2. Timeline
• Radioactivity was discovered by Antonio Henri Becquerel in
1898
• Radium was isolated by Pierre & Marie Curie also in 1898
• First recorded radiobiology experiment was performed by
Becquerel - He inadvertently left a Radium container in his
pocket, resulting in skin erythema about 2 weeks later
• In 1901, Pierre Curie repeated this experiment on his forearm
and produced an ulceration and charted its healing
• Field of Radiobiology began in 1901
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3. Energy Absorption In Radiobiology
• Energy is absorbed in form of small discrete
packets – PHOTONS
• The energy deposition is uneven, discrete &
non-uniform
• Leads to chemical & biological changes
• Absorption of radiation may lead to ionization
or excitation of atoms
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5. Biological Interactions of Radiation
• The interaction of radiation with cells is a probability
function or a matter of chance. i.e., it may or may
not interact and if interaction occurs, damage may or
may not be produced
• Radiation interaction in a cell is non‐selective.i.e., the
energy from ionization radiation is deposited
randomly in the cell. No areas of the cell are
“preferred” or “chosen” by the radiation
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6. • The initial deposition of energy occurs very
rapidly in a period of ~ 10-17 seconds
• The biologic changes from radiation occur only
after a period of time (latent period) which
depends on the initial dose and varies from
minutes to weeks, or even years
Biological Interactions of Radiation
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7. Biological Interactions of Radiation
• When ionizing radiation interacts with a cell,
possibility of either ionization or excitation
exists in macromolecules (e.g., DNA) or in the
medium
• Based on the site of these interactions, the
action of radiation on cell can be classified as:
o Direct action
o Indirect action
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10. Direct Action
• Occurs when an ionizing
particle interacts with and is
absorbed by a biologic
macromolecule such as DNA,
RNA or enzyme in the cell
• These ionized macromolecules
are now abnormal structures
• They lose their normal
functioning unless repaired in
time which is infrequent with
direct damage
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11. Indirect Action
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• Refers to absorption of
ionizing radiation in the
medium when the molecules
are suspended primarily in
water
• Absorption of radiation by
water molecule results in
production of ion pairs
•The ion pairs react and cause
damage to cellular
macromolecules
14. • As there exists more water in the cell than any structural
component, the probability of radiation damage occurring
through indirect action is >> than the probability of damage
occurring through direct action
• In addition, indirect action occurs primarily but not exclusively
from free radicals resulting from ionization of water
• The ionization of other cellular constituents, particularly fat,
also can result in free radicals formation
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15. Oxygen Fixation Hypothesis
• If molecular oxygen is present, DNA reacts with the free
radicals to yield DNA radical (R·)
• The DNA radical can be chemically restored to its reduced
form through reaction with a sulfhydryl (SH) group
• However, the formation of RO2 ·, an organic peroxide,
represents a non-restorable form of the target material
• Oxygen may be thus said to “fix” or make permanent the
radiation lesion
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20. Absorption of Radiation
• Absorption of X-rays
• Absorption of Neutrons
• Absorption of Protons
• Absorption of Heavy Ions
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21. Absorption of X-Rays
• Depends on energy of concerned photon and
chemical composition of absorber
• Mainly two processes involved :
Compton effect
Photo-electric effect
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22. Absorption of Neutrons
• Interact with nuclei of atoms,not planetary electrons
• Direct damage to DNA is the predominant mode of action
• Produce fast recoil particles
• In case of higher energy neutrons - “spallation products” are
formed
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23. Absorption of Protons
• Protons interact with both planetary electrons and with
nuclei of atoms
• Planetary electrons – Fast recoil electrons
• Nuclei of atoms – Heavy secondary particles
• As proton energy increases, nuclear disintegration
increases
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24. Absorption of Heavy Ions
• Direct action of radiation is predominant
• As the density of ionisation increases, probability of
direct interaction between particle track and target
molecule increases
• Radioprotective compounds are effective for x-rays
and gamma rays but are of little use in case of
heavier ions, neutrons or alpha particles
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29. SSB and DSB in DNA
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• Little biologic significance
• Repaired readily using
opposite strand as template
and defect may result in
mutation
• More common
• 1000 SSB per cell after 1- 2
Gy
• Most important lesions
produced by radiation
• Defect in repair may result
in cell killing, carcinogenesis
or mutation
• Less common
• 40 DSBs per cell
Single Stranded Break Double Stranded Break
30. Measurement of DNA breaks
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Pulsed field gel electrophoresis(PFGE)
Single cell electrophoresis(comet assay)
DNA damage induced nuclear foci assay
31. • Most widely used method to detect the induction and repair of
DNA DSBs
• It is based on the electrophoretic elution of DNA from agarose
plugs within which irradiated cells have been embedded and
lysed
• PFGE allows separation of DNA fragments according to size with
the assumption that DNA DSBs are induced randomly
• The fraction of DNA released from the agarose plug is directly
proportional to dose
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Pulsed Field Gel Electrophoresis(PFGE)
32. Single Cell Electrophoresis (Comet assay)
• It has the advantage of detecting differences in DNA
damage and repair at the single-cell level
• Cells are exposed to ionizing radiation, embedded in
agarose, and lysed under neutral buffer conditions to
quantify induction and repair of DNA DSBs
• To assess DNA SSBs and alkaline-sensitive sites, lysis
is performed with an alkaline buffer
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33. Single Cell Electrophoresis(Comet assay)
• As a result of the lysis and
electrophoresis conditions,
the fragmented DNA
migrates
• Unirradiated cells possess
a near spherical
appearance, whereas the
fragmented DNA in
irradiated cells gives the
appearance of a comet
when stained with
ethidium bromide
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34. DNA damage induced nuclear foci
assay
• In response to ionizing radiation, complexes of
signaling and repair proteins localize to sites of DNA
strand breaks
• It can be carried out on both tissue sections and
individual cell preparations
• Cells/tissues are incubated with a specific antibody
raised to the signaling/repair protein of interest
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35. DNA damage induced nuclear foci assay
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• Binding of the antibody is
then detected with a
secondary antibody, which
carries a fluorescent tag
• Fluorescence microscopy
detects the location and
intensity of the tag, which is
then quantified
36. Radiation Induced DNA Crosslinks
• DNA – Protein crosslinks
• DNA-DNA intrastand crosslinks
• DNA-DNA interstrand crosslinks
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37. Crosslinking of DNA
• Occurs under conditions of oxidative stress, in which free
oxygen radicals generate reactive intermediates
• They react with two nucleotides of DNA, forming a covalent
linkage between them
• This crosslink can occur within the same strand (intrastrand)
or between opposite strands of double-stranded DNA
(interstrand) or between an oxidised protein and DNA (DNA -
protein crosslink)
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38. Crosslinking of DNA
• These adducts interfere
with cellular metabolism,
such as DNA replication and
transcription, triggering cell
death
• Crosslinks can, however,
be repaired through
excision pathways
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39. Radiation Induced Chromosomal
Aberrations
• Occurs when cell is irradiated early in interphase, before the
chromosome material has been duplicated
• Radiation-induced break is in a single strand of chromatin
• During the DNA synthesis, this strand of chromatin lays down
an identical strand next to itself and replicates the break that
has been produced
• Leads to a chromosome aberration visible at the next mitosis
because there is an identical break in the corresponding
points of a pair of chromatin strands
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40. Dicentric Chromosome
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•Occurs by irradiation of
prereplication chromosomes
•Break is produced in each of
two separate chromosomes
•The “sticky” ends may join
incorrectly to form an
interchange between the two
chromosomes
•Replication then occurs in the
DNA synthetic period
•One chromosome has two
centromeres: a dicentric
41. Ring Aberration
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•Formation of a ring occurs
by irradiation of a
prereplication (i.e., G1)
chromosome
•A break occurs in each arm
of the same chromosome
•The sticky ends rejoin
incorrectly to form a ring and
an acentric fragment
•Replication then occurs
42. Radiation Induced Chromatid
Aberrations
• Occurs when cell is irradiated in late interphase after the DNA
material has doubled
• In regions away from the centromere, chromatid arms are
well separated, and the radiation might break one chromatid
without breaking its sister chromatid
• A break that occurs in a single chromatid arm after
chromosome replication and leaves the opposite arm of the
same chromosome undamaged leads to chromatid
aberrations
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43. Anaphase Bridge
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• By irradiation of a postreplication
chromosome
• Breaks occur in each chromatid of
the same chromosome
• Incorrect rejoining of the sticky ends
- sister union
• At next anaphase, the acentric
fragment is lost, one centromere of
the dicentric goes to each pole, and
the chromatid is stretched between
the poles
• This aberration is likely to be lethal
44. Non-lethal Chromosomal Lesions
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Symmetric translocation :
•Radiation produces breaks in two different
prereplication chromosomes
•The broken pieces are exchanged between
•the two chromosomes, and the “sticky”
ends rejoin
•Might lead to activation of an oncogene
Deletion
• Radiation produces two breaks in the same
arm of the same chromosome
45. Chromosomal Aberrations in Human
Lymphocytes
• In blood samples obtained for cytogenetic evaluation within a
few days to a few weeks after total body irradiation, the
frequency of asymmetric aberrations (dicentrics and rings) in the
lymphocytes reflects the dose received
• The dose can be estimated by comparison with in vitro cultures
exposed to known doses
• Cytogenetic evaluations in cultured lymphocytes readily can
detect a recent total body exposure of as low as 0.25 Gy in the
exposed person
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46. • Useful in distinguishing between “real” and “suspected”
exposures involving film badges
• Dicentrics are “unstable” aberrations because their number
declines with time after irradiation
• Symmetric translocations are “stable” aberrations because they
persist for many years
• If many years have elapsed, dicentrics underestimates the dose
and only stable aberrations such as translocations give an
accurate picture
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Chromosomal Aberrations in
Human Lymphocytes
49. Key Points
• The timescale of radiation effects can be divided into 3 phases –
physical, chemical and biological
• DNA within the cell nucleus is the primary target for radiation
effects
• Free radicals produced by water radiolysis contribute to 70 % of the
effect
• A range of DNA lesions are introduced by radiation including SSBs,
DSBs, base damage, nucleotide damage, crosslink formation
• DNA DSBs yields correlate best with cellular effects
• Oxygen and radiation quality are important response modifiers
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