Genetic risk and anti-inflammatory effects of low doses of different types of ionizing irradiation (GREWIS-project)

Transcription

1 Genetic risk and anti-inflammatory effects of low doses of different types of ionizing irradiation (GREWIS-project) Studies using cell and animal models and samples of radon patients Claudia Fournier M. Löbrich, C. Cardoso, P. Layer, G. Thiel University of Darmstadt U. Gaipl F. Rödel University of Erlangen University of Frankfurt C. Fournier, G. Kraft, S. Ritter, M. Durante GSI

2 Emission of α-particles during Radon decay: densely ionizing radiation Noble gas chemically inert; evaporates from rocks Radioactive decay chain α α α Photons γ-, X-rays Sparsely ionising Dose contribution/ photon very low Homogeneous distribution in the tissue All cells are irradiated Charged particles α-particles, protons, HI Densely ionising major dose contribution Dose deposition/ α-particle/nucleus Gy 0.5 Gy = 20 % of the cells in the tissue are irradiated Enhanced biological efficiency Increased risk for lung cancer at high doses 2

3 Entries of radon in the organism Organs with epithelial tissue 1. Lung 2. Skin 3. Gastrointestinal tract Diffusion/ transport (radon) Fast passage of Rn through the body Only 0.2 % of the Rn atoms decay during the passage Relevant half-life time of the daughter nuclei ~ 50 Min. + Deposition of short lived progeny α-particles: µm ca. 100 µm 3

4 Radon exposure: genetic risk? Epidemiological evidence for an increased Indoor No enhanced exposure risk? Mining risk for lung cancer Wohnräume Therapy Bath, galleries Anti-inflammatory Therapy 3w effects? µsv Bq/m 3 kbq/m msv 3 MBq/m Sv 3 Effective Activity dose/ year Pain releave? WP3: N. Paz, E. Nasonova, S. Ritter Reference dose-response curves for α-exposure (in vitro) α-source (Americium) high number of aberrant cells high number of break points/ cell complex aberrations (A,B: 4 breaks in 3 chromosomes) Healthy donor Patients Bad Steben Bad Gastein Mice bone marrow A A B B 4

5 Project structure Dosimetry and risk assessment Inflammation and associated processes (cell death, pain) functional changes and signal transduction (humoral/ cholinergic) Physical dosimetry penetration depth and diffusion of radon AP 1 Kraft Humoral mechanisms cellular and molecular interactions in blood vessels and bones AP 4 Fournier Cholinergic mechanisms anti-inflammatory reactions AP 6 Layer Biological dosimetry formation of yh2ax foci AP 2 Löbrich Intracellular signal transduction regulation of adhesion molecules role of NFκB AP 5 Cardoso Inhibition of pain ion channel activities AP 7 Thiel Estimation of dose and radiation risk cytogenetic analyses AP 3 Ritter Discontinuous dose-dependency AP 8 Rödel Immunological danger signals and inflammation AP 9 Gaipl mouse Model systems human wild type htnf transgenic rheumatoid arthritic healthy donors patients Gewebe-Schnitte tissue sections (Lunge, (lung, skin, Haut, bones) Knochen) primary Primärzellen cells (e.g. (Blut, mono- blood, Knochenmark and bone co-cultures marrow) u. a.) primary cells (e.g. blood, bone marrow) mono- and co-cultures primary cells (blood) mono- and co-cultures Radiation low and high dose ionizing irradiation (in vitro exposure) photons radon (AP 1 Kraft) α-particles (AP 2 Löbrich) carbon ions (AP 1 Kraft) 5

6 before Modulation of the chronic inflammation after radon exposure: immune status of the patients 9 x radon bath in 3 weeks 3 weeks 12 weeks 18 weeks 30 weeks 30 subtypes of immun and stem cells (surface marker) Markers of inflammation (cytokines, chemokines), cell death 100 patients with musco-skeletal diseases (Bad Steben) Blood samples Medical examination (CVD + pain) Medication Shift of the balance between immune cells with antagonistic functions? WP 9: P. Rühe, B. Frey, U. Gaipl Human primary cells CD4+ Photons (X-ray) + TGF-β 7days T H17 cells unchanged (inflammatory stimulation) T reg cells: increase after irradiation WP4: A. Groo, C. Fournier 6

7 Radon exposure under definied conditions Radon chamber is ready to be used (stability tests, cell growth curves) Irradiation of cells started+ small animals (rodents, start November 2013) Activity: 40 kbq/m 3 (galleries) 600 kbq/m 3 Dose rate ~1,3 mgy/h (will be increased source with higher activity) Adjustable First measurements parameters of (galleries, activity: cell culture) Absorption Temperature of Radon (20-45 C) on 4,8g carbon/ water (under work) γ-spektrum activity Humidity (0-100% rh) CO 2 -concentration (0-30%) Activity Anzahl Number 1.5 e e e e 3 Bi-214 Pb-214 Radon source Air humidification system Kammer Water bath WP1: A. Maier, G. Kraft Carbon filter Thermostat Energie (kev) Energy (kev) 7

8 Noble gas radon: distribution in the organism? Passiv transport Diffusion Respiratory tract (inhalation) Skin (contact) Gastrointestinal tract (ingestion) Active transport Vascular system Veins/ Arteries Organs Physical dosimetry WP1: A. Maier, G. Kraft Alveoli 1. Measurements of activity after exposure of different tissues (under work) Solubility coefficients (ratio of activity in different tissues) Retention time (activity in a tissue over time) 2. Dose estimations: Lung >> Alveolary capillaries > Red bone marrow > Fatty tissue [Nussbaum und Hursh, Science, 1957; Jacobi und Eisfeld, 1980; Harley und Robbins, 1992] Biological dosimetry WP2: M. Steinlage, M. Löbrich 1. In vitro exposure of HeLa cells to α-particules ( 241 Americium) 2. In vivo exposure of C57BL/6 mice to carbon ions (Radon scheduled for Nov. 2013) 8

9 Rheumatoid arthritis: disease of the bones and joints Bones and cartilage target of Radon exposure? Loss of ligament Inflammation of synovium Accumulation of immune cells Erosion of bone and cartilage [Morbus Bechterew Journal 2006 No. 107] Healthy joint Rheumatoid arthritis Changes in differentiation and activity of bone resorbing cells? Osteoblasts Osteoclasts Factors for differentiation (T-cells, synovial fibroblasts) 3D 9

10 Noble gas radon: accumulation in bone tissue? DAPI Immunfluorescence staining of DNA damage markers (CLSM) Adipozyten Erythrozyten Lymphozyten WP2: M. Steinlage, M. Löbrich periosteum compact bone DAPI γh2ax 53BP1 γh2ax/53bp1 focus periosteum 10

11 Low dose exposure influences bone erosion and density WP9: L. Deloch, B. Frey, U. Gaipl Mouse modell for human polyarthritis Improvement of joint function? X-ray (α-particles radon). C57/Bl6 htnf-α tg grip strength Inflammation? H&E staining Bone erosion? TRAP assay Reduction of inflamed areas and bone erosion Increase in bone density (not shown) 11

12 Radiation exposure modifies differentiation of osteoblasts and activation of osteoclasts Human primary cells: MSC/ monocytes WP4: A. Groo, D. Kraft, C. Fournier Inactivation of osteoclasts by irradiation? TRAP pos. > 3 nuclei / F-Actin ring /integrin Resorptive Activity Cell number and differentiation unchanged Release of factors modifying osteoclast activity by osteoblasts? Ca-deposition (Alizarin Red) Accelerated differentiation Release of OPG 12

13 Tanja Huhn Andreas Maier Daniela Kraft (Postdoc) Aljona Groo Nerea Paz Elena Nasonova Monika Steinlage Lisa Deloch Sylvia Ritter Gerhard Kraft Markus Löbrich Christina Cardoso Gerhard Thiel Paul Layer Franz Rödel Benjamin Frey Udo Gaipl..and for your attention! DoReMi Workshop, Budapest, 5 th of November