Cell and Molecular Effects of Low Doses of Radiation

1. Cell and Molecular Effects of Low Doses of Radiation Antone L. Brooks Washington State University Tri-Cities Richland WA, 99352 Health Physics Meeting Portland, Oregon July 8-12, 2007

2. Overview of Presentation • Frame the problem • Low Dose Radiation Research at the Cell and molecular level • Low Dose-Rate Radiation Effects (DDREF) • Data for Standard Setting • Communicating Radiation Risks

3. LNTH Assumption with Dose Low dose x large number of subjects High dose x small number of subjects Energy to system

4. LNTH Linear Hypersensitive response Threshold Adaptive response Radiation Dose-Response Models Data and radiation-induced cancer No data Cancer Frequency Background Cancer Rate Dose

5. 2.0 0.5 1. 0 1.5 A-Bomb Experience LNT High Dose High Dose-Rate 5%/Sv increase in Cancer Mortality 35 DDREF DOE Low Dose Radiation Research Program %Cancer Mortality 30 LNT Low Dose Low Dose-Rate 25 23 ? Background Cancer Mortality ? 0.1 Background Radiation in 70 years Dose (Sv)

6. Dose-Response Curve Following Low Dose Exposures • DDREF non-linear extrapolation from high dose region • BEIR VII Linear after the DDREF • Model one-hit, one DNA Damage, one mutation, one cancer • New Data suggest that it is non-Linear at low doses after applying DDREF

7. DOE Low-Dose Radiation Research Program • A 10 year program • Focused on biological mechanisms of low-dose (< 0.1 Gy) and low dose-rate (< 0.1 Gy / Yr) radiation • International in scope (currently about 80projects) • To develop a scientific basis for radiation standards http://www.lowdose.energy.gov

8. Why now? • Standards set from high dose effects, but low dose effects have not been measurable until now • New technological developments and biological discoveries have made it possible for mechanistic studies of low dose effects

9. Key Research Areas • Technological Advances • Biological Advances

10. CCD camera Lamp Epi-fluorescent microscope Micropositioning stage Optical shutter Zone-plate assembly X-ray mirror Carbon target Electron gun Focused X-ray Microbeam Spatial Resolution of the Microfocus Source Cell OSA 9mm Apodized Spot Zone-plate 200µm Michael et al. Gray Laboratory

11. Viewing Light Scintillation Detector Scintillation Plastic Piezoelectric Shutter Manually Adjustable Collimeter Faraday Cup Beam Control Slits Beam from Accelerator Vertical Bending Magnet Alpha-Particle Radiation System Video Camera Microscope Objective Lens Newport Positioning Stage Mylar Bottom Petri Dish Texas A&M

12. Microbeam Hit Accuracy

13. Microbeams- Recent findings Localized DNA damage observed after both focussed soft X-ray production and charged particle induction using gH2AX Single 3 MeV Helium ion Focused CK X-rays 5 mm

14. Cellular Changes • Adaptive Response • Small dose alters response to large dose • Small dose decreases spontaneous damage • Bystander Effects • Cells respond without energy deposition • Cell-cell communication • Materials into the media • Genomic Instability • Loss of genetic control many cell generations after the radiation exposure

15. Biological Responses Induced by Low Doses of Radiation AdaptiveResponse Genomic Instability Bystander Effects Genetic Sensitivity

16. Bystander Effects in vitro

17. Micronuclei in Non-Exposed Cells Geard

18. DNA Damage and Signaling • Alterations in gene expression • Changing redox status of the cells • Modifying signaling pathways • Modification of cell cycle • Induction of apoptosis • Alterations in differentiation

19. Role of DNA Damage and Epigenetics in Bystander Effects • DNA damage in “hit cells” is not the trigger to induce bystander responses • DNA damage and repair is important in the “bystander cells” for the induction of bystander endpoints. (Prise et al. 2006) • Epigenetic changes in “bystander tissue”: increase in some types of DNA methyl-transferase and a decrease in other types • Methyl-binding proteins known to be involved in transcriptional silencing increased in “bystander tissue”. (Koturbash et al. 2006)

20. Calcium Fluxes as Bystander Signals(24 h post-irradiation with 5 alpha particles) 10 % cells Nuclear 100 % cells Cytoplasmic 100% cells 10% cells treated with DMSO Control Shao et al 2006

21. Bystander Effects in vivo • Bystander effects after single, high-dose, acute radiation exposure. • Tissue exposed • Non-exposed organs or tissues • Bystander effects following internally deposited radioactive materials. • Tissues exposed • Non-exposed organs or tissues

22. Shielded Cells 400 No Cells Evaluated Micronuclei/1000 Cells 800 Lower half of lungs irradiated with 10 Gy The Influence of Communication on Radiation-induced Micronuclei in Lung Exposed Cells Khan et al 1998

23. Bystander Effects in vivo Following Acute Radiation Exposure • Clastogenic factors • Abscopal effects

24. Clastogenic Factors • A-bomb survivors • Chernobyl clean-up • Radiation therapy • Experimental animals

25. Radiation Exposure Induction of Clastogenic Factors in Experimental Animals Blood Sample Blood Sample Plasma Blood Lymphocytes Culture for chromosome analysis Morgan 2003

26. Bystander Effects in vivo Following Low Dose-rate Exposures • Exposure to non-uniform radiation fields • Exposure to internally deposited radioactive materials that target selected organs • Exposure to internally deposited radioactive particles

27. Hot Particle Hypothesis • Cellular dose and response are linked • Non-uniform dose distribution results in increased risks • Large doses to a few cells result in large risks • Risk from inhaled plutonium particles too low by a factor of 100,000-200,000. • Studies undertaken to test this hypothesis

28. Non-uniform Dose Distribution from Plutonium Inhalation

29. The Influence of 239PuO2 Particle Size on the Dose-Distribution in the Liver of Chinese Hamsters Citrate 0.44µm 0.84µm 0.17µm

30. The Influence of 239Pu Dose-Distribution on Chromosome Aberration Frequency Aberrations/Cell Brooks et al

31. Cumulative Liver Tumor Incidence After 239PuO2 or 239Pu Citrate Exposure

32. Low Dose Rate Exposures: No Bystander Effects in Unexposed Tissues or Organs • Cancer from internal emitters are at the site of radionuclide deposition • Secondary cancers from radio-therapy located at the exposure site • At low dose rates there is little evidence for cancer in non-exposed tissues

33. Biological Responses Induced by Low Doses of Radiation AdaptiveResponse Genomic Instability Bystander Effects Genetic Sensitivity

34. What Genes are Responsible for the Adaptive Response ? 90 80 70 60 Aberrations 50 Observed 40 Expected 30 20 10 0 0 0.5 150 0.5 + 150 Dose cGy Shadley and Wolff 1987

35. Radiation Induced Changes in Gene Expression: Adaptive and Non-adaptive Cells At 50 mGy Radiation Exposure 12,000 Genes Non-adaptive Cells All Cells Adaptive Cells 57 47 45 Coleman et al 2005

36. 50mGy NA A A Group 1 Genes up-regulated by radiation in all cells Group 2 Genes down-regulated by radiation in all cells Group 3 Genes up-regulated by radiation in adaptive cells, but down regulated in non-adaptive Group 4 Genes down -regulated by radiation in adaptive cells, but up-regulated in non-adaptive Adaptive vs Non-adaptive Radiosensitive Genes

37. Gene Response Adaptive Non-Adaptive vs DNA Repair Stress Response Apoptosis Cell Cycle Coleman et al 2005

38. Intervention Adaptive ResponseSub-linear Dose Response Transformation Frequency 0 10 20 30 40 50 60 70 80 90 100 Dose (cGy) Redpath et al. 2001

39. Transformation Frequency as a Function of Radiation Dose and Dose-rate Elmore et al. 2006

40. Adaptive Response Induced in Prostate by Very Low Radiation Doses Day et al. 2006

41. DNA damage from low-dose exposures reduced by changing oxidative metabolism de Toledo et al. 2006

42. Biological Responses Induced by Low Doses of Radiation AdaptiveResponse Genomic Instability Bystander Effects Genetic Sensitivity

43. Radiation-induced Genetic Damage Old Paradigm After a cell is mutated by radiation, all of its prodigy are mutated Mutation is a rare event

44. Micronuclei Cell death Gene mutation Mitotic failure-aneuploidy Chromosome aberration Genomic Instability New Paradigm After a cell is exposed to radiation, different things can happen …sometimes after many cell divisions. This is a frequent event.

45. Sensitive BALB/c mice Resistant C57BL/6 mice Genomic Instability can be Demonstrated in Some Strains of Mice 0.35 0.3 0.25 0.2 Aberrations/Cell 0.15 0.1 0.05 0 4 8 12 16 20 24 28 Population Doublings B. Ponnaiya & R.L. Ullrich, 1998

46. Radiation-induced Genomic Instability • High frequency event • 3%/Sv low LET • 4%/Sv high LET • Independent of dose rate at high total dose • Related to inflammation and the Redox status of the cell • Produced both in vitro and in vivo

47. Genomic Instability is Modified by Adaptive Response GFP+/- Colonies Genomic Instability 1 2 3 10 1 2 3 10 Dose (cGy) Huang et al. 2007

48. High Doses Cancer Changes in gene expression Mutations Chromosome aberrations Genomic instability Cell killing Stimulate cell proliferation Tissue and matrix disruption Inflammation Change in ROS status of cells Low Doses Cancer? Changes in gene expression Mutations Chromosome aberrations Adaptive response Bystander effects Differential apoptosis Mechanisms for Cancer Induction

49. Mechanisms Involved in New Phenomena • Altered gene expression • Impact of oxidative status of the cell • Radiation-induced changes in apoptosis • Cell/cell, cell/matrix interactions • Nutrition and radioprotectants

50. Numbers of Genes Differentially Regulated in HLB Cells 4 hr after IR 703 Genes with Significant F-ratio 245 Up-regulated at 2Gy 135 Down-regulated at 2Gy 182 Up-regulated at 0.1Gy Down-regulated at 0.1Gy 187 Yin 2003 DIFFERENCES IN TRANSCRIPTION PROFILES BETWEEN LOW AND HIGH DOSE IRRADIATION IN MURINE BRAIN CELLS 0.1 Gy 2Gy 191 299 213 Total gene set contains nearly 10,000 genes