Topic 6 - Biological Effects and Risk

Biological Effects

• Hall, Radiobiology for the Radiologist
• Cember, Ch. 7

Radiobiology

• The study of the action of ionizing radiations on living things. 
• Biology primer see http://www.biology.arizona.edu/cell_bio/tutorials/cell_cycle/main.html

• Absorption of energy in biological material leads to excitation or ionization.
  • excitation: raises electron to higher energy state without ejecting it from the atom. Radiations can be emitted from this process
  • ionization: ejects electron from the atom. Radiation is also released in the process

Energy absorption in Radiobiology

• Photons absorbed
• Energy deposited in cells and tissues
  • unevenly in discrete packets
  • typical energy per ionizing event ~ 33 eV
  • Typical C=C bond is 4.9 eV
  • Where does remainder go?

Energy Absorption

• Consider 70 kg person
  • LD_50/60 = 4 Gy (400 rad)
  • Energy absorbed: 70 kg · 4 J kg^-1 = 280 J
• What is the caloric equivalent of 280 J?
  • Calorie, metric unit of heat measurement. The small, or gram, calorie (cal) is the amount of heat required to raise the temperature of 1 g of water from 14.5° to 15.5° C.
  • 1 cal equals 4.1840 joules (J)
  • 280 J / 4.186 = 67 calories
• What would be the temperature rise in the body from this energy deposition?
• Why is this a lethal dose?

Direct and Indirect Action

• Biological effects of radiation result principally from damage to DNA
  • DNA the “critical target”
  • Target can be directly damaged by radiation
  • Target can be indirectly damaged

Direct and Indirect Action of Radiation in Biological Systems

Indirect Action in Detail

• H₂O ->  H₂O⁺ + e^-
• H₂O⁺ is an ion (electrically charged)
• H₂O⁺ is also a free radical
  • unpaired electron in outer shell
  • very reactive
• H₂O + H₂O ⁺->  H₃O⁺ + OH^·
• OH^· (hydroxyl radical)
  • 9 electrons, very reactive
  • free radical

Chain of events in X-ray absorption

Incident X-ray beam
ß
Fast electron (e-)
ß
Ion Radical
ß
Free Radical
ß
Chemical changes from bond breakage
ß
Biological Effects

Time Scale of Events

• Initial ionization: 10^-15s
• Ion radical lifetime: 10^-10s
• Free radical lifetime: 10^-5s
• Breakage of bonds and expression of biological effects: hours, days, months, years
  • cell killing: hours to days
  • oncogenic: years
  • mutation in germ cell: generations

Contrast Between Neutrons & Photons

• X and g-rays
  • indirectly ionizing
  • produce fast moving secondary electrons
• Neutrons
  • indirectly ionizing 
  • produce recoil protons, alphas, and heavier atoms

Neutron Interactions

• Direct action dominates for densely ionizing radiations
  • 2⁰ charged particles result in a dense column of ionizations more likely to interact with the DNA
  • Free radicals are also produced
• Recoil protons, alphas, heavy fragments
  • massive -similar size to medium
  • densely ionizing
  • positively charged

Direct and Indirect Action of Radiation in Biological Systems - Neutrons

Summary of Pertinent Conclusions

• X- and g-rays
  • indirectly ionizing
  • produce fast recoil electrons
• Neutrons are indirectly ionizing
  • produce fast recoil protons, a-particles, hcp
• Biological effects due to
  • direct action
  • indirect action
  • about 2/3 of X-ray damage is by indirect action
• Photons - Indirect action dominates
• Heavy particles - direct action dominates

Radiobiological Studies

• Methods Used to Assess Radiation Effects

Radiation Effects: Determining the  Mechanism of Cell Killing

• Sensitive sites are
  • The nucleus
  • Not the cytoplasm
• Early studies
  • microbeams
  • nonmammalian cells
  • cytoplasm or nucleus irradiated

Nucleus vs Cytoplasm - Radiosensitivity

• Habrobracon  (wasp) eggs
• Average # of incident a’s needed to reduce hatchability to 37%:
  • nucleus: 1
  • cytoplasm: 17.6 x 10⁶

Irradiation of Cytoplasm

• Irradiation of part of cytoplasm of a cultured Chinese Hamster cell by a-particles from a polonium-tipped micro-needle
• The irradiated volume is limited by the range of the particles

Other Evidence for Chromosomes As Primary Target in Cell Killing

• Cells killed by radioactive tritiated thymidine incorporated into DNA
• Structural analogues of thymidine when incorporated substantially increase radiosensitivity of cells
• Transplantation of irradiated nucleus into unirradiated cell is lethal at doses that an unirradiated nucleus can survive

Other Methods in Radiation Effects Research

• Methods for assessing dose response
  • in vitro:
    • cell survival curves
      • irradiate seeded cells
      • assess colony formation
      • determine surviving fraction
  • in situ -
    • clonogenic endpoints
      • clones regrowing on irradiated skin
    • Whole animal studies

In Situ Tests - Clonogenic Endpoints

Skin Studies - Example Data

Another “functional” endpoint

• Expose animals to graded dose
• Wait 3 days
• Sacrifice animal
• Thin-section jejunum
• Score for regenerating crypts per circumference

Fractionating dose extends survival

What Have We Learned in Radiation Response of Tissues

• Response is a function of
  • Cell sensitivity
  • Cell population kinetics
• Cell survival curves irrelevant in highly differentiated cells (no mitotic future)
• Closed population of mature cells very resistant
• Self-renewing tissues: dividing cells are weak link

Cell Sensitivity - Cell Type Matters

Cell Types

Tissue response, cont’d

• Loss of reproductive capacity occurs after a few Gy
• Impact to tissue/organ after dosing depends on how well it can function with reduced number of cells
• Time between dose & expression of damage is variable
  • lifetime of mature cell
  • time for stem cell to mature to functional state

Radiation Response, cont’d

• Blood cells
  • lifetime 3-4 months
  • damage to stem cells not evident until red blood cell pool dies off
• Intestinal epithelium
  • mature villi - short lifespan
  • impact obvious within few days

Parenchymal Cells & Connective Tissue

• Parenchymal cells
  • perform unique function of particular tissue
  • supported & held in place by connective tissue
  • supplied with O₂ & nutrients by blood vessels
• Casaret’s classification
  • connective tissue, blood vessels intermediate in sensitivity between most sensitive and most resistant parenchymal cells.
• Radiation effects parenchyma, connective tissues, vasculature, controversy on which responsible for “late effects”  

Casaret’s Classification of Mammalian Parenchymal Cell Sensitivity

• Type
  • I - VIM
  • II- DIM
  • III RPM
  • IV FPM
• Example
  • crypt
  • myelocytes
  • liver
  • nerve
• Sensitivity
  • High
  • Intermediate
  • Moderate
  • Low

Supporting structures (connective tissues, endothelial cells)

Noted exception to the classification - small lymphocyte, which disappears after small doses. It doesn’t divide, yet is one of the most sensitive mammalian cells.

Cell Sensitivity - Population Kinetics

The Cell Cycle

• An ordered set of events, culminating in cell growth and division into two daughter cells
• T_c, full mitotic cycle
• Only mitosis can be distinguished when examining cells under a scope -
  • chromosomes are condensed
  • mitosis ~ 1 hour
• Radiography used to study

Mitosis

Cell Cycle Times

• Radiography, other techniques
  • used to view cells
  • help identify cell cycle length
• All proliferating mammal cells have
  • mitotic cycle
  • followed by G₁
  • period of DNA synthesis (S)
  • G₂
  • mitosis repeats

Length of Cell Cycle

• M, S, G₂ vary little between cells
•   T_c varies 10 hours - 100’s of hours - mainly due to G₁

*In all cell lines cultured or in vivo, S never exceeds 15 h

X-Ray Sensitivity of Synchronous Cells

• Chinese Hamster cells in culture
  • Irradiated with 6.6 Gy after mitosis
    • time of exposure varied
    • cells irradiated at different stages in cell cycle
    • Shown in next figure
  • Key points
    • mid-late S least sensitive

X-Rays & Synchronously Dividing Cell Cultures after 6.6 Gy

Fraction of HeLa cells surviving a dose of 3 Gy

Key Differences between HeLa & Chinese Hamster Cells

• Cycle time
  • T_m
  • T_s
  • T_c
  • T_G2
  • T_G1
• Hamster
• 1
• 6
• 11
• 3
• 1
• HeLa
  • 1
  • 8
  • 24
  • 4
  • 11
• For S phase, HeLa & Hampster are similar
• Major Difference is Length of G1
  

Radiosensitivity & Mitotic Cycle

• Sensitivity
  • Cells most sensitive close to mitosis
  • Resistance greatest in latter part of S
  • For long G₁’s, there is an early resistance period followed by sensitive one at the end of G₁
  • G₂ ~ M in sensitivity
• Probably Repair is the key

Why’s & Wherefores of Sensitivity

• Cellular progression controlled by “checkpoint” genes
  • ensure completion of events prior to progression through cell cycle,
  • at G₂ cells are halted to inventory & repair damage before mitosis
  • cells where checkpoint gene inactivated
    • these cells move directly to mitosis with damaged chromosomes
    • are more sensitive to UV or g radiation or any DNA damaging agent

Cell Cycle Summary

• Cell cycle components
  • M, G₁, S, G₂
• Cycles in culture
  • crypt cells, 9 - 10 hours
  • stem cells (mouse skin) 200 hr
  • due to G₁
• Cells most radiosensitive in M, G₂
• Resistant in late S

Implications of Cell Cycle Sensitivity & Tumor Therapy

• Tumor cells initially asynchronous
  • (distributed throughout cycle)
  • Deliver dose
  • Kill sensitive cells (M)
• Radiation ~synchronizes population
• Allow cells to progress
  • “Sensitized” cycling population
• Time next dose to sensitive phase
  • Good in theory
  • Need tumour kinetics to accomplish

Age-Response Implications, cont’d

• Time next dose to correspond to sensitive phase of tumor - maximize cell killing
• Sensitization due to reassortment
• Therapeutic gain (tumors are rapidly dividing as opposed to most normal tissues)

Effect of Oxygen on Cells

• OER
  • oxygen enhancement ratio
  • aerated cells more sensitive
  • typical values 2.5 - 3 for g and x-rays
  • oxygen reacts with free radicals to produce peroxide - non repairable damage
• G₁: 2.3 - 2.4
• S: 2.8 - 2.9
• G₂ intermediate
• Implications are in radiation therapy

Oxygen Fixation Hypothesis

In general, the free radical reactions go like this:

O₂ Concentration & X ray Impacts

•   Bacteria & mammalian systems show similar effects
• A: air (210,000 ppm)
• B: 2200 ppm O₂(0.25%)
• C: 355 ppm O₂
• D: 100 ppm O₂
• E: 10 ppm O₂

Radiosensitivity & O₂ Concentration

• O₂ levels
  • 20 % normal
  • 16% dizziness
  • 10% immediate unconsciousness
• 2% is ~ plateau
• 0.5% is 50% effect
• Doesn’t take much for impact

Oxygen Movement Through Tissues

• Oxygen passes from blood to capillaries to tissue cells.
• CO₂ passes from tissue cells to capillaries to blood
• Hemoglobin binds with O₂ in O₂ rich environment (lung)
• Gases diffuse from high to low concentration
  • O₂ diffuses from blood to tissue fluid
  • Same, in reverse for CO₂

Diffusion of Oxygen Through a Capillary

Oxygen Movement, cont’d

• O₂ is on plasma & hemoglobin
  • small fraction on plasma
  • immediately available to diffuse to cells
  • remainder is with hemoglobin
• Many cells are borderline hypoxic
  • Liver
  • Skeletal cells

Conditions of Hypoxia

• Chronic hypoxia
  • Limited diffusion distance of O₂ through respiring tissues
  • Cells may be hypoxic for a long time
  • Tumors may outgrow blood supply, have O₂ starved regions
• Acute
  • Blood vessels can be temporarily shut down
  • Rapidly reopen to resupply tissues

Significance of O₂ Effect and Solid Tumors

• Solid tumor growth limited by oxygen supply
  • Often with necrotic (dead) center
  • As tumor grows, necrotic center expands
  • Thickness of healthy “sheath” remains constant
    • Estimated diffusion distance of O₂ in respiring tissue is 70mm

Thomlinson & Gray (1955) Key Paper

Reoxygenation in Tumors

• Mouse Sarcoma
  • Initial survey 14% of tumor cells hypoxic
• Series of dose fractions 5 dose, 1.9 Gy/day
  • Reassessed fraction hypoxic cells (18%)
• Similar experiment,
  • M-Th, 4 dose fractions,
  • Next day hypoxic cells 14%
• Implies fraction of hypoxic cells before/after therapy are same

Reoxygenation

• What is going on?
• Treatment kills oxygenated tumor cells
• Hypoxic ones become oxygenated
• This is a good thing
• Hypoxic cells reoxygenate after radiation therapy and become radiation sensitive

Process of Reoxygenation

• If reoxygenation occurs, then the presence of hypoxic cells won’t significantly impact the outcome of the multi-fraction regime

Mechanism of Reoxygenation

• Reoxygenation variable
  • Fast  (hours)
  • Slow (days)
• Some tumors exhibit two component process
  • related to type of hypoxia being reversed
  • chronic
  • acute
• Slow, long term
  • Revascularization occurs (dead cells are broken down and removed)
  • Tumor shrinks so all within O₂ diffusion zone
  • Applies to chronically hypoxic cells
• Fast, short term
  • Reoxygenation of acutely hypoxic cells
  • Blood supply reopens & rapid reoxygenation occurs

Significance for Radiotherapy

• Tumors (animal) include
  • Aerated
  • Hypoxic cells
• Hypoxia confers protection from
  • X-rays
  • Chemotherapeutic agents (e.g., bleomycin)
• Presence of O₂ enhances cell killing
• Reoxygenation of hypoxic cells could enhance cancer treatment through radiation therapy
• Reoxygenation pattern not well known for many systems
• If human tumors reoxygenate quickly
  • Multifraction therapy could deal with “resistant” subpopulation
  • Dosing at later intervals would maximize killing
• Human tumor data not available
  • Reoxygenation suggested from multifraction therapies
  • 60 Gy in 30 treatments eradicates many tumors where “cure” not expected

Other Factors Affecting Dose Response

• Relative Biological Effectiveness
  • General rule - high LET radiation more damaging than low LET
  • Different radiations can be compared:
  • RBE = D_x/D
  • D is dose of a given radiation required to produce a specific biological endpoint
  • D_xis the X-ray dose needed under same conditions to produce same endpoint
• RBE results used to produce Q, w_R values
• Dose Rate

Other Factors Affecting Dose Response

• Chemical Modifiers
  • Sensitizers
    • Oxygen
  • Radioprotectors
    • Sulfhydryl compounds (free radical scavengers)
• Heat

Radiation Effects on Living Systems

• Varies with:
  • dose magnitude
  • duration of exposure
  • region exposed
• Responses categorized:
  • stochastic:  mutagenic/carcinogenic/ teratogenic
  • nonstochastic (deterministic) - threshold

Radiation Effects

• Cellular Level
  • cell function
  • cell division
  • chromosomal damage
  • neoplastic transformation
• Organs
  • prompt (death)
  • delayed (fibrosis)
• Individual

Factors Affecting Cellular Radiosensitivity

• Cells that divide more rapidly are more sensitive to the effects of radiation ...
• … essentially because the resulting effect is seen more rapidly.

Factors Influencing Biological Effect

• Total absorbed energy (dose)
• Dose rate
  • Acute (seconds, minutes)
  • Chronic (days, years)
• Type of radiation
• Source of radiation
  • External
  • Internal
• Age at exposure

Factors Influencing Biological Effect

• Time since exposure
• Area or location being irradiated
  • Localized (cells, organ)
  • Extremities (hands, forearms, feet, lower legs)
  • Entire body (trunk including head)
  • Superficial dose (skin only - shallow)
  • Deep tissue (“deep dose”)

Categorizing Exposure

• Acute
  • Single, large, short-term whole-body dose
  • Characterized by four sequential stages
    • Initial (prodromal) - lasts 48 hrs
    • Latent - 48 hr to 3 weeks
    • Manifest illness - 6 to 8 weeks postexposure
    • Recovery (if death does not occur) weeks-months
• Chronic
  • Lower, protracted dose

Terms

Acute exposure

dose received in a short time (seconds, minutes)

Acute effects

symptoms occur shortly after exposure

Chronic exposure

dose received over longer time periods (hrs, days)

Delayed effects

symptoms occur after a latent (dormant) period

Somatic effects

those which occur in the person exposed

Genetic effects

those which occur in the offspring of exposed persons

Stochastic effects

likelihood of effect is random, but increases with increasing dose

Non-stochastic effects

likelihood of effect is based solely on dose exceeding some threshold

Symptoms of Acute Radiation Sickness

• Three categories (E. Hall, 1994)
  • Hemopoietic:  3-8 Gy LD_50/60
    • radiation damages precursors to red/white blood cells & platelets
    • prodromal may occur immediately
    • symptoms: septicemia,
    • survival mixed
    • examples include Chernobyl personnel (203 exhibited symptoms, 13 died)
  • Gastrointestinal : >10 Gy
    • radiation depopulates GI epithelium (crypt cells)
    • abdominal pain/fever, diarrhea, dehydration
    • death 3 to 10 days (no record of human survivors above 10 Gy)
    • examples include Chernobyl firefighters
  • Cerebrovascular : > 100 Gy
    • death in minutes to hours
    • examples include criticality accidents

Delayed Effects

• Some biological effects, either from acute or chronic exposure may take a long time to develop & become evident in exposed individual. 
• Called delayed or late, somatic effects
• Cancer
  • Leukemia
  • Bone Cancer
  • Lung Cancer
• Life Shortening
• Cataracts

Other Effects

• Mental Retardation
• Genetic Effects

Sources of Human Data

• Considerable body of data exists
  • Atomic-bomb survivors
    • RERF
    • 93,000 survivors
    • 27,000 nonexposed comparators
• Medical patients
  • Therapeutic
    • Thymus treatments - increase in thyroid tumors
    • Tinea capitis (ringworm of the scalp)- 10,000 children- 6 fold increase in malignant tumors of thyroid
    • Ankylosing spondylitis (inflammatory arthritis that affects the spinal joints) - 14,000 patients - increase in leukemia
  • Diagnostic
• Body of data, cont’d
  • Radium dial painters - several hundred, bone cancer
  • Uranium miners - thousands - lung cancer
  • Accidents
    • Critical assemblies
    • Particle accelerators
    • Radiation devices
    • Weapons fallout
    • Chornobyl

Examples of Dose-Response Relationships

Non-Stochastic (Deterministic) Effects

• Occurs above threshold dose
• Severity increases with dose
  • Alopecia (hair loss)
  • Cataracts
  • Erythema (skin reddening)
  • Radiation Sickness
  • Temporary Sterility

Stochastic (Probabilistic) Effects

• Occurs by chance
• Probability increases with dose

Dose Response: Radiation Carcinogenesis

• Two effects of radiation exposure:
  • deterministic (threshold)
  • stochastic: cancer
• Radiation Standards
  • set below threshold
  • set to limit stochastic risk
• Controversy on “Risk”
  • possibility of threshold
  • low-dose data limited

Linear non-threshold hypothesis

• Fits data
• Single hit effect
• Accepted by most regulatory bodies
• Conservative
• Basis of current regulations

Biological Effects Summary:

• What We Know and What We Don’t Know About Radiation Health Effects
• Borrowed from a talk by G. Roessler “An Educational Briefing By The HEALTH PHYSICS SOCIETY Specialists In Radiation Safety March 28, 2001”

Health Effects of Ionizing Radiation

• More known about radiation effects than effects from any other potentially toxic substance -- more than chemicals

Sources of Information

• Molecules and Cells
• Animals
• Humans (Epidemiological Studies)
  • Medical
  • Occupational
  • Hiroshima and Nagasaki

Time Frame of Effects

• Physical -- less than seconds
• Chemical -- seconds
• Biological -- seconds to many years
  • Reactions with molecules, cells
  • Tissue changes
  • Cancer, leukemia

Effects

• The radiation may enter the body but miss important targets
• The radiation may not cause any damage to a target
• The damage may be repaired
• A damaged cell may die
• A damaged cell may be changed (mutated)
• High Doses
  • May Lead to Early Effects or Death
• Low Doses
  • Cancer and Leukemia
  • Inherited Effects
  • Embryo and Fetus

What We Know

• Radiation is a weak carcinogen
• The probability of getting cancer is a function of the dose
• No evidence of any cancer effects below about 10 rem

What We Don’t Know

• If there are:
  • any bad effects below about 10 rem
  • beneficial effects below about 10 rem
  • any effects other than cancer and leukemia
  • any inherited effects at any dose

Important Points

• High normal incidence of cancer (about 30%)
  • Can’t prove relationship on an individual basis -  only an increased relative risk on a large group basis
• Long latent period
  • Leukemia - 2 to 7 years from exposure
  • Cancer - 10 to 40 or 50 years from exposure
• Dose to tissue
  • Cancer won’t occur in an organ of the body unless that organ has received a dose

Another Important Point

• Some cancers are not associated with low-to-moderate doses of ionizing radiation     
  • Hodgkin's Disease
  • Non-Hodgkin's Disease
  • Chronic Lymphocytic Leukemia
  • Cutaneous Malignant Melanoma
  • Uterus
  • Prostate

Links

http://www.nas.edu/ssb/besrmenu.html
http://www.rerf.or.jp/eigo/experhp/rerfhome.htm