk factors are still poorly understood. To push forward the research on the causes of CL, the German Federal Office for Radiation Protection has been organizing recurring international workshops since 2008 every two to three years. In November 2019 the 6th International Workshop on the Causes of CL was held in Freising and brought together experts from diverse disciplines. The workshop was divided into two main parts focusing on genetic and environmental risk factors, respectively. Two additional special sessions addressed the influence of natural background radiation on the risk of CL and the progress in the development of mouse models used for experimental studies on acute lymphoblastic leukemia, the most common form of leukemia worldwide. The workshop presentations highlighted the role of infections as environmental risk factor for CL, specifically for acute lymphoblastic leukemia. Major support comes from two mouse models, the Pax 5 +/- and Sca1- ETV6-RUNX1 mouse model, one of the major achievements made in the last years. Mice of both predisposed models only develop leukemia when exposed to common infections. These results emphasize the impact of gene-environment-interactions on the development of CL and warrant further investigation of such interactions - especially because genetic predisposition is detected with increasing frequency in CL. This article summarizes the workshop presentations and discusses the results in the context of the international literature., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schmidt, Hornhardt, Erdmann, Sánchez-García, Fischer, Schüz and Ziegelberger.)

Ionizing radiation interacts with the immune system in many ways with a multiplicity that mirrors the complexity of the immune system itself: namely the need to maintain a delicate balance between different compartments, cells and soluble factors that work collectively to protect, maintain, and restore tissue function in the face of severe challenges including radiation damage. The cytotoxic effects of high dose radiation are less relevant after low dose exposure, where subtle quantitative and functional effects predominate that may go unnoticed until late after exposure or after a second challenge reveals or exacerbates the effects. For example, low doses may permanently alter immune fitness and therefore accelerate immune senescence and pave the way for a wide spectrum of possible pathophysiological events, including early-onset of age-related degenerative disorders and cancer. By contrast, the so called low dose radiation therapy displays beneficial, anti-inflammatory and pain relieving properties in chronic inflammatory and degenerative diseases. In this review, epidemiological, clinical and experimental data regarding the effects of low-dose radiation on the homeostasis and functional integrity of immune cells will be discussed, as will be the role of immune-mediated mechanisms in the systemic manifestation of localized exposures such as inflammatory reactions. The central conclusion is that ionizing radiation fundamentally and durably reshapes the immune system. Further, the importance of discovery of immunological pathways for modifying radiation resilience amongst other research directions in this field is implied., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Normal tissue toxicity is a dose-limiting factor in radiation therapy. Therefore, a detailed understanding of the normal tissue response to radiation is necessary to predict the risk of normal tissue toxicity and to development strategies for tissue protection. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of peripheral blood mononuclear cells (PBMC). PBMCs are highly sensitive to ionizing radiation (IR); however, little is known about how IR affects the PBMC response on a systemic level. It was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles (EVs) secreted from PBMCs after radiation exposure to different doses. Therefore, whole blood samples from healthy donors were exposed to X-ray radiation in the clinically relevant doses of 0, 0.1, 2 or 6 Gy and PBMC-secreted EVs were isolated 72 h later. Proteome and miRNome analysis of EVs as well as functional studies were performed. Secreted EVs showed a dose-dependent increase in the number of significantly deregulated proteins and microRNAs. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. Integrated pathway analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated molecules with apoptosis, cell death and survival. Functional studies identified endothelial cells as an efficient EV recipient system, in which irradiation of recipient cells further increased the uptake. Furthermore an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells was detected. In summary, this study demonstrates that IR modifies the communication between PBMCs and endothelial cells. EVs from irradiated PBMC donors were identified as transmitters of protective signals to irradiated endothelial cells. Thus, these data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs as a novel systemic communication pathway between irradiated normal, non-cancer tissues.

Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.

Despite substantial experimental and epidemiological research, there is limited knowledge of the uranium-induce health effects after chronic low-dose exposures in humans. Biological markers can objectively characterize pathological processes or environmental responses to uranium and confounding agents. The integration of such biological markers into a molecular epidemiological study would be a useful approach to improve and refine estimations of uranium-induced health risks. To initiate such a study, Concerted Uranium Research in Europe (CURE) was established, and involves biologists, epidemiologists and dosimetrists. The aims of the biological work package of CURE were: 1. To identify biomarkers and biological specimens relevant to uranium exposure; 2. To define standard operating procedures (SOPs); and 3. To set up a common protocol (logistic, questionnaire, ethical aspects) to perform a large-scale molecular epidemiologic study in uranium-exposed cohorts. An intensive literature review was performed and led to the identification of biomarkers related to: 1. retention organs (lungs, kidneys and bone); 2. other systems/organs with suspected effects (cardiovascular system, central nervous system and lympho-hematopoietic system); 3. target molecules (DNA damage, genomic instability); and 4. high-throughput methods for the identification of new biomarkers. To obtain high-quality biological materials, SOPs were established for the sampling and storage of different biospecimens. A questionnaire was developed to assess potential confounding factors. The proposed strategy can be adapted to other internal exposures and should improve the characterization of the biological and health effects that are relevant for risk assessment.

Gene expression time-course experiments allow to study the dynamics of transcriptomic changes in cells exposed to different stimuli. However, most approaches for the reconstruction of gene association networks (GANs) do not propose prior-selection approaches tailored to time-course transcriptome data. Here, we present a workflow for the identification of GANs from time-course data using prior selection of genes differentially expressed over time identified by natural cubic spline regression modeling (NCSRM). The workflow comprises three major steps: 1) the identification of differentially expressed genes from time-course expression data by employing NCSRM, 2) the use of regularized dynamic partial correlation as implemented in GeneNet to infer GANs from differentially expressed genes and 3) the identification and functional characterization of the key nodes in the reconstructed networks. The approach was applied on a time-resolved transcriptome data set of radiation-perturbed cell culture models of non-tumor cells with normal and increased radiation sensitivity. NCSRM detected significantly more genes than another commonly used method for time-course transcriptome analysis (BETR). While most genes detected with BETR were also detected with NCSRM the false-detection rate of NCSRM was low (3%). The GANs reconstructed from genes detected with NCSRM showed a better overlap with the interactome network Reactome compared to GANs derived from BETR detected genes. After exposure to 1 Gy the normal sensitive cells showed only sparse response compared to cells with increased sensitivity, which exhibited a strong response mainly of genes related to the senescence pathway. After exposure to 10 Gy the response of the normal sensitive cells was mainly associated with senescence and that of cells with increased sensitivity with apoptosis. We discuss these results in a clinical context and underline the impact of senescence-associated pathways in acute radiation response of normal cells. The workflow of this novel approach is implemented in the open-source Bioconductor R-package splineTimeR.

Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in "DNA damage response", "direct p53 effectors" and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols.

The potential health impacts of chronic exposures to uranium, as they occur in occupational settings, are not well characterized. Most epidemiological studies have been limited by small sample sizes, and a lack of harmonization of methods used to quantify radiation doses resulting from uranium exposure. Experimental studies have shown that uranium has biological effects, but their implications for human health are not clear. New studies that would combine the strengths of large, well-designed epidemiological datasets with those of state-of-the-art biological methods would help improve the characterization of the biological and health effects of occupational uranium exposure. The aim of the European Commission concerted action CURE (Concerted Uranium Research in Europe) was to develop protocols for such a future collaborative research project, in which dosimetry, epidemiology and biology would be integrated to better characterize the effects of occupational uranium exposure. These protocols were developed from existing European cohorts of workers exposed to uranium together with expertise in epidemiology, biology and dosimetry of CURE partner institutions. The preparatory work of CURE should allow a large scale collaborative project to be launched, in order to better characterize the effects of uranium exposure and more generally of alpha particles and low doses of ionizing radiation.

It has been suggested that a mechanistic understanding of the cellular responses to low dose and dose rate may be valuable in reducing some of the uncertainties involved in current risk estimates for cancer- and non-cancer-related radiation effects that are inherited in the linear no-threshold hypothesis. In this study, the effects of low-dose radiation on the proteome in both human fibroblasts and stem cells were investigated. Particular emphasis was placed on examining: 1. the dose-response relationships for the differential expression of proteins in the low-dose range (40-140 mGy) of low-linear energy transfer (LET) radiation; and 2. the effect on differential expression of proteins of a priming dose given prior to a challenge dose (adaptive response effects). These studies were performed on cultured human fibroblasts (VH10) and human adipose-derived stem cells (ADSC). The results from the VH10 cell experiments demonstrated that low-doses of low-LET radiation induced unique patterns of differentially expressed proteins for each dose investigated. In addition, a low priming radiation dose significantly changed the protein expression induced by the subsequent challenge exposure. In the ADSC the number of differentially expressed proteins was markedly less compared to VH10 cells, indicating that ADSC differ in their intrinsic response to low doses of radiation. The proteomic results are further discussed in terms of possible pathways influenced by low-dose irradiation.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Cataracts are the major eye disorder and have been associated mainly with mutations in lens-specific genes, but cataracts are also frequently associated with complex syndromes. In a large-scale high-throughput ENU mutagenesis screen we analyzed the offspring of paternally treated C3HeB/FeJ mice for obvious dysmorphologies. We identified a mutant suffering from rough coat and small eyes only in homozygotes; homozygous females turned out to be sterile. The mutation was mapped to chromosome 7 between the markers 116J6.1 and D7Mit294;4 other markers within this interval did not show any recombination among 160 F2-mutants. The critical interval (8.6 Mb) contains 3 candidate genes (Apoe, Six5, Opa3); none of them showed a mutation. Using exome sequencing, we identified a c.2209T>C mutation in the Xpd/Ercc2 gene leading to a Ser737Pro exchange. During embryonic development, the mutant eyes did not show major changes. Postnatal histological analyses demonstrated small cortical vacuoles; later, cortical cataracts developed. Since XPD/ERCC2 is involved in DNA repair, we checked also for the presence of the repair-associated histone γH2AX in the lens. During the time, when primary lens fiber cell nuclei are degraded, γH2AX was strongly expressed in the cell nuclei; later, it demarcates clearly the border of the lens cortex to the organelle-free zone. Moreover, we analyzed also whether seemingly healthy heterozygotes might be less efficient in repair of DNA damage induced by ionizing radiation than wild types. Peripheral lymphocytes irradiated by 1Gy Cs137 showed 6 hrs after irradiation significantly more γH2AX foci in heterozygotes than in wild types. These findings demonstrate the importance of XPD/ERCC2 not only for lens fiber cell differentiation, but also for the sensitivity to ionizing radiation. Based upon these data, we hypothesize that variations in the human XPD/ERCC2 gene might increase the susceptibility for several disorders besides Xeroderma pigmentosum in heterozygotes under particular environmental conditions.

Context: Radio-sensitivity in normal tissue is characterized by heterogeneity throughout the population and the absence of pre-diagnostic biomarkers., Objective: We conducted a proteomic approach to search for radiation characteristic protein regulation., Materials and Methods: Cell lines were 10 Gy irradiated and analysed by 2D-DIGE after 24 h. RESULTS were analysed intra- and inter-individually. The principal component analysis and hierarchical clustering was applied to all datasets., Results: Differences in intra-individual spot abundance prior and post irradiation exactly show the separation of sample classes in two groups: sham-irradiated and irradiated. The inter-individual datasets clustered according to the cell line. The intra-individual differences on protein level after gamma-irradiation are very low, compared with the inter-individual differences among cell lines derived from the same tissue., Conclusion: The application of 2-D DIGE may offer a realistic chance for a better molecular characterization of radio-sensitivity and for the discovery of candidate biomarkers.

Recent findings related to childhood leukaemia incidence near nuclear installations have raised questions which can be answered neither by current knowledge on radiation risk nor by other established risk factors. In 2012, a workshop was organised on this topic with two objectives: (a) review of results and discussion of methodological limitations of studies near nuclear installations; (b) identification of directions for future research into the causes and pathogenesis of childhood leukaemia. The workshop gathered 42 participants from different disciplines, extending widely outside of the radiation protection field. Regarding the proximity of nuclear installations, the need for continuous surveillance of childhood leukaemia incidence was highlighted, including a better characterisation of the local population. The creation of collaborative working groups was recommended for consistency in methodologies and the possibility of combining data for future analyses. Regarding the causes of childhood leukaemia, major fields of research were discussed (environmental risk factors, genetics, infections, immunity, stem cells, experimental research). The need for multidisciplinary collaboration in developing research activities was underlined, including the prevalence of potential predisposition markers and investigating further the infectious aetiology hypothesis. Animal studies and genetic/epigenetic approaches appear of great interest. Routes for future research were pointed out.

Cancer risk and radiation sensitivity are often associated with alterations in DNA repair, cell cycle, or apoptotic pathways. Interindividual variability in mutagen or radiation sensitivity and in cancer susceptibility may also be traced back to polymorphisms of genes affecting e.g. DNA repair capacity. We studied possible associations between 70 polymorphisms of 12 DNA repair genes with basal and initial DNA damage and with repair thereof. We investigated DNA damage induced by ionizing radiation in lymphocytes isolated from 177 young lung cancer patients and 169 cancer-free controls. We also sought replication of our findings in an independent sample of 175 families (in total 798 individuals). DNA damage was assessed by the Olive tail moment (OTM) of the comet assay. DNA repair capacity (DRC) was determined for 10, 30 and, 60min of repair. Genes involved in the single-strand-repair pathway (SSR; like XRCC1 and MSH2) as well as genes involved in the double-strand-repair pathway (DSR; like RAD50, XRCC4, MRE11 and ATM) were found to be associated with DNA damage. The most significant association was observed for marker rs3213334 (p=0.005) of XRCC1 with basal DNA damage (B), in both cases and controls. A clear additive effect on the logarithm of OTM was identified for the marker rs1001581 of the same LD-block (p=0.039): BCC=-1.06 (95%-CI: -1.16 to -0.96), BCT=-1.02 (95%-CI: -1.11 to -0.93) and BTT=-0.85 (95%-CI: -1.01 to -0.68). In both cases and controls, we observed significantly higher DNA basal damage (p=0.007) for carriers of the genotype AA of marker rs2237060 of RAD50 (involved in DSR). However, this could not be replicated in the sample of families (p=0.781). An alteration to DRC after 30min of repair with respect to cases was observed as borderline significant for marker rs611646 of ATM (involved in DSR; p=0.055), but was the most significant finding in the sample of families (p=0.009). Our data indicate that gene variation impacts measurably on DNA damage and repair, suggesting at least a partial contribution to radiation sensitivity and lung cancer susceptibility., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them., Methods: A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients., Results: The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h., Conclusion: So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels.

In this pilot study we compared for the first time the radiation sensitivity of mouse lens epithelial cells (LECs) and mouse lymphocytes. We freshly prepared LECs and lymphocytes and irradiated them with γ-rays ((137)Cs; doses ranging from 0.25 to 2 Gy). DNA damage and repair were evaluated by alkaline comet assay and γH2AX foci assay. Using the comet assay, we observed a dose-dependent increase in DNA damage in both cell types. The faster formation of single- and double-strand breaks in LECs of C57BL/6 mice at doses below 1 Gy needs to be confirmed in other mouse strains. Immunofluorescence for γH2AX foci showed a higher degree of lesions in LECs from C57BL/6J mice compared to those of JF1 mice and to lymphocytes of both strains. Correspondingly, repair of DNA damage proceeded faster in LECs of C57BL/6J mice compared to LECs of JF1 mice and lymphocytes of both strains. It is obvious that the lymphocytes of both strains repaired DNA lesions more slowly than the corresponding LECs. In conclusion, our results demonstrate that LECs of C57Bl/6 mice show a steeper dose-response than lymphocytes in both types of experiments. It shows that both test systems are able to be used also at doses below 0.25 Gy. The observed difference in DNA repair between the LECs from C57BL/6J mice compared to the LECs from JF1 mice and to the lymphocytes of both strains warrants further experiments to identify the underlying molecular mechanisms.

Western blots are used to specifically measure the relative quantities of proteins of interest in complex biological samples. Quantitative measurements can be subject to error due to process inconsistencies such as uneven protein transfer to the membrane. These non-sample-related variations need to be compensated for by an approach known as normalization. Two approaches to data normalization are commonly employed: housekeeping protein (HKP) normalization and total protein normalization (TPN). In this study, we evaluated the performance of Stain-Free technology as a novel TPN tool for Western blotting experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a representative of the HKP normalization strategy. The target protein (TP) used for this study was MCM7, a DNA licensing replication factor, which was shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell lines (LCLs). We studied the regulation of MCM7 with a multiplex Western blotting approach based on fluorescently labeled secondary antibodies and found that Stain-Free technology appears to be more reliable, more robust, and more sensitive to small effects of protein regulation when compared with HKP normalization with GAPDH. Stain-Free technology offers the additional advantages of providing checkpoints throughout the Western blotting process by allowing rapid visualization of gel separation and protein transfer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Radiation sensitivity is assumed to be a cancer susceptibility factor due to impaired DNA damage signalling and repair. Relevant genetic factors may also determine the observed familial aggregation of early onset lung cancer. We investigated the heritability of radiation sensitivity in families of 177 Caucasian cases of early onset lung cancer. In total 798 individuals were characterized for their radiation-induced DNA damage response. DNA damage analysis was performed by alkaline comet assay before and after in vitro irradiation of isolated lymphocytes. The cells were exposed to a dose of 4 Gy and allowed to repair induced DNA-damage up to 60 minutes. The primary outcome parameter Olive Tail Moment was the basis for heritability estimates. Heritability was highest for basal damage (without irradiation) 70% (95%-CI: 51%-88%) and initial damage (directly after irradiation) 65% (95%-CI: 47%-83%) and decreased to 20%-48% for the residual damage after different repair times. Hence our study supports the hypothesis that genomic instability represented by the basal DNA damage as well as radiation induced and repaired damage is highly heritable. Genes influencing genome instability and DNA repair are therefore of major interest for the etiology of lung cancer in the young. The comet assay represents a proper tool to investigate heritability of the radiation sensitive phenotype. Our results are in good agreement with other mutagen sensitivity assays.

The International Agency for Research on Cancer (IARC) has classified high as well as low-frequency fields as "possibly carcinogenic to humans" (Group 2B). For high frequency fields the recent assessment is based mainly on weak positive associations described in some epidemiological studies between glioma and acoustic neuroma and the use of mobile and other wireless phones. Also for lowfrequency fields the evidence is based on epidemiological findings revealing a statistic association between childhood leukemia (CL) and low-level magnetic fields. The basic findings are already 10 years old. They have since been supported by further epidemiological studies. However, the knowledge on the main/crucial question of causality has not improved. This fact and in addition the small, but statistically significant increased incidence of CL in the surrounding of German nuclear power plants have motivated the German Office for Radiation Protection (BfS) to work toward a better understanding of the main causes of CL. A long-term strategic research agenda has been developed which builds on an interdisciplinary, international network and aims at clarifying the aetiology of childhood acute lymphoblastic leukemia., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The COMET assay is recognized as a rapid and sensitive method in quantifying radiation induced DNA damage. We investigated the distorting influence of endogenous, assay-inherent factors onto base (single cell level) and primary outcome measures (experimental/slide level), such as olive tail moment (OTM) and percentage DNA in the tail (%tail-DNA). From 2003 to 2008, we performed the assay on lymphocytes isolated from the blood samples of 355 lung cancer patients, 170 controls, and 610 relatives, as well as one single reference individual, repeated 170 times. In total, the data from 10,016 single experiments containing around 1,750,000 cells have been included in this study. This is the first time that the endogenous variability of the COMET assay has been validated systematically on such a huge data set over a 5 year period. Assuming that the reference sample reflects assay specific white noise, we estimated a proportion of 7-95% of the variability of the outcome measures due to assay variation (white noise) depending on parameter, exposure level, and study group. The proportion of white noise was largest for the initial radiation damage. The specific endogenous factors considered attribute to 14.8% of the total variability in the primary outcome measurements of the OTM and 6.9% of the %tail-DNA. OTM turns out to be a sensitive parameter to detect variation, but is also more susceptible to disturbance caused by endogenous factors than %tail-DNA. To reduce the experimental variability in COMET assays, we recommend a highly standardized operation protocol as well as inspecting and/or adjusting the primary outcome measures according to endogenous factors before calculating secondary outcome measures, e.g. DNA repair capacity (DRC) or testing statistical inference. A human reference (HR) sample is also useful to inspect homogeneity in the temporal progression of long lasting investigations, but not for calibrating primary outcome measurements., (Copyright © 2010 Elsevier B.V. All rights reserved.)

An easy, fast and reliable method was developed to screen hundreds of Epstein-Barr virus-transformed cell lines (lymphoblastoid cell lines, LCLs) for radiation sensitivity that were generated from lymphocytes isolated from young lung cancer patients. The WST-1 test explores the metabolic activity of the mitochondria as an indicator for the vital status of cells. Cell proliferation as well as indirect cell death can be quantified by this method on a large scale in microtiter plates. Cell survival was measured at 24- and 48-h post-irradiation with 10 Gy ((137)Cs source) by the WST-1 assay and Trypan blue staining. To set up the experimental screening conditions and to establish a positive and a negative control, an ATM-mutated cell line from a radiation-sensitive ATM patient and an ATM proficient cell line from a healthy brother were compared. An optimal differentiation between the two cell lines was demonstrated for 10 Gy and 24- and 48-h cell growth after irradiation. Upon screening 120 LCLs of young lung cancer patients under these conditions, 5 of them were found to be radiation sensitive to a high degree of statistical significance. The results have been confirmed by a second laboratory by means of Trypan blue testing. The WST-1 test represents an efficient and reliable method by means of screening for radiation-sensitive cell lines.

In the field of radiation protection the combined exposure to radiation and other toxic agents is recognised as an important research area. To elucidate the basic mechanisms of simultaneous exposure, the interaction of the carcinogens and environmental toxicants cadmium and two arsenic compounds, arsenite and arsenic trioxide, in combination with gamma-radiation in human lymphoblastoid cells (TK6) were investigated. Gamma-radiation induced significant genotoxic effects such as micronuclei formation, DNA damage and apoptosis, whereas arsenic and cadmium had no significant effect on these indicators of cellular damage at non-toxic concentrations. However, in combination with gamma-radiation arsenic trioxide induced a more than additive apoptotic rate compared to the sum of the single effects. Here, the level of apoptotic cells was increased, in a dose-dependent way, up to two-fold compared to the irradiated control cells. Arsenite did not induce a significant additive effect at any of the concentrations or radiation doses tested. On the other hand, arsenic trioxide was less effective than arsenite in the induction of DNA protein cross-links. These data indicate that the two arsenic compounds interact through different pathways in the cell. Cadmium sulphate, like arsenite, had no significant effect on apoptosis in combination with gamma-radiation at low concentrations and, at high concentrations, even reduced the radiation-induced apoptosis. An additive effect on micronuclei induction was observed with 1muM cadmium sulphate with an increase of up to 80% compared to the irradiated control cells. Toxic concentrations of cadmium and arsenic trioxide seemed to reduce micronuclei induction. The results presented here indicate that relatively low concentrations of arsenic and cadmium, close to those occuring in nature, may interfere with radiation effects. Differences in action of the two arsenic compounds were identified.

Experiments were designed and performed in order to investigate whether or not the different cellular energy deposition patterns of photon radiation with different energies (29 kV, 220 kV X rays; Co-60, Cs-137-gamma-rays) and alpha-radiation from an Am-241 source differ in DNA damage induction capacity in human cells. For this purpose, the alkaline comet assay (single cell gel electrophoresis) was applied to measure the amount of DNA damage in relation to the dose received. The comet assay data for the parameters '% DNA in the tail' and 'tail moment' for human peripheral lymphocytes did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiations, 220 kV X rays and the gamma rays, whether for the total mean dose range of 0-3 Gy nor in the low-dose range. In contrast, when the 'tail length' data were analysed saturation of the fitted dose response curve appeared for X rays at about 1.5 Gy but was not apparent for gamma rays up to 3 Gy. Preliminary data for alpha exposures of HSC45-M2 cells showed a significant increase in DNA damage only at high doses (>2 Gy Am-241), but the damage at 2 Gy exceeded the damage induced at 2 Gy by Cs-137-gamma-rays by a factor of 2.5. In contrast, other experiments involving different cell systems and DNA damage indicators such as chromosomal aberrations have detected a significant increase in DNA damage at much lower doses, that is at 0.02 Gy for Am-241 and depicte a higher biological effectiveness. These results indicate that differences in biological effects arise through downstream processing of complex DNA damage.

Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, 137Cs gamma rays and 60Co gamma rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F(d) = A/(t + A), where F(d) is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the gamma rays of 137Cs and 60Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150-250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.

Arsenic present in drinking water and mining environments in some areas has been associated with an increased rate of skin and internal cancers. Contrary to the epidemiological evidence in humans, arsenic does not induce cancer in animal models, but is able to enhance the mutagenicity of other agents. In order to achieve a better understanding of the interaction between arsenic and ionising radiation, an investigation was conducted to detect differences at the proteome level of human TK6 lymphoblastoid cells exposed to these agents. Cells were exposed to either a single dose of 1-Gy 137Cs-gamma-rays or to 1 microM arsenite (As(III)) or to both agents in combination. Two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) were employed for the screening and identification of proteins, respectively. It proved possible to identify seven proteins with significantly affected abundance, three of which showed increased levels and the remaining four showed decreased levels under at least one of the exposure conditions. Following arsenite treatment or irradiation, a significant increase compared with that of the control was observed for glutathione (GSH) transferase omega 1 and proteasome subunit beta type 4 precursor. The combined exposure did not result in an induction of the enzymes. The expression of electron-transfer flavoprotein subunit alpha was found to be enhanced under all three-exposure conditions. Ubiquinol-cytochrome C reductase complex core protein I, adenine phosphoribosyl transferase and endoplasmic reticulum protein hERp29 showed decreased levels after irradiation or arsenite treatment, but not after the combined exposure. The level of serine/threonine protein phosphatase 1 alpha decreased with all treatments. The main conclusions are that both arsenite and gamma-radiation influence the levels of several proteins involved in major metabolic and regulatory pathways, either directly or by triggering the defence mechanisms of the cell. The combined effect of both exposures on the level of some essential proteins such as glutathione transferase, proteasome or serine/threonine phosphatase may contribute to the co-carcinogenic effect of arsenic.

Various benzylic alcohols are metabolically activated to electrophilic, potentially mutagenic and carcinogenic sulphuric acid esters. The involved sulphotransferases are not expressed in the cell lines in culture which are commonly used for mutagenicity testing. The liver of adult female rats is very efficient in the bioactivation of 1-hydroxymethylpyrene. The major enzyme involved was purified and identified as hydroxysteroid sulphotransferase a. Its cDNA was stably expressed in Chinese hamster V79 cells, which are particularly suited for the quantitative detection of various types of mutations and other genotoxic and cytotoxic effects. The mRNA, protein and enzyme activity levels in the constructed cell lines (V79rSTa-1 and V79rSTa-2) were measured, and the cells were also used in mutagenicity and cytotoxicity investigations with benzylic alcohols. 1-Hydroxymethylpyrene, 9-hydroxymethylanthracene and 6-hydroxymethylbenzo[a]pyrene showed enhanced cytotoxicity in V79rSTa-1 and V79rSTa-2 cells, as compared with sulphotransferase-deficient control cells. In addition, 1-hydroxymethylpyrene induced sister chromatid exchanges, and 6-hydroxymethylbenzo[a]pyrene induced gene mutations in V79rSTa-1 cells. We intend carrying out more investigations with other chemicals on these cell lines. Their advantages, as compared with systems with external metabolising systems, include the formation of the active metabolites within the target cell, as in ST-proficient cells in vivo, eliminating the problems which may result from restricted intercellular transport of reactive and ionized sulphuric acid conjugates. Furthermore, cells expressing other sulphotransferases, including human enzymes, may be constructed and used for comparative investigations.

Various environmental chemicals are metabolised to chemically reactive sulfuric acid esters, which may covalently bind to cellular macromolecules and induce mutations and tumours. This activation pathway is usually not taken into account in external xenobiotic-metabolising systems used in short-term tests. We therefore analysed the abilities of cytosols from mammalian cell lines to activate benzylic alcohols (1-hydroxymethylpyrene and 9-hydroxymethylanthracene) to mutagens detectable in Salmonella typhimurium TA98. No activation was observed in cell lines which are commonly used in mutagenicity and cell transformation assays, and only low activities were found in epithelial cell lines in culture. We have therefore constructed Chinese hamster V79-derived cell lines which stably express a heterologous sulfotransferase, rat hydroxysteroid sulfotransferase a. Cytosol of these cells effectively activated 1-hydroxymethylpyrene and 9-hydroxymethylanthracene to mutagens detected in S. typhimurium. The hepatocarcinogen 6-hydroxymethylbenzo[a]pyrene induced gene mutations in sulfotransferase-expressing V79-derived cells, whereas it elicited only marginal effects in sulfotransferase-deficient control cells. The new cell lines may allow the detection of novel classes of mutagens, since some externally generated reactive sulfuric acid esters may not readily penetrate target cells due to their short life span and their ionization.

n Chang liver cells we studied the influence of polychlorinated biphenyls (PCB) on the expression of different protooncogenes. In cells incubated with medium supplemented with PCB we observed an early effect after 3 h on c-erbA and c-erbB RNA level. This reduction of RNA was due to a delayed transcriptional activation of the genes. The PCB congener 3,3',4,4',5-pentachlorobiphenyl (5-CB) in a 1,000-fold lower concentration influenced protooncogene expression in the same manner. In contrast c-raf RNA level increased transiently after 24 h. The fact that persistent chemicals like PCB interfere with protooncogene expression is particularly interesting in view of their tumor promoting activity.

Two ethylnitrosourea-induced heterozygous mouse mutants with approximately 58 and 50% of wild-type lactate dehydrogenase (LDH) activity and a gamma-ray-induced heterozygous mutant with 50% of wild-type LDH activity in blood, liver and spleen (expressing predominantly the Ldh-1 gene) were recovered in mutagenicity experiments following spermatogonial treatment. Physiological and genetic studies revealed no indications for differences in fertility as well as hematological or other physiological traits between heterozygotes of each mutant line and wild types. This suggests that neither the mutations in the heterozygous state per se nor the resulting approximate 42 to 50% LDH deficiency affect metabolism and fitness. Physicochemical and immunological studies clearly demonstrated that the two mutations with 50% deficiency in heterozygotes result from null alleles of the Ldh-1 structural locus, generating neither enzyme activity nor immunological cross-reacting material. In contrast, the heterozygous mutant with approximately 58% of normal blood LDH activity was shown to be due to a Ldh-1 allele creating protein subunits, which in random assortment with wild-type subunits in vivo exhibit a reduced specific activity and further alterations of kinetic and physicochemical characteristics. All the mutations in the homozygous state were found to be lethal at an early postimplantation stage of embryonic development, probably due to a block of glycolysis with the corresponding loss of the main source of metabolic energy during this ontogenetic stage. The distinct physiological consequences of the total absence of a functioning LDH-A subunit in mice and humans are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The expression of 10 protooncogenes was studied in control rat liver and at various times after exposure to polychlorinated biphenyls (PCB), a known tumour promoter. 2. The expression of protooncogenes in liver is more pronounced in those rats treated with PCB beginning at weaning ('weanlings') than in adult rats. 3. The RNA levels of c-Ha-ras, c-raf, c-yes, c-erbA and c-erbB are elevated after PCB feeding. 4. Nuclear run-on transcription analysis revealed that the altered expression of the protooncogenes is transcriptionally regulated. 5. In one group the prompt rise of the protooncogene transcription rate is followed by a decline (c-Ha-ras, c-raf c-yes). In a second group a further increase in transcription at later feeding times (c-erbA, c-erbB) was observed. 6. A correlation between the altered expression of these protooncogenes and the action of PCB as a tumour promotor remains to be determined.

By crossed immunoelectrophoresis with antibodies against the NAD-linked hydrogenase the presence of three hydrogenase protein species was demonstrated in crude extracts of Alcaligenes eutrophus H16. Protein 1 (antigen 1) exhibited NAD-reducing activity and was shown to be identical with the native heterotetrameric enzyme. Protein 2 (antigen 2) was catalytically inactive in the antibody-precipitated form and corresponded to the beta subunit (56 kDa) of the holoenzyme. Protein 3 (antigen 3) was serologically distinct from antigen 2 and catalyzed NADH-oxidizing (diaphorase) activity, suggesting that it either consists of the alpha peptide or of the alpha and gamma subunits of the diaphorase dimer. Tandem immunoelectrophoresis revealed that antigen 2 was the predominant protein species in cells cultivated under nickel deficiency. Low concentrations of the diaphorase-active antigen 3 were also detected under these conditions. Extracts from mutants defective in the catalytic activity of NAD-reducing hydrogenase still contained the four polypeptides. This was shown by immunodiffusion and immunoblotting with antibodies raised against the individual subunits. However, as observed with nickel-deficient cells, no complete tetrameric protein could be identified, and the dominant subunit species (70-80%) was the beta peptide.

The cytoplasmic, NAD-linked hydrogenase of Alcaligenes eutrophus H16 consists of four non-identical subunits. From the mutant strain HF14, defective in this enzyme, a protein was isolated that reacted with specific antibodies raised against the wild-type hydrogenase; the reaction type was of partial identity. The same protein was also tested with specific antibodies raised against each of the four denatured subunits of the wild-type hydrogenase and was found to be completely identical with the second largest subunit; it reacted weakly with the antibody against the largest subunit and not at all with the antibody against the small subunits. In SDS-polyacrylamide gel electrophoresis the protein of the mutant migrated as a single polypeptide and corresponded to the second largest subunit of soluble hydrogenase with Mr = 56,000. The mutant enzyme strongly differed from the wild-type hydrogenase in its binding behaviour to chromatographic gels. It had pronounced hydrophobic properties and bound strongly to phenyl-Sepharose; it had high affinity to triazin dye gels. Enzymatically the polypeptide was totally inactive with NAD as electron acceptor, but showed weak residual activities with methylene blue, ferricyanide and cytochrome c. The protein also contained nickel; however, because of the instability of this polypeptide the amount varied between 0.2-1.4 nickel per enzyme molecule. As shown by ESR studies, the iron is organized in a [4Fe-4S] cluster but is partially present also in the 3Fe-form. No nickel signal could be detected. The role of the polypeptide subunit for hydrogen activation in the intact hydrogenase is discussed.