DNA DAMAGE RESPONSE OF EX-VIVO PORCINE EYE LENSES IN ORGAN-CULTURE AND IN-VITRO CULTURED LENS EPITHELIAL CELLS TO IONIZING RADIATION.

Astronauts on space missions, especially on long-term missions to Moon or Mars have a higher risk for the expression of radiation late effects such as cancer or sub-capsular cortical eye lens opacities. This is due to higher dose and different patterns of cellular energy deposition from high-linear-energy-transfer (LET) components of galactic cosmic radiation in space than that of terrestrial low-LET radiation on Earth. The eye lens is considered to be a radiation sensitive organ with radiation induced cataract to occur with a threshold absorbed dose of 0.5 Gy of sparsely ionizing radiation. For terrestrial occupational radiation lens exposure limit is set to yearly 20 mSv by the International Commission on Radiological Protection (1). Doses perceived by astronauts are much higher: in average 150 mSv per year on the International Space Station (ISS) and 1.2 to 1.4 mSv per day on Apollo and Skylab missions (2)

Validation of biological recognition elements for signal transduction as first step in the development of whole cell biosensors

Choosing the proper combination of receptor element, cell type and measurable signal requires major consideration for developing cell-based biosensors. In order to use physiologically relevant cellular responses towards (geno)toxic conditions, information on the mechanism of action and of the expected outcome of exposure needs to be considered

UNRAVELING ASTROCYTE BEHAVIOUR IN THE SPACE BRAIN: RADIATION RESPONSE OF PRIMARY ASTROCYTES

Exposure to ionizing radiation as part of space radiation, is a major limiting factor for crewed space exploration. Astronauts will encounter different types of space radiation, which may cause cognitive damage causing detrimental effects on learning and attention, elevated anxiety and depression. Due to its limited regenerative potential, especially the central nervous system (CNS) is very vulnerable towards radiation-induced damage. Astrocytes, the most abundant glial cells of the CNS, have different crucial functions in the CNS, e.g. maintaining normal brain function. In this work, the response of astrocytes towards low linear energy transfer (LET) X-rays and high-LET carbon ions was compared to unravel possible specific effects of space-relevant high-LET radiation. [...

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

Effects of ionizing radiation on human adipose derived mesenchymal stem cells and their differentiation towards the osteoblastic lineage

Space travel presents many challenges to human health, including radiation exposure and musculoskeletal disuse. In addition, astronauts lose calcium from bones due to the uncoupling of bone formation and bone resorption. Bone forming osteoblasts are derived from undifferentiated MSCs. In this study, the ability of human adipose tissue derived stem cells (ATSC) to differentiate into the osteoblastic lineage was examined after radiation exposure as well as supplementation with osteogenic medium additives. After exposure to ionizing radiation changes in cellular morphology, cell cycle progression, as well as cellular radiosensitivity was characterized. Exposure to ionizing radiation resulted in an accumulation of cells in the G2/M phase of the cell cycle, concerning both cell lines. Alizarin Red S staining as well as quantitative determination of bone cell differentiation was performed by analyzing the hydroxyapatite content of the ECM. The results demonstrated LW24 deposited less calcium compared to SAOS-2. Additionally, gene expression analysis during differentiation process revealed expression of genes that are required for skeletal development, bone mineral metabolism and transcription factors. Detailed investigation of stem cell differentiation after radiation exposure is required to assure health of astronauts in upcoming space missions

Response of mammalian eye lenses to space radiation qualities in vitro and in organ culture

The eye lens is known to be a radiosensitive mammalian organ, and ionizing radiation is considered to be a widely known risk factor inducing lens opacities. During space missions, astronauts are constantly exposed to galactic cosmic radiation, which contains energetic heavy ions of high linear energy transfer (LET). Due to higher dose and different patterns of cellular energy deposition from the high-LET ions, astronauts have higher risk for developing cataract compared to low-LET radiation exposure on earth. Although the exact mechanism of opacification is not known in detail, it is hypothesized that it initiates from the post-irradiation proliferative activity of genetically damaged lens epithelial cells. [...

DNA DAMAGE RESPONSE OF EX-VIVO PORCINE EYE LENSES IN ORGANCULTURE AND IN-VITRO CULTURED LENS EPITHELIAL CELLS TO IONIZING RADIATION

Astronauts on space missions, especially on long-term missions to Moon or Mars have a higher risk for the expression of radiation late effects such as cancer or sub-capsular cortical eye lens opacities. This is due to higher dose and different patterns of cellular energy deposition from high-linear-energy-transfer (LET) components of galactic cosmic radiation in space than that of terrestrial low-LET radiation on Earth. The eye lens is considered to be a radiation sensitive organ with radiation induced cataract to occur with a threshold absorbed dose of 0.5 Gy of sparsely ionizing radiation. For terrestrial occupational radiation lens exposure limit is set to yearly 20 mSv by the International Commission on Radiological Protection (ICRP, Statement on tissue reactions, Ottawa, Canada, 2011). Doses perceived by astronauts are much higher: in average 150 mSv per year on the International Space Station (ISS) and 1.2 to 1.4 mSv per day on Apollo and Skylab missions (Cucinotta FA, Manuel FK, Jones J, Iszard G, Murrey J, Djojonegro B, Wear M. Space radiation and cataracts in astronauts. Radiat Res. 156:460-466, 2001)

RADIATION RESPONSE OF PORCINE LENS EPITHELIAL CELLS AND EYE LENSES IN ORGAN-CULTURE

Astronauts on long-term space missions have a higher risk for the expression of radiation late effects such as cancer or sub-capsular cortical eye lens opacities. This is due to higher dose and different patterns of cellular energy deposition from high-linear-energy-transfer (LET) components of galactic cosmic radiation in space than that of terrestrial low-LET radiation on Earth. The eye lens is a radiation sensitive organ with radiation induced cataract to occur with a threshold absorbed dose of 0.5 Gy (0 - 1 Gy) of sparsely ionizing radiation. Doses perceived by astronauts on the International Space Station (ISS) are in average 150 mSv per year (Cucinotta et al. (2001) Radiat Res. 156:460-466). Radiationinduced lens opacification is assumed to initiate from post irradiation proliferative activity of genetically damaged lens epithelial cells with alterations in cell cycle control, apoptosis, differentiation, and cellular disorganization, or other pathways controlling lens fiber cells’ differentiation. As the porcine eye lens is similar to the human lens in size and anatomy, DNA damage response was investigated in ex-vivo porcine lenses in organ culture, in in-vitro cultivated lens epithelial slabs (ES) and in porcine lens epithelial cells (pLEC). Cell survival of proliferative cells was calculated from colony forming ability (CFA) assay. The phosphorylated form of H2AX (γH2AX) was used as a molecular marker to visualize DNA double strand breaks (DSB) and their repair. Propidium iodide based DNA staining for cellular DNA content marked radiation-induced cell cycle disturbances. In pLEC the cell survival curve of immediate plated cells and after a recovery period of 24 h follow the equation S=1.40xD+ln 1.47 and S=1.59xD+ln 1.79, respectively. DNA DSB are induced in a dose-dependent manner ( 18 DSB/cell/Gy) and repaired during successive recovery ( 5 DSB/cell/Gy residual damage after 24 h). For doses >2 Gy a cell cycle arrest in G2 phase occurred 24 h after X-irradiation and persisted up to 72 h post-irradiation. DNA DSB induction and repair could as well be documented for ES and whole lenses after X-irradiation. In whole lenses, the amount of residual damage (after 24 h and 48 h) was highest in the equatorial zone while in the central epithelial zone DSB repair seemed to proceed with time in a manner comparable to in-vitro cultivated pLEC. Lens organ culture allows cellular metabolism and DNA synthesis in whole lenses. Repair of DNA DSB takes place in the central epithelial layer and is reduced in the equatorial region of cultivated lenses

NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia

International audienceCellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (12C)-LET radiation on NF-κB responsive genes’ upregulation, as well as its target cytokines’ synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h, followed by irradiation with 8 Gy X-rays or 12C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to 12C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells

Response of primary astrocytes to ionizing radiation exposure

Introduction: Exposure to space conditions during crewed long-term exploration missions can cause several health risks for astronauts. Space radiation, isolation and microgravity are major limiting factors. The role of astrocytes in cognitive disturbances by space radiation is unknown. Astrocytes’ response towards low linear energy transfer (LET) X-rays and high-LET carbon (¹²C) and iron (⁵⁶Fe) ions was compared to reveal possible effects of space-relevant high-LET radiation. Methods: Primary murine cortical astrocytes were irradiated with different doses of X-rays, ¹²C and ⁵⁶Fe ions at the heavy ion accelerator GSI. DNA damage and repair (γH2AX, 53BP1), cell proliferation (Ki-67), astrocytes’ reactivity (GFAP) and NF-κB pathway activation (p65) were analyzed by immunofluorescence microscopy. Cell cycle progression was investigated by flow cytometry of DNA content. Gene expression changes after exposure to X-rays were investigated by mRNA-sequencing. RT-qPCR for the genes of interest was performed with X-rays- and heavy-ion-irradiated astrocytes: Cdkn1a, Cdkn2a, Gfap, Tnf, Il1β, Il6 and Tgfβ1. Levels of the pro-inflammatory cytokine IL-6 were determined using ELISA. Results: Astrocytes showed distinct responses towards the three different radiation qualities. Induction of radiation-induced DNA double strand breaks (DSB) and the respective repair was dose-, LET- and time-dependent. Proliferation and cell cycle progression were not affected by radiation qualities examined in this study. Astrocytes expressed IL-6 and GFAP with constitutive NF-κB activity independent of radiation exposure. mRNA sequencing of X-irradiated astrocytes revealed downregulation of 66 genes involved in DNA damage response and repair, mitosis, proliferation and cell cycle regulation. Conclusion: Primary murine astrocytes are DNA repair proficient irrespective of radiation quality. Only minor gene expression changes were observed after X-ray exposure and reactivity was not induced. Co-culture of astrocytes with microglial cells, brain organoids or organotypic brain slice culture experiments might reveal whether astrocytes show a more pronounced radiation response in more complex network architectures in the presence of other neuronal cell types. Acknowledgement: We thank our liaison scientists at GSI, Insa Schröder and Denise Eckart, for their excellent technical assistance in preparation of and during the beamtimes. Ulrich Weber and Thomas Friedrich at GSI are acknowledged for their dedicated and precise irradiation of our samples at GSI. Our thanks also go to the beam operators at GSI for operating the accelerator during our experiments