Hypoxia Modulates Radiosensitivity and Response to Different Radiation Qualities in A549 Non-Small Cell Lung Cancer (NSCLC) Cells.

Hypoxia-induced radioresistance reduces the efficacy of radiotherapy for solid malignancies, including non-small cell lung cancer (NSCLC). Cellular hypoxia can confer radioresistance through cellular and tumor micro-environment adaptations. Until recently, studies evaluating radioresistance secondary to hypoxia were designed to maintain cellular hypoxia only before and during irradiation, while any handling of post-irradiated cells was carried out in standard oxic conditions due to the unavailability of hypoxia workstations. This limited the possibility of simulating in vivo or clinical conditions in vitro. The presence of molecular oxygen is more important for the radiotoxicity of low-linear energy transfer (LET) radiation (e.g., X-rays) than that of high-LET carbon (12C) ions. The mechanisms responsible for 12C ions' potential to overcome hypoxia-induced radioresistance are currently not fully understood. Therefore, the radioresistance of hypoxic A549 NSCLC cells following exposure to X-rays or 12C ions was investigated along with cell cycle progression and gene expression by maintaining hypoxia before, during and after irradiation. A549 cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h and then irradiated with X-rays (200 kV) or 12C ions (35 MeV/n, LET ~75 keV/µm). Cell survival was evaluated using colony-forming ability (CFA) assays immediately or 24 h after irradiation (late plating). DNA double-strand breaks (DSBs) were analyzed using γH2AX immunofluorescence microscopy. Cell cycle progression was determined by flow cytometry of 4',6-diamidino-2-phenylindole-stained cells. The global transcription profile post-irradiation was evaluated by RNA sequencing. When hypoxia was maintained before, during and after irradiation, hypoxia-induced radioresistance was observed only in late plating CFA experiments. The killing efficiency of 12C ions was much higher than that of X-rays. Cell survival under hypoxia was affected more strongly by the timepoint of plating in the case of X-rays compared to 12C ions. Cell cycle arrest following irradiation under hypoxia was less pronounced but more prolonged. DSB induction and resolution following irradiation were not significantly different under normoxia and hypoxia. Gene expression response to irradiation primarily comprised cell cycle regulation for both radiation qualities and oxygen conditions. Several PI3K target genes involved in cell migration and cell motility were differentially upregulated in hypoxic cells. Hypoxia-induced radioresistance may be linked to altered cell cycle response to irradiation and PI3K-mediated changes in cell motility and migration in A549 cells rather than less DNA damage or faster repair.

Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies.

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson's disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5Y.

The Use of ProteoTuner Technology to Study Nuclear Factor κB Activation by Heavy Ions.

Nuclear factor κB (NF-κB) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-κB binding sites (HEK-pNFκB-DD-tdTomato-C8). Using this reporter cell line, NF-κB activation after exposure to different energetic heavy ions (16O, 95 MeV/n, linear energy transfer-LET 51 keV/µm; 12C, 95 MeV/n, LET 73 keV/µm; 36Ar, 95 MeV/n, LET 272 keV/µm) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-κB-dependent DD-tdTomato expression. Approximately 44 hits of 16O ions and ≈45 hits of 12C ions per cell nucleus were required to double the NF-κB-dependent DD-tdTomato expression, whereas only ≈3 hits of 36Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of 36Ar ions doubled NF-κB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-κB activation detection by a factor of three, allowing the detection of single particle hits' effects.

Radiation Response of Murine Embryonic Stem Cells.

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies.

The Role of the Nuclear Factor κB Pathway in the Cellular Response to Low and High Linear Energy Transfer Radiation.

Astronauts are exposed to considerable doses of space radiation during long-term space missions. As complete shielding of the highly energetic particles is impracticable, the cellular response to space-relevant radiation qualities has to be understood in order to develop countermeasures and to reduce radiation risk uncertainties. The transcription factor Nuclear Factor κB (NF-κB) plays a fundamental role in the immune response and in the pathogenesis of many diseases. We have previously shown that heavy ions with a linear energy transfer (LET) of 100⁻300 keV/µm have a nine times higher potential to activate NF-κB compared to low-LET X-rays. Here, chemical inhibitor studies using human embryonic kidney cells (HEK) showed that the DNA damage sensor Ataxia telangiectasia mutated (ATM) and the proteasome were essential for NF-κB activation in response to X-rays and heavy ions. NF-κB's role in cellular radiation response was determined by stable knock-down of the NF-κB subunit RelA. Transfection of a RelA short-hairpin RNA plasmid resulted in higher sensitivity towards X-rays, but not towards heavy ions. Reverse Transcriptase real-time quantitative PCR (RT-qPCR) showed that after exposure to X-rays and heavy ions, NF-κB predominantly upregulates genes involved in intercellular communication processes. This process is strictly NF-κB dependent as the response is completely absent in RelA knock-down cells. NF-κB's role in the cellular radiation response depends on the radiation quality.

Linear Energy Transfer Modulates Radiation-Induced NF-kappa B Activation and Expression of its Downstream Target Genes.

Nuclear factor kappaB (NF-κB) is a central transcription factor in the immune system and modulates cell survival in response to radiotherapy. Activation of NF-κB was shown to be an early step in the cellular response to ultraviolet A (UVA) and ionizing radiation exposure in human cells. NF-κB activation by the genotoxic stress-dependent sub-pathway after exposure to different radiation qualities had been evaluated to a very limited extent. In addition, the resulting gene expression profile, which shapes the cellular and tissue response, is unknown. Therefore, in this study the activation of NF-κB after exposure to low- and high-linear energy transfer (LET) radiation and the expression of its target genes were analyzed in human embryonic kidney (HEK) cells. The activation of NF-κB via canonical and genotoxic stress-induced pathways was visualized by the cell line HEK-pNF-κB-d2EGFP/Neo L2 carrying the destabilized enhanced green fluorescent protein (d2EGFP) as reporter. The NF-κB-dependent d2EGFP expression after irradiation with X rays and heavy ions was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after irradiation with X rays (significant NF-κB activation for doses >4 Gy) and heavy ions (significant NF-κB activation at doses as low as 1 Gy), it was expected that radiation quality (LET) played an important role in the cellular radiation response. In addition, the relative biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival were compared for heavy ions having a broad LET range (∼0.3-9,674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real-time reverse transcriptase quantitative PCR (RT-qPCR). The maximal RBE for NF-κB activation and cell killing occurred at an LET value of 80 and 175 keV/µm, respectively. There was a dose-dependent increase in expression of NF-κB target genes NF-κB1A and CXCL8. A qPCR array of 84 NF-κB target genes revealed that TNF and a set of CXCL genes (CXCL1, CXCL2, CXCL8, CXCL10), CCL2, VCAM1, CD83, NF-κB1, NF-κB2 and NF-κBIA were strongly upregulated after exposure to X rays and neon ions (LET 92 keV/µm). After heavy-ion irradiations, it was noted that the expression of NF-κB target genes such as chemokines and CD83 was highest at an LET value that coincided with the LET resulting in maximal NF-κB activation, whereas expression of the NF-κB inhibitory gene NFKBIA was induced transiently by all radiation qualities investigated. Taken together, these findings clearly demonstrate that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ∼50-200 keV/µm. The upregulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, CXCL8/IL-8 and TNF) could be important for cell-cell communication among hit as well as nonhit cells (bystander effect).

An in-vitro approach for water quality determination: activation of NF-κB as marker for cancer-related stress responses induced by anthropogenic pollutants of drinking water.

Epidemiological studies show that there is a link between urban water pollution and increase in human morbidity and mortality. With the increase in number of new substances arising from the chemical, pharmaceutical, and agricultural industries, there is an urgent need to develop biological test systems for fast evaluation of potential risks to humans and the environmental ecosystems. Here, a combined cellular reporter assay based on the cellular survival and the stress-induced activation of the survival-promoting factor nuclear factor κB (NF-κB) and its use for the detection of cytotoxicity and cancer-related stress responses is presented. A total of 14 chemicals that may be found in trace-amounts in ground water levels are applied and tested with the presented assay. The project is embedded within the joint research project TOX-BOX which aims to develop a harmonized testing strategy for risk management of anthropogenic trace substances in potable water. The assay identified carbendazim as a NF-κB-activating agent in mammalian cells.

Molecular Signaling in Response to Charged Particle Exposures and its Importance in Particle Therapy.

Energetic, charged particles elicit an orchestrated DNA damage response (DDR) during their traversal through healthy tissues and tumors. Complex DNA damage formation, after exposure to high linear energy transfer (LET) charged particles, results in DNA repair foci formation, which begins within seconds. More protein modifications occur after high-LET, compared with low-LET, irradiation. Charged-particle exposure activates several transcription factors that are cytoprotective or cytodestructive, or that upregulate cytokine and chemokine expression, and are involved in bystander signaling. Molecular signaling for a survival or death decision in different tumor types and healthy tissues should be studied as prerequisite for shaping sensitizing and protective strategies. Long-term signaling and gene expression changes were found in various tissues of animals exposed to charged particles, and elucidation of their role in chronic and late effects of charged-particle therapy will help to develop effective preventive measures.

Transcription Factors in the Cellular Response to Charged Particle Exposure.

Charged particles, such as carbon ions, bear the promise of a more effective cancer therapy. In human spaceflight, exposure to charged particles represents an important risk factor for chronic and late effects such as cancer. Biological effects elicited by charged particle exposure depend on their characteristics, e.g., on linear energy transfer (LET). For diverse outcomes (cell death, mutation, transformation, and cell-cycle arrest), an LET dependency of the effect size was observed. These outcomes result from activation of a complex network of signaling pathways in the DNA damage response, which result in cell-protective (DNA repair and cell-cycle arrest) or cell-destructive (cell death) reactions. Triggering of these pathways converges among others in the activation of transcription factors, such as p53, nuclear factor κB (NF-κB), activated protein 1 (AP-1), nuclear erythroid-derived 2-related factor 2 (Nrf2), and cAMP responsive element binding protein (CREB). Depending on dose, radiation quality, and tissue, p53 induces apoptosis or cell-cycle arrest. In low LET radiation therapy, p53 mutations are often associated with therapy resistance, while the outcome of carbon ion therapy seems to be independent of the tumor's p53 status. NF-κB is a central transcription factor in the immune system and exhibits pro-survival effects. Both p53 and NF-κB are activated after ionizing radiation exposure in an ataxia telangiectasia mutated (ATM)-dependent manner. The NF-κB activation was shown to strongly depend on charged particles' LET, with a maximal activation in the LET range of 90-300 keV/µm. AP-1 controls proliferation, senescence, differentiation, and apoptosis. Nrf2 can induce cellular antioxidant defense systems, CREB might also be involved in survival responses. The extent of activation of these transcription factors by charged particles and their interaction in the cellular radiation response greatly influences the destiny of the irradiated and also neighboring cells in the bystander effect.

Simulated Microgravity Modulates Differentiation Processes of Embryonic Stem Cells.

BACKGROUND/AIMS: Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of altered gravity on the embryonic development processes we established an in vitro methodology allowing differentiation of mouse embryonic stem cells (mESCs) under simulated microgravity within a fast-rotating clinostat (clinorotation) and capture of microarray-based gene signatures. METHODS: The differentiating mESCs were cultured in a 2D pipette clinostat. The microarray and bioinformatics tools were used to capture genes that are deregulated by simulated microgravity and their impact on developmental biological processes. RESULTS: The data analysis demonstrated that differentiation of mESCs in pipettes for 3 days resultet to early germ layer differentiation and then to the different somatic cell types after further 7 days of differentiation in the Petri dishes. Clinorotation influences differentiation as well as non-differentiation related biological processes like cytoskeleton related 19 genes were modulated. Notably, simulated microgravity deregulated genes Cyr61, Thbs1, Parva, Dhrs3, Jun, Tpm1, Fzd2 and Dll1 are involved in heart morphogenesis as an acute response on day 3. If the stem cells were further cultivated under normal gravity conditions (1 g) after clinorotation, the expression of cardiomyocytes specific genes such as Tnnt2, Rbp4, Tnni1, Csrp3, Nppb and Mybpc3 on day 10 was inhibited. This correlated well with a decreasing beating activity of the 10-days old embryoid bodies (EBs). Finally, we captured Gadd45g, Jun, Thbs1, Cyr61and Dll1 genes whose expressions were modulated by simulated microgravity and by real microgravity in various reported studies. Simulated microgravity also deregulated genes belonging to the MAP kinase and focal dhesion signal transduction pathways. CONCLUSION: One of the most prominent biological processes affected by simulated microgravity was the process of cardiomyogenesis. The most significant simulated microgravity-affected genes, signal transduction pathways, and biological processes which are relevant for mESCs differentiation have been identified and discussed below.

Space experiment "Cellular Responses to Radiation in Space (CellRad)": Hardware and biological system tests.

One factor contributing to the high uncertainty in radiation risk assessment for long-term space missions is the insufficient knowledge about possible interactions of radiation with other spaceflight environmental factors. Such factors, e.g. microgravity, have to be considered as possibly additive or even synergistic factors in cancerogenesis. Regarding the effects of microgravity on signal transduction, it cannot be excluded that microgravity alters the cellular response to cosmic radiation, which comprises a complex network of signaling pathways. The purpose of the experiment "Cellular Responses to Radiation in Space" (CellRad, formerly CERASP) is to study the effects of combined exposure to microgravity, radiation and general space flight conditions on mammalian cells, in particular Human Embryonic Kidney (HEK) cells that are stably transfected with different plasmids allowing monitoring of proliferation and the Nuclear Factor κB (NF-κB) pathway by means of fluorescent proteins. The cells will be seeded on ground in multiwell plate units (MPUs), transported to the ISS, and irradiated by an artificial radiation source after an adaptation period at 0 × g and 1 × g. After different incubation periods, the cells will be fixed by pumping a formaldehyde solution into the MPUs. Ground control samples will be treated in the same way. For implementation of CellRad in the Biolab on the International Space Station (ISS), tests of the hardware and the biological systems were performed. The sequence of different steps in MPU fabrication (cutting, drilling, cleaning, growth surface coating, and sterilization) was optimized in order to reach full biocompatibility. Different coatings of the foil used as growth surface revealed that coating with 0.1 mg/ml poly-D-lysine supports cell attachment better than collagen type I. The tests of prototype hardware (Science Model) proved its full functionality for automated medium change, irradiation and fixation of cells. Exposure of HEK cells to the ß-rays emitted by the radiation source dose-dependently decreased cell growth and increased NF-κB activation. The signal of the fluorescent proteins after formaldehyde fixation was stable for at least six months after fixation, allowing storage of the MPUs after fixation for several months before the transport back to Earth and evaluation of the fluorescence intensity. In conclusion, these tests show the feasibility of CellRad on the ISS with the currently available transport mechanisms.

Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology.

The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/µm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.

Imaging of nuclear factor κB activation induced by ionizing radiation in human embryonic kidney (HEK) cells.

Ionizing radiation modulates several signaling pathways resulting in transcription factor activation. Nuclear factor kappa B (NF-κB) is one of the most important transcription factors that respond to changes in the environment of a mammalian cell. NF-κB plays a key role not only in inflammation and immune regulation but also in cellular radiation response. In response to DNA damage, NF-κB might inhibit apoptosis and promote carcinogenesis. Our previous studies showed that ionizing radiation is very effective in inducing biological damages. Therefore, it is important to understand the radiation-induced NF-κB signaling cascade. The current study aims to improve existing mammalian cell-based reporter assays for NF-κB activation by the use of DD-tdTomato which is a destabilized variant of red fluorescent protein tdTomato. It is demonstrated that exposure of recombinant human embryonic kidney cells (HEK/293 transfected with a reporter constructs containing NF-κB binding sites in its promoter) to ionizing radiation induces NF-κB-dependent DD-tdTomato expression. Using this reporter assays, NF-κB signaling in mammalian cells was monitored by flow cytometry and fluorescence microscopy. Activation of NF-κB by the canonical pathway was found to be quicker than by the genotoxin- and stress-induced pathway. X-rays activate NF-κB in HEK cells in a dose-dependent manner, and the extent of NF-κB activation is higher as compared to camptothecin.

Cell cycle delay in murine pre-osteoblasts is more pronounced after exposure to high-LET compared to low-LET radiation.

Space radiation contains a complex mixture of particles comprised primarily of protons and high-energy heavy ions. Radiation risk is considered one of the major health risks for astronauts who embark on both orbital and interplanetary space missions. Ionizing radiation dose-dependently kills cells, damages genetic material, and disturbs cell differentiation and function. The immediate response to ionizing radiation-induced DNA damage is stimulation of DNA repair machinery and activation of cell cycle regulatory checkpoints. To date, little is known about cell cycle regulation after exposure to space-relevant radiation, especially regarding bone-forming osteoblasts. Here, we assessed cell cycle regulation in the osteoblastic cell line OCT-1 after exposure to various types of space-relevant radiation. The relative biological effectiveness (RBE) of ionizing radiation was investigated regarding the biological endpoint of cellular survival ability. Cell cycle progression was examined following radiation exposure resulting in different RBE values calculated for a cellular survival level of 1 %. Our findings indicate that radiation with a linear energy transfer (LET) of 150 keV/µm was most effective in inducing reproductive cell killing by causing cell cycle arrest. Expression analyses indicated that cells exposed to ionizing radiation exhibited significantly up-regulated p21(CDKN1A) gene expression. In conclusion, our findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression.

Activation of the nuclear factor κB pathway by heavy ion beams of different linear energy transfer.

PURPOSE: Risk assessment of radiation exposure during long-term space missions requires the knowledge of the relative biological effectiveness (RBE) of space radiation components. Few data on gene transcription activation by different heavy ions are available, suggesting a dependence on linear energy transfer. The transcription factor Nuclear Factor κB (NF-κB) can be involved in cancerogenesis. Therefore, NF-κB activation by accelerated heavy ions of different linear energy transfer (LET) was correlated to survival. MATERIALS AND METHODS: NF-κB-dependent gene induction after exposure to heavy ions was detected in stably transfected human embryonic kidney 293 cells (HEK-pNF-κB-d2EGFP/Neo cells carrying a neomycin resistance), using the destabilized Enhanced Green Fluorescent Protein (d2EGFP) as reporter. RESULTS: Argon (LET 272 keV/µm) and neon ions (LET 91 keV/µm) had the highest potential to activate NF-κB, resulting in a RBE of 8.9 in comparison to 150 kV X-rays. The RBE for survival also reached its maximum in this LET range, with a maximal value of 2. CONCLUSIONS: NF-κB might be involved in modulating survival responses of cells hit by heavy ions in the LET range of 91-272 keV/µm and could therefore become a factor to be considered for risk assessment of radiation exposure during space travel.

Carbon-ion-induced activation of the NF-κB pathway.

Carbon-ion cancer therapy offers several physical and radiobiological advantages over conventional photon cancer therapy. The molecular mechanisms that determine cellular outcome, including the activation of transcription factors and the alteration of gene expression profiles, after carbon-ion exposure are still under investigation. We have previously shown that argon ions (LET 272 keV/µm) had a much higher potential to activate the transcription factor nuclear factor κB (NF-κB) than X rays. NF-κB is involved in the regulation of cellular survival, mostly by antiapoptosis and cell cycle-regulating target genes, which are important in the resistance of cancer cells to radiotherapy. Therefore, activation of the NF-κB pathway by accelerated carbon ions (LET 33 and 73 keV/µm) was examined. For comparison, cells were exposed to 150 kV X rays and to accelerated carbon ions. NF-κB-dependent gene induction after exposure was detected in stably transfected human 293 reporter cells. Carbon ions and X rays had a comparable potential to activate NF-κB in human cells, indicating a comparable usefulness of pharmacological NF-κB inhibition during photon and carbon-ion radiotherapy.

X-irradiation-induced cell cycle delay and DNA double-strand breaks in the murine osteoblastic cell line OCT-1.

Radiation response of bone cells, especially the bone-forming osteoblasts, is an important issue for radiotherapy in young age. A radiation-induced cell cycle arrest may enhance or accelerate osteoblastic differentiation. To analyze radiation response of osteoblastic cells, the correlation between DNA double-strand break induction (DSB), cell cycle alterations and gene expression modifications after X-irradiation was investigated in the osteoblast-like cell line OCT-1. As marker of the cellular response to DSB, the temporal appearance of gamma-H2AX foci after X-irradiation was visualized. Gene expression profiles of the key cell cycle regulatory protein p21 (CDKN1A), and the most abundant growth factor in human bone, transforming growth factor beta 1 (TGF-beta1) were recorded using quantitative real-time reverse transcription PCR (qRT-PCR). The distribution of cells in the cell cycle phases G1, S and G2 was determined by propidium iodide (PI) staining and flow cytometry. Initial studies show a strong dose dependency in the number of gamma-H2AX foci shortly after X-irradiation. Exposure to 1 Gy yields approximately 36 small foci in OCT-1 cells after 30 min that became larger after 1 h of incubation; after 24 h most of the foci had disappeared. X-rays provoked a dose-dependent arrest in G2 phase of the cell cycle, accompanied by a dose-dependent gene expression regulation for p21 and TGF-beta1. As TGF-beta1 is known to affect osteoblast differentiation, matrix formation and mineralization, modulation of its expression could influence the expression of the main osteogenic transcription factor Runx2 (Cbfa1) and other osteoblast differentiation markers.

Cytotoxicity and genotoxicity reporter systems based on the use of mammalian cells.

With the dramatic increase in the number of new agents arising from the chemical, pharmaceutical, and agricultural industries, there is an urgent need to develop assays for rapid evaluation of potential risks to man and environment. The panel of conventional tests used for cytotoxicity and genotoxicity and the strategies to progress from small scale assays to high content screening in toxicology are discussed. The properties of components necessary as sensors and reporters for new reporter assays, and the application of genetic strategies to design assays are reviewed. The concept of cellular reporters is based on the use of promoters of chemical stress-regulated genes ligated to a suitable luminescent or fluorescent reporter gene. Current reporter assays designed from constructs transferred into suitable cell lines are presented.

Toxicity of ethylene combustion condensates is directly proportional to their carbon content.

Numerous epidemiological studies have shown a strong link between air pollution and human morbidity and mortality. Combustion sources are most significant contributors to the urban air pollution. So far, toxicological research has focused predominantly on combustion generated particulate matter, thereby neglecting chemical complexity of combustion exhausts. The aim of this study was to assess toxic potential of ethylene combustion condensates, containing both particulate and gaseous combustion by-products, by means of a recombinant bacterial assay called the SWITCH (Salmonella Weighting of Induced Toxicity (Genotoxicity) and Cytotoxicity for Human Health) test. Thereby, the suitability of total organic carbon (TOC) as a parameter for toxicity assessment was also investigated. Ethylene was combusted in a low-pressure burner under controlled laboratory conditions by only varying the carbon/oxygen ratio (C/O=0.63-0.93). Ethylene combustion condensates were generated by drawing 10 l of combustion exhaust at constant flow rate (0.4 l/min) and collecting it in condensated form in glass bottles cooled by liquid nitrogen. Genotoxic and cytotoxic potency of combustion condensates was analyzed with the SWITCH test, based on sequential measurements of luminescence, absorbance and fluorescence outputs of treated bacterial cultures. Our results show correlation between TOC content of combustion condensates and their genotoxicity/cytotoxicity. Moreover, combustion condensates of same TOC concentration exert the same toxic effect regardless of the used C/O ratios during their generation. Our results revealed that toxicologically relevant component(s) of the ethylene combustion exhausts is/are being produced during highly, mildly and non-sooting combustion conditions, only in different proportions. Thereby, total organic carbon proved to be a suitable parameter for the assessment of the toxicity of combustion condensates.

The combined bacterial Lux-Fluoro test for the detection and quantification of genotoxic and cytotoxic agents in surface water: results from the "Technical Workshop on Genotoxicity Biosensing".

The bioassay Lux-Fluoro test was developed for the rapid detection and quantification of environmental pollutants with genotoxic and/or cytotoxic potential. This bacterial test system uses two different reporter genes whose gene products and their reactions, respectively, can be measured easily and simultaneously by optical methods. Genotoxicity is measured by the increase of bioluminescence in genetically modified bacteria which carry a plasmid with a complete lux operon for the enzyme luciferase from the marine photobacterium P. leiognathi under the control of a DNA-damage dependent so-called SOS promoter. If the deoxyribonucleic acid in these bacteria is damaged by a genotoxic chemical, the SOS promoter is turned on and the lux operon is expressed. The newly synthesized luciferase reacts immediately with its substrate thereby producing bioluminescence in a damage-proportional manner. In the second part of the system, genetically modified bacteria carry the gene for the green fluorescent protein (gfp) from the jellyfish Aequora victoria downstream from a constitutively expressed promoter. These bacteria are fluorescent under common growth conditions. If their cellular metabolism is disturbed by the action of cytotoxic chemicals, the fluorescence decreases in a dose-proportional manner. The combined Lux-Fluoro test is shown to be well suited for the biological assessment of the geno- and cytotoxicity of a series of model agents and environmental samples at the Technical Workshop on Genotoxicity Biosensing (TECHNOTOX).