1. Basics/Teminology
   1. Ionization
      1. Strips electrons fromshell
      2. Leaves charged particle
   2. LET
      1. amount of energy lost per unit distance
      2. High LET = more damage
      3. High LET deposits more energy per cell
      4. Low LET = less damage
   3. Critical Target
      1. serious consequences for cell if inactivated
      2. DNA - may result in abnormal cellular response
   4. Specific Ionization (SI)
      1. number of ion pairs produced per unit track length
      2. High LET = low SI
      3. Low LET = high SI
   5. Direct Effect
      1. High LET radiation
      2. critical target directly ionized and damaged
      3. altered structually or functionally
   6. Indirect Effect
      1. Low LET radiation
      2. water molecules frequency hit
      3. formation of free radicals
         1. OH responsible for 2/3 of indirect damage
         2. free radical interacts
            1. with another free radical (recombination
            2. H + OH -> H2O (safe)
            3. H + H -> H2 (tolerable/safe)
            4. OH + OH -> H2O2 (poisonous)
            5. with own reaction productss
            6. H2 + OH -> H2O + H
            7. H2O2 + OH -> H2O + HO2
            8. with oxygen
            9. H + O2 = HO2
            10. H + H2- -X->
            11. with organic molecules/forms bioradicals
            12. RH + OH -> R + H2O
            13. RH + H -> R + H2
            14. reconstitution with hydrogen radical
            15. R + H -> RH
            16. dimerization reactions
            17. R + R -> R-R
            18. disproportionaltion reactions
            19. addition of O2 or HO2 producing chemical changes
            20. R + O2 -> ROOH
            21. R + HO2 -> ROOH
            22. hydrogen transfer reactions
            23. R + RH -> RH + R
2. Four Temporal Stages of Biological Interaction
   1. 1. Physical Stage
      1. energy absorbed and activated species formed
      2. eg. fast electrons & ionized molecules
      3. inhomogeneous spatial distribution, limited distance
      4. 1E-17 to 1E-15
   2. 2. Physico-chemical Stage
      1. secondary reactions involving primary products
      2. fast electrons produce further ionizations
      3. ions produced or free radicals
      4. 1E-15 to 1E-12
   3. 3. Chemical Stage
      1. ions/free radicals react with each other and biomolecules
      2. bioradicals formed
      3. stable altered molecular products formed
      4. 1E-12 to 1E-6
   4. 4. Biological Stage
      1. multi-faceted response of irradiated organism
      2. repair/recovery
      3. immediate effects
         1. cell death
         2. tissue damage
         3. organ dysfunction
      4. late somatic effects
         1. tissue damage
         2. leukemia
         3. cancer
      5. manifest genetic damage
         1. present in offspring
3. Cell Cycle
   1. typically lasts 10-24hr
   2. regulated by checkpoints
   3. Mitosis
      1. ~1h in length
      2. Events
         1. RNA synthesis ceases
         2. protein synthesis decreases
         3. cell division occurs
   4. G1
      1. gap/growth 1
      2. interval after mitosis before DNA replication
      3. arrest point near beginning, can enter G0 or G1
      4. quiescent G1 state: progression through cell cycle halted by commited to replicate DNA
      5. checkpoint near end: proliferation competency assessed state of DNA integrity assessed
         1. may initiate cell cycle arrest and DNA repair
         2. may undergo apoptosis if unprepared
      6. Events
         1. synthesis of enzymes for DNA replication
         2. all species of RNA transcribed
         3. synthesis of metabolic enzymes, structural proteins
         4. end of G1 marked by: appearance of inducer factor for DNA synthesis beginning of synthesis of histones and thymidine kinase
   5. S
      1. DNA synthesis phase
      2. 6 - 8 hours
      3. Events
         1. replication of chromosomal DNA
         2. maturation of daughter centrioles
         3. centriole reproduction begins
         4. production of proteins to maintain DNA synthesis
         5. rate of RNA synthesis similar to G1 and G2
   6. G2
      1. gap2 or growth2
      2. interval between DNA replication and mitosis
      3. ~1-5 hours in length
      4. Events
         1. RNA and protein synthesis for mitosis
         2. centrioles separate into two pairs
         3. growth and development
         4. DNA/chromatin checkpoint
            1. can initiate cell cycle arrest and repair
            2. apoptosis if unprepared for mitosis
   7. G0
      1. gap0 or growth0
      2. represents mature, highly differentiated function cell state
      3. cells have left mitotic cycle
      4. can be induced to re-enter cell cycle
   8. D
      1. differentiated phase
      2. mature terminally differentiated stage
      3. cannot enter mitotic cycle
   9. Diagram
      1. <html><img src="untitled.JPG">
4. Cell Classification
   1. developed by Rubin and Casarett
   2. catergorized by
      1. mitotic activity
      2. function/metabolic activity
      3. response to radiation
   3. Vegatative Intermitotic (VIM)
      1. short lived individual cells
      2. primitive, undifferentiated
      3. divides regularily and rapidly
      4. one daughter cell becomes DIM, one VIM
      5. examples
         1. primitive stem cells - blast cells
         2. dividing cells in intestinal crypts
   4. Differentiating Intermitotic (DIM)
      1. overlaps with DIM depending on frequency of division
      2. relatively short lived as individuals
      3. produced by division of VIM
      4. divide regularily for limited number of divisions
      5. examples
         1. spermatocytes
         2. myelocytes
   5. Reverting Postmitotic (RPM)
      1. relatively long lived as individuals
      2. do not undergo regular ot periodic mitosis
      3. can be stimulated to divide again
      4. stimuli involves damage to cells
      5. examples
         1. epithelial cells
         2. endocrine glands
         3. interstitial glands
         4. smooth muscle cells
   6. Fixed Postmitotic (FPM)
      1. do not divide
      2. highly differentiated
      3. specialized functions
      4. replaced by VIM/DIM
      5. examples
         1. RBC
         2. sperm
         3. epithelial cells of HI
         4. neurons
         5. muscle cells
   7. Multipotential Connective Tissue (MCT)
      1. cells of support and connective tissues
      2. life span variable with type
      3. more differentiated than VIM/DIM cells
         1. endothelial cells
         2. active fibroblasts
5. Cellular Response
   1. damage expressed at mitosis
   2. classified according to time at which tissues express damage
   3. response in tissues due to depletion of critical target cells
   4. differences in time depend on turnover kinetics
   5. Early
      1. manifest injury within a short time
      2. relatively radio-resistant, short lived mature cells not directly damaged
      3. but active, radiosensitive stem cells cannot maintain replacement requirements
      4. few days to a few weeks/month
      5. repair/recovery very rapid
   6. Late
      1. injury does not manifest for three month or longer
      2. more slowly dividing cell populations
   7. Response of any cell after irradiation may be:
      1. no damage to any critical target
         1. unaffected by dose of radiation
      2. accumulated non-lethal damage
         1. sublethal damage
         2. repair and cell can recover completely
         3. can accumulate enough damage to be lethal
      3. accumulated enough damage to be letal
         1. lethal or potentially lethal damage
         2. die immediately or in mitosis
   8. Four ways to respond
      1. division delay
         1. progress towards mitosis delayed
         2. occurs in lethal and non-lethal damage
         3. delayed mechanism introduces opportunity for repair
         4. cell in mitosis completes division and delays in early G1
         5. irradiated in G1 delay at checkpoint in late G1/S
         6. irradiated in S or G2 delay at late G2 checkpoint
         7. post G2 delayed entry into mitosis
         8. delay increases as dose increases
         9. max delay before G2 checkpoint
      2. reproductive failure
         1. mitotic death, cells die attempting to divide
         2. inability of cells to undergo repeated divisions after irradiation
         3. ability to reproduce directly related to integrity of DNA/chromosomes
      3. apoptosis
         1. programmed cell death
         2. deliberate suicide by unwanted cell in multicellular organisms
         3. occurs naturally but can be induced by radiation
         4. cells respond to DNA damage by increasing production of p53
         5. p53 is a potent inducer of apoptosis
      4. interphase death
         1. instant cell death
         2. can occur in all cell types
         3. acute dose dependent on cell type
         4. not primary response to occupational radiation exposure
         5. rapidly dividing undifferentiated cells exhibit interphase death at lower doses
6. Classification of radiation induced damage
   1. no damage
      1. survival of normal reproductive integrity
   2. sublethal damage
      1. injures but does not kill
      2. can be repaired
      3. may accumulate to lethal levels
   3. potentially lethal damage
      1. damage which is lethal if not repaired before M
      2. probability of repair depends on post irradiation environment
      3. survival increases in nutrient poor conditions
   4. non-lethal damage
      1. induction of heritable lesion that slows proliferation but does not prevent
      2. cells more responsive to second radiation dose
   5. lethal damage
      1. interphase or mitotic deaeteh
      2. may be result of accumulation of sublethal damage
      3. unrepair potentially lethal damage
7. Radiosensitivity
   1. Bergonie and Tribondeau 1906
      1. radiosensitivty depends on
         1. mitotic activity high mitotic activity more radiosensitive
         2. degree of differentiation less well differentiated cells more radiosensitive
         3. level of metabolic activity high metabolic activity cells more radiosensitive
   2. Ancel and Vitemberger 1925
      1. expression of radiation damage influenced by
         1. biological stress on cell
            1. mitotic activty
            2. functional/synthetic activity
         2. conditions of cell pre/post irradiation
            1. modifiers, O2, chemicals
            2. opportunities for repair
            3. conditions favouring or opposing mitotic activity
      2. radioresponse different, not inherent radiosensitivity
   3. most radiosensitive cells
      1. rapid division
      2. primitive in their degree of maturity
      3. ability to divide for long periods of time
   4. important in
      1. determination of radiation dose levels
      2. evaluation of relative risks associated with chronic and acute exposures
      3. relative risks to pregnant or potentially pregnant workers
8. Factors modifying cellular response
   1. Physical
      1. amount of radiation expressed as Gy
      2. equal doses do not produce equal biological effects
      3. quality of radiation depends on LET
      4. Relative Biological Effectiveness
         1. depends on
            1. endpoint selected
            2. exposed cell or tissue type
            3. LET
            4. very high LET deposits more energy than required
            5. very low does not deposit energy with enough density
            6. dose rate
            7. rate at which radiation is delivered
            8. low dose rates less efficient
            9. cell able to repair at low dose rates
            10. affects survival curve
            11. significant with low LET
            12. number of dose fractions
      5. Survival curve
   2. Biological
      1. detoxification
         1. natural mechanisms exist for removal of ROS
         2. indirect effect produces hundreds to thousands of ROS per cell
         3. increase in ROS signals a stimulation of enzyme activity to remove them
         4. maximally expressed after 100mGy to 500mGy
         5. peaks ~4hr after irradiation
         6. lasts several hours to days
      2. repair of radiation induced DNA damage
         1. must give enough time for cell to repair
         2. structural forms of DNA damage must be repair for functional repair
            1. base changes -> repaired on an individual basis
            2. inter & intra strand crosslinks -> removed and repaired
            3. single strand breaks -> sister chromatid used as a template
            4. double strand breaks -> sister chromatid used as template
         3. low doses of low LET radiation induces improved protection
      3. removal of heavily damaged cells
         1. apoptosis
            1. form of signal induced cell death
            2. non-inflammatory process
            3. occurs within hours after high dose radiation
         2. low dose induced apoptosis a mechanism for removal of pre-damaged cells
      4. position within cell cycle
         1. cells more radiosensitive during M phase
         2. late G2phase most sensitive
         3. radioresistance greatest in S phase
         4. cells with short G1 -> single hump
         5. cells with long G1 -> double hump
         6. radiosensitivity
            1. S -> most radioresistant
            2. Late G1 -> radiosensitive
            3. Early G1 -> radioresistant
            4. M -> radiosensitive
            5. G2 -> radiosensitive
   3. Chemical
      1. damage results from radiation induced chemical alterations
      2. chemicals can alter cellular response
      3. radioprotectors
         1. effectiveness indicated by dose reduction factor (DRF)
         2. most effective for low LET
         3. effectiveness decreases as LET increases (overkill)
         4. psudo radioprotectors
            1. do not directly influence cellular radioresponse
            2. function by reducing O2 content
            3. vasoconstriction
            4. alteration of metabolic processes
            5. hypoxic cells more radioresistant
            6. examples
            7. sodium cyanide (NaCN)
            8. carbon monoxide (CO)
            9. epinephrine
            10. histamine
            11. seratonin
         5. true radioprotectors
            1. directly influences cellular radioresponse
            2. sulphydryl (-SH) most significant group, affinity for free radical
            3. mechanism of action
            4. free radical scavenging
            5. hydrogen atom donor
            6. naturally occuring (cysteine)
            7. naturally occuring amino acid
            8. toxic at effective levels if administered
            9. synthetic (glutathione)
            10. much research during Cold War
            11. >4000 compounds research at Walter Reed institute
            12. WR 343, 638, 2721
      4. radiosensitizers
         1. effectiveness indicated by sensistizer enhancement ratio (SER)
         2. most effective for low LET
         3. effectiveness decreases as LET increases (overkill)
         4. pseudo radiosensitizers
            1. do not directly influence cellular radioresponse
            2. function by inhibiting repair of damage
            3. do not have to be present during irration to exert effect
            4. example
            5. actimomycin D - depresses DNA dependent RNA synthesis
            6. puromycin - depresses protein synthesis
            7. methrotrexate - interferes with DNA synthesis thus inhibiting repair
         5. true radiosensitizers
            1. actively increases probability of damage occuring
            2. must be present in cell during irradiation to exert effect
            3. ex: oxygen
            4. oxygen enhancement ratio (OER)
            5. degree by which oxygen increases efficiency of ionizing radiation
            6. compares doses required to produce a given effect under hypoxic and aerobic conditions
            7. OER = hypoxic/aerobic
9. Categories of radiation bioeffects
   1. Stochastic effects
      1. random in nature, effects tend to increase with increasing dose
      2. Linear dose response
         1. certain effects observed at zero radiation dose
         2. probability of observing increases with dose
         3. basis of rad pro programs
   2. Deterministic effects
      1. effects whose severity increases with increasing dose
      2. a threshold dose above which effects are seen
      3. Non-linear threshold dose response
         1. more accurately describes effects with high doses
         2. assumes recovery from bioeffects at low doses
   3. Linear quadratic dose response
      1. assumes linear response at low doses and nonlinear at high
      2. underestimate low dose effects
      3. assumes relatively low risk at low doses
      4. hybrid of linear and non-linear
   4. additional factors influencing dose effect models
      1. time period over which dose is delivered
      2. age of exposed individual
      3. genetic constitution
      4. state of health of exposed
      5. time period between exposures
      6. extreme temperature
10. Late effects
    1. arises in cells who survive but retain memory of exposure
    2. induced by radiation
    3. Somatic effects
       1. risk depends on
          1. type of cancer considered
          2. age or sex of person exposed
          3. magnitude of dose to organ
          4. quality of radiation and nature of exposure
          5. prescense of other factors
       2. leukemia
          1. most apparantly long term effect
       3. cancer
          1. excess risk exhibits ~5-35 years post exposure
          2. difficult to tell if cancer related to radiation exposure
          3. radiation induced cancers tend to be aggressive
          4. tumors located near area of exposure
    4. Genetic effects
       1. no longer considered a major potential effect
       2. increase in incidence of spontaneous natural mutations
       3. doubling dose - dose required to produce a # of mutagens equal to the spontaneous #
          1. measures effectiveness of mutagenic agent
          2. low LET: 1.56 Gy for acute, 4.86 for chronic
       4. Genetically Signifant Dose (GSD): index of presumed genetic impact of radiation exposure for general population
       5. Chromosomal mutations
          1. gross, obvious changes in chromosome
          2. Change in number of whole chromosomes, alters amount of DNA
             1. euploidy - normal number of chromosomes
             2. polyploidy - increase in number of whole sets of chromosomes
             3. aneuploidy - change in number of one of more specific chromosomes of set
             4. hypodiploid - less than diploid number of chromosomes
             5. hyperdiploid - one or more additional chromosome
          3. Chromosome breaks
             1. arises when radiation breaks a chromosome at one or more points along its length
             2. involves gross structural changes in chromosome
             3. broken ends may
             4. rejoin so no lesion results
             5. heal without restitution
             6. join with broken ends of other chromosomes
             7. can result in
             8. change in number of genes (amt of DNA)
             9. change in linear sequence of DNA
             10. Single-hit chromosome aberrations
             11. single break
             12. called a terminal deletion
             13. centric fragment with centromere will move to pole during mitosis
             14. acentric fragment remain in cytoplasm and is eventually reabsorbed
             15. Multi-hit chromosome aberrations
             16. single chromosome sustains multiple hits
             17. 1. Interstitial Deletion
             18. loss of chromosome segment between two breaks
             19. 2. Inversion
             20. chromosome segment between two breaks turn end for end
             21. 3. Ring chromosomes
             22. two broken ends join with each toher to form ring
             23. 4. Dot deletion
             24. breaks very close so small portion of chromosome deleted
             25. Translocation
             26. one break in two different chromosomes, exchange of genes
             27. low doses, single hit, high doses, multi hit
       6. Point mutations
          1. aka gene mutations
          2. permanent change in primary structure of sequence of DNA
          3. Base substitution
             1. direct mispairing of bases during replication
             2. caused by
             3. Ionization of bases
             4. Tautomeric shifts in chemical form of bases
             5. Sense codon - codes for the same amino acid
             6. Missense codon - codes for a different amino acid
             7. Nonsense codon - one that does not code for an amino acid
          4. Frameshift mutation
             1. results from gain or loss of a few nucleotides
             2. linear sequence of nucleotides is changed
             3. frame of reading shifted so triplet code are different
    5. Summary
       1. <html><img src="untitled1.JPG">
11. Radiation induced illness
    1. response of a mammal to a given, acute dose of radiation
    2. sources
       1. animal experiments
       2. Japanese survivors of Hiroshima & Nagasaki
       3. Marshall Islanders (1954)
       4. accidents at nuclear installations
       5. Uranium miners
       6. Case studies
    3. 3 stages in response
       1. Prodromal stage
          1. nausea, vomiting, diarrhea
       2. Latent stage
          1. general healthy appearance, but may surivive or die
       3. Clinical stage
          1. manifestation of the changes in latent period to decide survival or death
    4. A: Subclinical syndrome
       1. Exposure
          1. up to ~2Gy
          2. threshold as low as .5 Gy
          3. no detectable clinical effects under .5 Gy