CHROMOSOME ABERRATIONS

1
CHROMOSOME ABERRATIONS

• AND
• HUMAN LYMPHOCYTES

2
A LITTLE BACKGROUND

• Traditionally the study of radiation damage on
  chromosomes was principally conducted on plant
  cells (e.g. Tradescantia paludosa)
• Study on mammalian cells hampered by large number
  of chromosomes per cell and small size of nucleus
  
• Plants contain fewer and generally much larger
  chromosomes
• Study of chromosome aberrations through the
  effects of ionizing radiation described in terms
  of their appearance at first metaphase after
  exposure to radiation

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CELL CYCLE

• From Brum GD, et al (1989). BiologyExploring
  Life pg. 156

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HOW IT HAPPENS

• When cells are irradiated, breaks are produced in
  the chromosomes
• Broken ends appear to be sticky and can rejoin
  with any other sticky end
• Once breaks are produced, different fragments may
  behave in a variety of ways
• Breaks may rejoin in their original configuration
• Breaks may fail to rejoin and give rise to a
  deletion
• Broken ends may resort and rejoin other broken
  ends and give rise to chromosomes that appear to
  be grossly distorted

5
TYPES OF ABERRATIONS

• ABERRATIONS SEEN AT METAPHASE ARE OF 2 CLASSES
• Chromosome Aberrations
• - result if cell is irradiated early in
  interphase (G1),
• before chromosome material has been
  duplicated
• - during DNA synthetic phase that follows,
  this
• strand of chromatin lays down an identical
  strand
• next to itself
• Chromatid Aberration
• - result if cell is irradiated later in
  interphase after
• DNA has doubled (G2) and chromosome consist
  of
• 2 strands of chromatin
• - break that occurs in a single chromatid
  arm, leaves
• opposite arm of same chromosome undamaged

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EXAMPLES OF ABBERATIONS

• Many types of aberrations and rearrangements are
  possible
• 3 Types are lethal to the cell
• Dicentric, Centric Ring (chromosome aberrations)
  and Anaphase Bridge (chromatid aberration)
• 2 Important Non-Lethal Rearrangements
• Symmetric translocations and small deletions
  (interstitial and terminal)
• Associated with several human malignancies via
  activation of oncogenes or loss of suppressor
  genes

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LETHAL ABERRATIONS

• All 3 represent gross chromosomal distortions
• Dicentric (or Tricentric)
• Involves interchange between 2 separate
• chromosomes
• If break occurs in each one early in interphase
  and sticky ends are close together they may join
• this interchange is replicated during DNA
  synthesis and results in a distorted chromosome
  with 2 (or 3) centromeres and an acentric fragment

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DICENTRIC CHROMOSOME

• From Radiobiology for the Radiologist, pg 24

9

• From Radiobiology for the Radiologist, pg 25

10

• Centric Ring
• Break induced by radiation in each arm of a
  single chromatid early in cell cycle
• The sticky ends may rejoin to form a ring with a
  centromere and an acentric fragment
• Later during DNA synthetic phase the chromosome
  is replicated

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CENTRIC RING

• From Radiobiology for the Radiologist, pg 24

12

• From Radiobiology for the Radiologist, pg 26

13

• Anaphase Bridge
• Results from breaks that occur late in cell cycle
  (G2), after chromosome has replicated
• Breaks may occur in both chromatids of the same
  chromosome, sticky ends may rejoin incorrectly to
  form a sister union
• At anaphase, when the 2 sets of chromosomes move
  to opposite poles, the section of chromatin
  between the centromeres is stretched across
  between the poles, hindering separation into new
  daughter cells
• Difficult to see in human cell cultures since
  bridge is only evident at anaphase

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ANAPHASE BRIDGE

• From Radiobiology for the Radiologist, pg 24

15

• From Radiobiology for the Radiologist, pg 27

16

• Symmetric Translocation
• Involves break in 2 pre-replication chromosomes,
  with broken ends being exchanged between the 2
  chromosomes
• Divided into pericentric (includes centromere)
  and paracentric (confined to 1 chromosome arm)
  inversions
• Difficult to see in conventional preparation, but
  easy to observe with fluorescent in situ
  hybridization (FISH)
• A translocation is associated with several human
  malignancies by the activation of an oncogene
  (e.g. Burkitts lymphoma)

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• Small Deletion
• Terminal deletion
• Loss of genetic material from end of chromatid
• Not possible to distinguish between non-sister
  union isochromatid and chromosome-type
• Interstitial deletion
• Minute very small deletion show as small paired
  dots
• Acentric ring larger interstitial deletion show
  as acentric rings
• Deletion may be associated with carcinogenesis if
  loss of material includes a suppressor gene

18

• From Biological Dosimetry, pg 18

19
HUMAN LYMPHOCYTES AS A BIOMARKER

• Chromosomal aberrations in peripheral lymphocytes
  currently most fully developed biological
  indicator of exposure to ionizing radiation
• In vitro and in vivo irradiation of blood
  lymphocytes produces similar yields of chromosome
  damage per rad, so that the observed of
  aberrations in exposed persons can be related to
  the dose by comparison with an in vitro produced
  dose-response curve
• In circulation they are generally non-dividing
  and accumulate damage typical of that caused by
  irradiation in the G0/G1 cell cycle stage

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RADIATION EFFECTS ON LYMPHOCYTES

• Undamaged cells contain 46 chromosomes, each with
  1 centromere
• Damaged cells may display a number of types of
  aberrations including dicentrics, centric rings,
  acentric fragments and translocations, all of
  which may be related to radiation dose

21

• From Biological Dosimetry, pg 16

22
DICENTRICS AS A BIOMARKER

• For biological dosimetry the dicentric
  historically has been the aberration of choice
• most reliably scored, easily seen in chromosome
  spread
• low level of occurrence in unirradiated persons
  (about 1/1000 cells)
• 10x more common than rings and about as common as
  excess fragments
• Less reliably scored
• Have a higher occurrence (1/300 cell) in
  unirradiated persons

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SCORING LYMPHOCYTE ABERRATIONS

• Lymphocytes may be stimulated to divide in vitro
  by adding phytohemagglutinin (PHA)
• Stopped at their 1st metaphase by addition of
  Colcemid after about 45 hrs of culture
• Slides containing metaphase spreads stained with
  Giesma or FISH probes and scored
• Contamination with 2nd division cells made by
  incorporating bromodeoxyuridine (BrdU) into the
  culture
• In suspected exposure case, typically 500 cells
  are scored

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DOSE-RESPONSE CURVES

• Yc?D?D2
• where Y is yield of dicentrics/cell, D is
  dose(Gy) and c,?,? are fitted constants
• From Radiobiology for the Radiologist, pg 30

25

• From The use of Chromosomal Aberrations in Human
  Lymphocytes for Biological Dosimetry, pg S41

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LIMITATIONS OF SCORING DICENTRICS

• Major limitation of using dicentrics for
  dosimetry is loss of lymphocytes from the blood
• Yield of measured dicentrics after an
  irradiation would decrease with time (1/2 life is
  about 3 yrs), although this is uncertain
• When replaced by stem cell division, new
  lymphocytes will tend not to contain dicentrics
  due to elimination at anaphase
• unstable aberration since it is lethal to the
  cell

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FISH AND TRANSLOCATIONS

• Cells which contain balanced translocations tend
  not to be eliminated at cell division
• Difficult to see in a conventional preparation,
  but easy to observe with FISH
• Probes now available for every human chromosome
  that make them fluorescent in bright colours
• Since this is a stable aberration, the advantage
  of this method is its application to persons
  exposed to radiation at some time in the past