Radiation Effects in Life Sciences

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					           RESMDD'02                  Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP




  Radiation Effects in
  Life Sciences
                                                        oocyte     eggs in uterus                 gonad
                                                                                    spermatheca




        Quality of Radiation                                                         vulva



        Biological Effects
        Applications of SSD in Life sciences
               Nanodosimetry
               Particle Microscope
               (pCT)
          RESMDD'02                     Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                      Radiation in Life Sciences

        Why Should Biologists Care about Radiation?
              Imaging and Therapy of Cancer
              Space Radiation Environment
              Our Origins ( Evolution)
              Our Future ??

        Radiation
           • targets DNA
           • influences the Gene Pool
           • can be used for Cancer Research to induce DNA Faults
        RESMDD'02                     Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP           Charged Particles in Radiobiology
    • Much of Radiobiology is done with Photons (UV, X-and γ-Rays)
    • Protons & Light Ions
       – Physical Properties are Superior for Cancer Treatment
           • Defined Range
           • Track Structure
           • Magnetic and Electrical Confinement
       – Main Component of Space Radiation
           • Trapped Belts, Sun and Galactic Sources
           • Energy Range Similar to Synchrotron

    • Responses of Normal Tissues to Radiation Limit Medical
      Treatment and Space Mission Operations
       – Cell Inactivation
       – Cell and Tissue Changes of State
            RESMDD'02                          Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


 SCIPP
                Energy Loss by Radiation in Matter




                                                        Mass attenuation coefficient (cm2g–1)
• Photons interact “catastrophically”
                                                                                                        1000
                                                                                                                                                                                     Silicon
                                                                                                                     100
   N(x)/No=e-µx                                                                                                               10
                                                                                                                                                                               At high E, small
      • N(x)/No is fraction of photons                                                                                            1      Rayleigh               total
                                                                                                                                                                               Conversion Prob.
                                                                                                                                        (coherent)                             in thin Materials
        left at depth x                                                                                                  0.1             Compton
                                                                                                                                                              photo-
      • µ is linear attenuation coefficient                                                                  0.01                                             electric                  pair production

                                                                                                                                  0.001      0.01       0.1        1          10        100      1,000 10,000
                                                                                                                                                        Photon energy (MeV)


• Charged Particles leave structured track
                 π
   1/ρ(dE/dx) = 4πro2 Nez2moc2/β2 [ln(2moc2β2/I(1-β2) -β2-Σi(ci/Z)]
         » Ne is electrons per gram of medium,
                                                                                                                                  50




                                                                                                Energy loss dE/dx (MeV cm 2g–1)
                                                                                                                                  30                               Bethe-Bloch
                                                                                                                                                Silicon
           ρ is density of medium, β is v/c,                                                                                      20
                                                                                                                                                β
                                                                                                                                             ~1/β1.5                                          Rad
           z is charge of particle                                                                                                10
                                                                                                                                   7
                                                                                                                                                         measure                   e±
                                                                                                                                   5                     p
         » The important component is :                                                                                            4                                                           µ±
                                                                                                                                   3                    MIP
           dE/dx ∝ ρ       β
                        z2/β2                                                                                                      2
                                                                                                                                                                      W it h o u t ra
                                                                                                                                                                                      d ia ti ve lo ss
                                                                                                                                                                                                       es

                                                                                                                                   1
                                                                                                                                       0.1          1            10            100         1000        10000
                                                                                                                                                                   p/m = βγ
        RESMDD'02              Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP       Photon and Proton: Dose vs. Depth


            D
X-rays &    o
Gamma Rays: s
Exponential e

                      Depth

                D
 Protons:
                o
 Bragg Curve s
 (derives from e
 negative slope
 of Bethe-Bloch)      Depth
         RESMDD'02                      Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
        Parameters to Describe Quality of Radiation

   • Dose & Dose Rate
   • Fluence & Fluence Rate
   • Linear Energy Transfer (LET)
   • Relative Biological Effectiveness (RBE) or Quality
     Factor (Q)

Cellular Responses to Charged Particles
Fluence (E) is Better than Dose as an Independent Variable
LET is Insufficient to Describe Track Structure
(Track dimensions vs. Target geometry)
           RESMDD'02                Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                  Proton Radiobiology in Perspective

                        D = 1 Gy        dE/dx per µm                           RBE*
    n = 416                                     1.3 keV
                                                                                   1.1
    50 MeV                                 36 ionizations
    protons


    n = 112                                    4.7 keV
                                                                                   1.4
    10 MeV                               134 ionizations
    protons

        n = 54                                   10 keV
                         10 µm                                                     2.0
        4 MeV                             276 ionizations
        protons                                         * rel to 60 Co γ rays
          RESMDD'02                        Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                           Nano Dosimetry
 • Are there different classes of damage depending on the Linear Energy
   Transfer (LET) and number of ionizations/DNA molecule?

          LET         # of Ionizations      Damage                       Example
                       (Cluster Size)
          Low               1-5            Repairable ?                Single Strand
                                                                          Break
          High             6-12            Irreparable ?              Double Strand
                                                                         Break


        Very High           >12          Recombination &                       ??
                                           Saturation ?
 • Effect of OH- radicals in the damage process
 • Improve dosimetry of proton beam for cancer therapy
            RESMDD'02                        Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


  SCIPP
                           Radiation Damage DNA

 Ionization event (formation of
         water radicals)
                                                    Light damage- reparable
Primary particle track



           delta rays

                         e-
                        OH•
   Water radicals attack
        the DNA                                               Clustered damage-
                                                                      irreparable


   The mean diffusion distance of OH radicals before they react is only 2-3 nm

Ionization in 1 mm of low pressure gas = Ionization in 2nm of DNA
        RESMDD'02                   Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP               Schematic of Nanodosimeter


                        particle
                                            SSD
        low pressure                                       vacuum
        gas                        δ electron
                                    ion
                                                        ion counter
                                      E2
                                      (strong)

                          E1
                                          SSD


                                   differential pumping
         RESMDD'02
                       ND Ion Cluster Spectra Hartmut F.-W. Sadrozinski , SCIPP
                                   Radiation in Life Sciences:


SCIPP                (LLUMC Weizmann group)
        Ion Cluster Spectra                      Ave. Clustersize as a function of LET
                                          100



                                           10



                                            1
                                                                                    P (22Mev)
                                                                                    He (4.5MeV)

                                           0.1                                      C (70MeV)
                                                                                    C (20MeV)



                                          0.01
                                              101          102               103      104
                                                                            2
                                                            dE/dx [MeV/(g/cm )]

    Ion cluster spectra depend on particle type and energy
    Need to correct for the position of the primary particle track
    The average cluster size increases with increasing LET
    Protons have small chance to generate large LET: lucky for us!
        RESMDD'02                                                             Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                                                         Quality of Radiation

              Comparison of unc-22 Mutation Rates
        for Low and High Energy Protons and Gamma Rays

                                                        unc-22 Mutation Frequency vs. Dose

                                         1.00e-3


                                         8.00e-4
         Arithmetic Mutation Frequency




                                                                      250 MeV Bragg Peak p+
                                         6.00e-4

                                                                                          250 MeV p+
                                         4.00e-4
                                                                                          60Co Gamma
                                         2.00e-4


                                             0.00


                                         -2.00e-4
                                                    0   10     20    30      40      50        60       70
                                                                    Dose in Gray
        RESMDD'02                          Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                     Rat Skin Tumorigenesis

                    Argon ions vs. electrons




                         From Burns & Albert, 1986
        RESMDD'02                      Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP        Low Dose: Atomic Bomb Risk Data
                    Departs from Linearity at Low Doses




                                                                    Pierce & Preston (2000)
                                                                    Rad Res. 154:178
        RESMDD'02                       Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP               Low-Dose Cell Survival


                                                           Dose (Gy)
• Low-dose studies with
  a proton microbeam                                                              3.2 MeV
                                                                                  protons
    – precise low-
      dose/fluence cell
      survival curves
    – hypersensitive region
      at low doses
    – more pronounced at
      higher proton energies
      (3.2 MeV vs. 1 MeV)
                               Schettino et al. Radiation Res. 156, 526-534, 2001
         RESMDD'02                     Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP         Adaptive Response & Bystander Effect



• Low-dose studies with an
  alpha particle microbeam
   – only 10% of cells exposed                                              --- expected

   – more cells inactivated than
     traversed (bystander effect)
   – previous exposure to low
     level of DNA damage
     increases resistance                           -o- 0 hrs after priming γ dose
     (Adaptive response)                            - - 6 hrs after priming γ dose




                                    Sawant et al. Radiation Res. 156, 177-180, 2001
        RESMDD'02               Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                    Cross Section vs. LET



                                      Probability of killing
                                      a cell per particle vs.
                                      a measure of track
                                      structure (LET) scales
                                      with geometric cross
                                      section of the target
                                      but is not a unique
                                      function.



                                       Kiefer
        RESMDD'02                          Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP

   Biological Effectiveness: Colony Formation Assay
SCIPP
                    The Standard Cell Killing Method




                                Colony formation method and example of CHO
                                cell colonies.

                                A 70 colonies in control culture resulting from 100 seeded
                                cells. Plating efficiency is 70%.

                                B 32 surviving colonies from 2000 cells irradiated with 8
                                Gy of x-rays. Surviving fraction is: 32/0.7*2000 = 0.023

                                 From Hall, Radiobiology for the Radiologist
        RESMDD'02                                    Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                Relative Biological Effectiveness


                                                                RBE is used to
                                                                compare different
                                                                “qualities” of
                                                                radiation.
           Densely ionizing
                                 Sparsely ionizing




                                         X-Rays

                    Heavy ions

                                                                  J. Kiefer (1990)
         RESMDD'02            Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
        Scales and Structures of DNA and Chromatin


                              DNA in cells is always
                              in the form of chromatin
                              which is a highly-ordered
                              DNA-protein complex
                              that is constantly being
                              remodeled.

                              Charged particle tracks
                              would be expected to
                              cause patterns of
                              molecular damage that
                              reflect the ordered
                              packing.
        RESMDD'02              Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP   Particle Tracks Place Clusters of Ionizations
           in Volumes on the Scale of Chromatin
          RESMDD'02                             Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP

    The Fundamental Concept of Dose is Misleading
SCIPP


           • Dose is defined as energy absorbed per unit mass
          (irrespective of the spatial distribution of the absorbed energy)
                1 Dose Unit                                 1 Dose Unit




        Low LET radiation deposits             High LET radiation deposits
        energy in a uniform pattern            energy in a non-uniform pattern


    Structured Energy Deposition Patterns Interact with Target Structure
    to Produce Correlated Damage
        RESMDD'02             Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                    Correlated Damage



                                     • The organization
                                       of chromatin
                                       favors the
                                       production of
                                       multiple damage
                                       sites within loops
                                       and nucleosomes.
                                     • Such patterns are
                                       unique to charged
                                       particle radiation.



                                            A. Chatterjee, LBNL
                                RESMDD'02                                                                        Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                                     Risk Mitigation through Shielding?

                                   Fragmentation May Enhance Damage
                               • Physical vs Biological Assessment of Shielding

                     GCR Dose Equivalent on ISS                                               Chromosome Aberrations on ISS




                                                                           Dicentrics per Cell per Quarter
                                                                                                             0.006
                                                                                                                                                         Track Model
                     0.8                                                                                                       Aluminum
                     0.7
 Dose Eq., mSv/day




                     0.6                                                                                     0.004
                                              Aluminum                                                                                           Water
                     0.5

                     0.4
                                                         Water
                                                                                                             0.002                                Polyethylene
                     0.3
                                                                                                                               Liquid hydrogen
                     0.2
                                 Liquid hydrogen   Polyethylene
                     0.1
                                                                                                             0.000
                     0.0
                           0    10       20         30           40   50                                             0    10        20           30        40          50

                                        Depth, g/cm2                                                                              Depth, g/cm2


                                                                                                                                                      F. Cucinotta
            RESMDD'02                          Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                Cell Reaction to Radiation Damage
        • Repair (redundant information in DNA), “Annealing”
        • Death
           – loss of genetic material
                        » metabolic death
                        » delayed apoptotic death
           – genetic instability
                        » delayed apoptotic death
        • Reproductive Death
           – long-term arrest (exit cell cycle, senesce, differentiate)
           – remain metabolically viable for long time

    Cell Death May Actually Be Controlled Self-Disassembly
    “Apoptosis” (Programmed Cell Death) eliminates Damaged Cells
        RESMDD'02              Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
             Physiologic Functions of Apoptosis


                                •    A. Fingers
                                •    B. Gut
                                •    C. Tadpole Tails
                                •    D. Sex Differentiation
                                •    E. Proliferating Cells
                                •    F. Self-reactive Lymphocytes
                                •    G. Irradiation




                                         CELL 88:350 1997
        RESMDD'02                      Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP     Microenvironment & Communication
                                    Cells Are Not Autonomous
                                    and Sometimes Aren’t Even Targets

                    α & p+




              Irradiated       Unirradiated
               Precision Field Experiments needed
        RESMDD'02              Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                    The Bystander Effect
        RESMDD'02              Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                    The Bystander Effect




   Too Many Cells Respond       Soluble Factors Implicated
        RESMDD'02                   Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP

    New Understanding of Cellular Radiation Damage
SCIPP

                    Conventional Wisdom New Principles
  Target in Cell Only DNA in the cell            Also cell membranes &
                 nucleus                         control systems
  Cell Death     Random                          Also controlled
                  ~ DSB in DNA                   disassembly(apoptosis
  Dose           ~ Dose                          May not ~ Dose
  Dependence                                     (Track Structure, RBE)
  Charged       Dose Equivalent                  Dose Equivalent &
  particles vs. Assumption is Valid              normalization factors
  X-ray effects                                  (RBE) not always valid
  Time scale of Immediate, to all                May not appear for up to
  change in     dependents                       50 cell generations
  Genome                                         (genomic instability)
        RESMDD'02                   Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
        New Understanding of Repair Mechanisms
                     Conventional New Principles
                     Wisdom
  Mitigation of Cell Repair of DNA Control of signal
  and Tissue         damage           transduction pathways
  Damage                              Apoptosis may be
                                      advantageous
  Management of      Autonomy of      Distribution of damage to
  Damage and         Individual Cells neighbors (Bystander
  Survival                            Effect)
                                      Cells in tissues respond
                                      differently than individuals
                                      (Microenvironment
                                      Effects)
           RESMDD'02                     Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP      Specific Experiments to track the damage
        Tracking Resolution limited to the few um level.
        Selected biological systems are of that size:
        LLU- SCIPP Particle Tracking Silicon Microscope
        a versatile and inexpensive broad-beam and microbeam particle
        tracking system for

            –protons and alpha particles
            –wide range of energies (1 MeV - 250 MeV protons)
            –in vitro and in vivo radiobiological studies
            –research studies for radiation therapy and protection
            –support of DOE and NASA low-dose research programs
         RESMDD'02                   Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP                Gametogenesis
        in the adult hermaphrodite of C. elegans




                     oocyte   eggs in uterus                                  gonad
                                                spermatheca




                                                  vulva
          RESMDD'02             Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
                Chromosome structures
        in the gonad of the adult hermaphrodite
        RESMDD'02                   Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP
          Silicon Strip Detectors in Life Sciences

   • X-rays are difficult for silicon detectors (Eff or Energy low)
   BUT charged particles are perfect:
   • Combined measurement of position, angle and energy or LET
     of single particles
   • High spatial resolution (microns)
   • Support high particle rate
   • Wide dynamic energy range
   • Fast response (self-trigger possible)
   • Radiation hardness
   • Adaptation to most geometries
   • Compatibility with physiological conditions of cells
   • Simple operation (e.g. low voltages)
   • No consumables, compact
        RESMDD'02                       Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP     Specific Experiments to track the damage

  244Cm   Alpha Irradiator + SSD

                                        Objective


                                                              Mylar Bottom
                             Aperture                         Petri Dish
                                                                He
                    Helium
                             32 mCi                                 Slider
                             244Cm


                                        Condenser
          RESMDD'02                         Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP   Particle Tracking Silicon Microscope (PTSM)
Protons produce damage AND identify damaged molecule

                                                         Transfer to Automated
                                                         Microscope when
                                                         Occupancy ~ 10%
Biological Sample
(directly on SSD)


Double-sided SSD:
x-y coordinate, Energy,      Assay with
“Cluster characteristics”.   Automated
                             Microscope using
                             stored x-y coordinates
                     RESMDD'02                                                                                                                   Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP                   Statistical vs. Specific Radiobiology
                Global (Nanodosimetry):
                Well in Hand ?                                                                                                                   Ionization
                                                                                                                                                 Cluster Spectra
                                                                                                                                                %90
                                                                                                                                                %88
                                                                                                                                                %86




                                                                                                                           Relative frequency
                                                                                                                                                                   protons 4 MeV
                                                                                                                                                %16                α 5 MeV


                                 Nanodosimeter
                                                                                                                                                %14
                                                                                                                                                %12
                                                                                                                                                %10
                                                                                                                                                 %8
                                                                                                                                                                        Correlation needed!
        Radiation




                                                                                                                                                 %6



                                                                                                                                                                        Tag individual Interaction,
                                                                                                                                                 %4
                                                                                                                                                 %2
                                                                                                                                                 %0
                                                                                                                                                                          101112 141516 181920
                                                                                                                                                      0 1 2 3 4 5 6 7 8 9 10 121314 161718 20
                                                                                                                                                                  Cluster size


                                                                                                                                                                        Investigate Damage
                                                                                                                  ssb

                                                                                                                 dsb
                                                                                                                                                            Frequency ofin detail on struck Structures
                                                         mobility
                                                                                                                  intact
                                                                                                                                                            lesions of different
                                                                                                                                                            complexities
                                                         0 minutes   15 minutes   30 minutes   60 minutes   120 minutes




                    Plasmid Sample
                                                         Gel
                                                         Electrophoresis
                                     Incubation with
                                     Base Excision     Local: Needs Improvement
                                     Enzymes
                                                       No Radiometry Measurement
                                                       Correlated with Damage on
                                                       individual DNA Molecule
         RESMDD'02                          Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP         Particle Tracking Silicon Microscope


   • Conventional radiobiological
     experiment: statistical
        – random traversal of cells by a broad
          particle beam
        – only average number of hits per cell         λ = 1.5 P(n) = λn/n! e-λ
          is known
   • Particle-tracking radiobiological                                                 SSD
     experiment: specific
        – number of particles per cell is           n=2          1 3         0
          exactly known                                                                collimator
        – broad beam or microbeam setup
                                                                                        SSD

                                                    n=0          0 3         0
         RESMDD'02                       Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP


SCIPP          Particle Tracking Silicon Microscope

                                                           MCM

• Conceptual design
   – biological targets located
                                  DSSD      ASIC       RO Control         Cables            DAQ
     on detector surface
   – single-particle tracking
   – energy or LET
     measurement
   – ASIC and controller
     design adapted to
     application
   – dedicated data acquisition
     system
            RESMDD'02                     Radiation in Life Sciences: Hartmut F.-W. Sadrozinski , SCIPP

    Radiation Effects in Life Sciences vs. in Materials
SCIPP


  • Many Similarities:
        –   Fluence or Dose
        –   Low Dose effects
        –   Rate effects
        –   LET & NIEL
        –   RBE & Kerma
        –   Microenvironment: Bystander Effect & Latch-up (SEE)
        –   Adaptive Response & Annealing
        –   Cell behavior vs. Circuit
  • Living cells are well engineered (many cycles of design rule
    and VHDL verification!) and have mechanisms to deal with
    radiation damage: Repair or Apoptosis.
  • In special cases, we will be able to supply specific data from
    particle-tracking measurements to replace the conventional
    statistical radiobiological approach.