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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5196_Библиотеки_им_академика_М_И_Перельмана.pdf
CHAPTER 7 Molecular and Cellular Radiation Biology
117
DIRECT ACTION
X-ray photon
DNA Helix
INDIRECT ACTION
Water
Reactive
DNA Helix
Free radical
X-ray photon
molecule
Fig. 7.3 The action of radiation on the cell can be direct or
indirect. It is direct when ionizing particles interact with a vital
biologic macromolecule such as DNA. The action is indirect
when ionizing particles interact with a water molecule, thus
resulting in the creation of ions and reactive free radicals that
eventually produce toxic substances that can create biologic
damage. (From Radiobiology and radiation protection: Mosby’s
radiographic instructional series, St. Louis, 1999, Mosby.)
produce a vast number of ionizations in a very short
distance of travel. This response is in stark contrast to
exposure to low-LET radiation, such as x-rays, which
are only sparsely ionizing.
Direct Action Characteristics
When ionizing particles interact directly with vital biologic macromolecules such as:
• DNA
• Ribonucleic acid (RNA)
• Proteins
• Enzymes
damage to these molecules occurs from the absorp-
tion of energy through photoelectric and Compton
interactions. The ionization, or even the excitation,
of the atoms of the biologic macromolecules can result
in breakage of the macromolecules’ intricate chemical
bonds, causing them to become abnormal structures.
This change could lead to inappropriate cellular chemical reactions. As an example, when enzyme molecules
are damaged by interaction with ionizing particles,
essential biochemical processes that depend on the
facilitating action of the enzymes may not occur in
the cell when needed. Should this occur during the
process associated with the synthesis of a particular
protein, the protein will not be manufactured, and
if this protein were intended to perform a specific function, its failure to exist would hinder or prevent that
function. If other cell operations depend on the suppressed function, these operations will be compromised
as well, and consequently, a negative biologic sequence
occurs.
Radiolysis of Water
Ionization of Water Molecules. Radiolysis refers to
the dissociation of molecules by ionizing radiation.
Thus, when x-ray photons interact with water molecules
contained within the human body, this can result in
their separation into other molecular components. For
example, one type of interaction could create an ion pair
consisting of a water molecule with a positive charge
(HOH1) and a single electron (e2). After the original
ionization of the water molecule, several other successive
reactions are possible. One such outcome is that the
positively charged water molecule (HOH1) may recombine with the electron (e2) to reform a stable water
molecule (HOH1 1 e2 5 H2O). If this happens, no
damage will occur. Alternatively, the electron (the negative ion) may join with another water molecule to
produce a negative water ion (H2O 1 e2 5 HOH2).
This result, however, can have unfavorable consequences.
Production of Free Radicals. The positive water mol-
ecule (HOH1) and the negative water molecule (HOH2)
are fundamentally unstable. Hence, they will soon break
apart into smaller molecules. HOH1 decomposes into a
hydrogen ion (H1) and the neutral oxygen–hydrogen
combination called the hydroxyl radical (OH*), whereas
HOH2 becomes a hydroxyl ion (OH2) and a hydrogen
radical (H*). The asterisk symbolizes a free radical. A
free radical is a molecular unit that has no net electrical
charge but, because of having an unpaired valence elec-
tron, it is an extremely reactive entity, which typically
exists as such for approximately one millisecond before
pairing up with another electron, even if to do this it
has to break a chemical bond in a vital molecule.
In summary, the interaction of radiation with water
ultimately results in the formation of an ion pair, H1
and OH2, and two free radicals, H* and OH* (Fig. 7.4).
Production of Adverse Chemical Reactions and Biologic Damage. Energetic hydrogen and hydroxyl free
radicals within the human body can initiate undesirable
118
CHAPTER 7 Molecular and Cellular Radiation Biology
Hydrogen atoms
Water
X-ray photon
molecule
Ion
pair
Water
O)
(H
2
and
−
e
−
e
combines with
Oxygen atom
Positively charged
water molecule =
positive ion
Electron = negative
ion
molecule
(H
O)
2
Hydrogen
ion
*
and
Hydroxyl
radical
Unstable
water molecule
with negative
charge
Fig. 7.4 Radiolysis of water. The final result of the interaction of radiation with water is the formation of an
ion pair (H1 and OH–) and two free radicals (H* and OH*).
chemical reactions. In the process, the free radicals’
excess energy is transferred to some biologic molecules,
thereby either breaking these molecules’ chemical
bonds, or at the very least, causing point lesions
(i.e., altered areas caused by the fracturing of a single
chemical bond) in the molecule. Approximately twothirds of all radiation-induced damage is believed to be
ultimately caused by the hydroxyl free radical (OH*).
Also, because free radicals have excess energy and can
travel through the cell, they are capable of destructively
interacting with other molecules located at some distance from the radicals’ place of origin.
*
and
Hydrogen
Hydroxyl
ion
radical
Production of Cell-Damaging Substances. Hydrogen
and hydroxyl radicals are not the only destructive substances produced during the radiolysis of water. A
hydroxyl radical (OH*) can bond with another hydroxyl
radical (OH*) and form hydrogen peroxide (OH* 1
OH* 5 H2O2), a substance that is very poisonous to
the cell. Additionally, hydroperoxyl radical (HO2*) is
formed when a hydrogen free radical (H*) combines
with molecular oxygen (O2). The hydroperoxyl radical
and hydrogen peroxide are believed to be among the
primary substances that produce biologic damage
directly after the interaction of radiation with water.
CHAPTER 7 Molecular and Cellular Radiation Biology
Organic Free Radical Formation. Absorption of radi-
ation can cause a healthy organic molecule (for simplicity, known as RH, in which H stands for hydrogen and
R can be any organic molecule) to form the free radicals
R* (an organic-neutral free radical) and H*. Without
oxygen or a force to attract an electron, these radicals
usually react with each other to reform the original
organic molecule (RH). When oxygen is present, however, R* and H* may react with oxygen molecules (O2)
to form the radicals RO2* and HO2*. Hence the original
organic molecule (RH) is destroyed and replaced by
the radicals RO2* and HO2*. These radicals can react
with other organic molecules to cause biologic damage.
Thus, a small-scale chain reaction of destructive events
results when radiation deposits energy within tissue in
the presence of oxygen.
Indirect Action Characteristics
To summarize, when free radicals previously produced
by the interaction of radiation with water molecules
act on a molecule such as DNA, the damaging action
of ionizing radiation is indirect in the sense that the
radiation is not the immediate cause of injury to
the macromolecule. The by-products of the radiation,
the free radicals, are the direct cause of this damage.
Because the human body is 80% water and less than 1%
DNA, essentially all effects of low-LET irradiation
in living cells result from indirect action.1 Fig. 7.5
depicts a useful flow chart of the radiobiologic process
of indirect action.
Specific Effects of Ionizing Radiation on DNA
Single-Strand Break. If ionizing radiation interacts
with a DNA macromolecule, the transferred energy
could rupture one of its chemical bonds and possibly
sever one of the sugar–phosphate chain side rails, or
strands, of the ladder-like molecular structure (singlestrand break) (Fig. 7.6). This type of injury to DNA is
called a point lesion. Such a single alteration along
the sequence of nitrogenous bases can result in a
gene abnormality. Point lesions commonly occur with
low-LET radiation. Repair enzymes, however, are often
capable of reversing this damage.
Double-Strand Break. Further exposure of the af-
fected DNA macromolecule to ionizing radiation will
likely lead to additional breaks in the sugar–phosphate
molecular chain(s). These breaks may also be repaired,
119
X-ray photon
H2O
molecule
Ions
H2O
re-formed
No biologic
damage
produced
Fig. 7.5 Indirect action of ionizing radiation on biologic mole-
cules. X-ray photons interact directly with a water (H2O) molecule. The H2O molecule breaks down into ions and free
radicals. The ions can recombine to form a water molecule,
thereby creating no biologic damage. The free radicals can
migrate to another molecule, such as a DNA molecule located
at some distance from the site of the initial ionization, and
destructively interact with it by ionizing it or rupturing some
chemical bonds. This creates molecular or point lesions in
the DNA macromolecule. Alternatively, free radicals can spread
biologic damage by combining with other molecules to form
toxic substances that also can migrate to distant DNA molecules and destructively interact.
macromolecule
radicals
I
n
d
i
r
DNA
Biologic
damage
produced
Free
o
i
e
t
c
c
t
a
Toxic
substances
n
but double-strand breaks (one or more breaks in each
of the two sugar–phosphate chains) (Fig. 7.7) are not
repaired as easily as single-strand breaks. If a repair does
not take place, further separation can occur in the DNA
chains, threatening the life of the cell. Double-strand
breaks occur more commonly with densely ionizing
120
CHAPTER 7 Molecular and Cellular Radiation Biology
S
A~T
P
S
A~T
P
P
C~G
S
P
S
A~T
P
S
T~A
G~C
P
T~A
S
P
S
C~G
C~G
P
S
P
S
P
S
C~G
A~T
P
S
G~C
P
T~A
S
S
P
S
P
A~T
S
P
G~C
T~A
S
P
S
P
Fig. 7.6 A single-strand break in the ladder-like DNA molecular
structure.
S
A~T
P
S
A~T
P
P
C~G
S
P
S
A~T
P
S
T~A
G~C
P
T~A
S
P
S
C~G
C~G
P
S
P
S
P
S
P
S
T~A
C~G
A~T
P
S
G~C
T~A
G~C
A~T
S
P
S
P
S
P
S
S
P
S
A~T
P
S
A~T
P
P
C~G
S
P
S
P
S
S
P
S
S
P
S
S
P
S
A
A~T
T~A
G~C
P
T~A
C~G
C~G
A~T
P
S
G~C
P
T~A
C~G
A~T
P
G~C
T~A
S
P
S
P
S
P
S
P
S
P
B
Fig. 7.8 A double-strand break in same rung of the (A) DNA
molecular structure causes complete chromosome breakage,
(B) resulting in a cleaved or broken chromosome.
both strands be broken at the same nitrogenous base
“rung” (Fig. 7.8A) the result will be the same as if
both side rails of the ladder were severed at the same
step, namely the DNA ladder would be chopped into
two pieces. This situation will result in the associated
chromosome to be broken. Thus, some types of chromosomal damage that are caused explicitly by high-LET
radiation are related to double-strand breaks of DNA.
Because the chance of reversing this type of injury is
meager, the possibility of a lethal alteration of nitrogenous bases within the genetic sequence is now far more
significant.
Fig. 7.7 A widely spaced double-strand break in the DNA
molecular structure.
(high-LET) radiation and often are associated with the
loss of one or more nitrogenous bases. Thus, when
high-LET radiation interacts with DNA molecules, the
ionization interactions may be so closely spaced that, by
chance, both strands of a DNA chain are broken. Should
Chromosome Effect After a Double-Strand Break in the Same Rung of DNA. As mentioned earlier, when
two interactions (hits), one on each of the two sugar–
phosphate chains, occur within the same rung of the
DNA ladder-like configuration (see Fig. 7.8A), the result
is a cleaved or broken chromosome (see Fig. 7.8B), with
each new portion generally containing an unequal
amount of genetic material. If this damaged chromosome divides, each new daughter cell will receive an
CHAPTER 7 Molecular and Cellular Radiation Biology
121
incorrect amount of genetic material. This defect will
culminate in either impaired functioning or death of
the newly created daughter cell.
Mutation. Interactions of ionizing radiation with
DNA molecules may cause the loss of or change in a
nitrogenous base on the DNA chain. The direct consequence of this damage is an alteration of the base
sequence (Fig. 7.9) within the DNA molecule. Because
the genetic information to be passed on to future generations is contained in the strict sequence of these
bases, the loss or change of a base in the DNA chain
represents a mutation. Damage may not be reversible
and may generate acute consequences for the cell, but,
more importantly, if the cell remains viable, incorrect
genetic information will be transferred to one of the two
daughter cells when the cell divides.
Covalent Cross-Links. Covalent cross-linking is the
process of chemically joining two or more molecules by
a covalent bond, which is the sharing of one or more
pairs of electrons between the molecules. Covalent
cross-links involving DNA comprise another effect directly initiated by high-LET radiation. With low-LET
interactions, however, covalent cross-links are most
likely caused by the process of indirect action. After irradiation, some molecules can fragment or change into
small, spur-like molecules that become very interactive
(“sticky”) when exposed to radiation. Such sticky
molecules can facilitate cross-linking by attaching or
connecting to other macromolecules or other segments
of the same macromolecule chain. Cross-linking can
occur in many different patterns. For example, it can
form between two places on the same DNA strand. This
joining is an intrastrand cross-link. Cross-linking may
also take place between complementary DNA strands
(Fig. 7.10) or between entirely different DNA molecules.
These joining’s are interstrand cross-links. Finally, DNA
molecules also may become covalently linked to a protein molecule.4 These linkages are potentially fatal to the
cell if they are not correctly repaired.
Effects of Ionizing Radiation on Chromosomes
Large-scale structural changes in a chromosome produced by ionizing radiation may be as serious for the cell
as are radiation-induced changes in DNA. When changes
occur in the DNA molecule, the chromosome exhibits
the variation. Because DNA modifications are discrete,
they do not inevitably result in observable structural
chromosome revisions. However, if these distinct effects
are numerous enough, such as may be brought about by
exposure to very high-LET radiation, then an observable
structural chromosome alteration is possible.
Radiation-Induced Chromosome Breaks. After irra-
diation and during cell division, some radiationinduced chromosome breaks may be viewed microscopically. These changes are revealed during the
T~A
Base change
A~U
G~C
Fig. 7.9 Alteration of the nitrogen base sequence on the DNA
chain caused by the action of ionizing radiation directly on a
DNA molecule.
T~A
G~C
C~G
Fig. 7.10 Interstrand covalent crosslink produced by high-LET
radiation acting directly on a DNA molecule.
Interstrand
cross-link
122
CHAPTER 7 Molecular and Cellular Radiation Biology
metaphase and anaphase stages of the cell division cycle,
when the length of the chromosomes is visible. Because
the events that precede these phases of cell division
are not observable, they can only be assumed to have
occurred. What can be seen, however, is the effect of
these events—the gross or apparent differences in the
structure of the chromosome. Both somatic cells and
reproductive cells are subject to chromosome breaks
induced by radiation.
Chromosomal Fragments. After chromosome break-
age, two or more chromosomal fragments are produced. Each of these fragments contains a fractured
extremity. These broken ends are chemically very active
and therefore have a strong tendency to adhere, or
chemically combine, to another similar end. The fractured fragments can:
• Rejoin in their original configuration
• Fail to rejoin and create an aberration (lesion or
anomaly)
• Join to other broken fragments and thereby create
new chromosomes that may not appear structurally
altered compared with the chromosome before
irradiation
Chromosome Anomalies. Two types of chromo-
some anomalies have been observed at metaphase.
They are:
• Chromosome aberrations and
• Chromatid aberrations
Chromosome aberrations result when irradiation
occurs early in interphase, before DNA synthesis takes
place. In this situation, the break caused by ionizing
radiation is in a single strand of chromatin, which is
the original chromosome. During the DNA synthesis
that follows, the resultant break is replicated when this
strand of chromatin lays down an identical strand adjacent to itself (called the sister chromatid) if repair is not
complete before the start of DNA synthesis. This situation leads to a chromosome aberration in which both
chromatids (the arms of the new chromosome) exhibit
the break. The break is visible at the next mitosis. Each
daughter cell generated will have inherited a damaged
chromatid as a consequence of a failure in the repair
mechanism. Solitary chromatid aberrations, conversely,
result when irradiation of individual chromatids occurs
later in interphase, after DNA synthesis has taken place.
Then only one chromatid of the X-shaped pair may
undergo a radiation-induced break. Therefore, only one
daughter cell is affected.
Summary of Structural Changes Caused by Ionizing Radiation. Ionizing radiation interacts randomly with
matter, expending energy in the process. Because of this
energy transfer, exposure to radiation can lead to the
occurrence of a variety of harmful effects in biologic
tissue, including the following in cell nuclei:
• A single-strand break in one chromosome
• A break in one chromatid
• A single-strand break in separate chromosomes
• A strand break in separate chromatids
• More than one break in the same chromosome
• More than one break in the same chromatid
• Chromosome stickiness, or clumping together
Consequences to the Cell From Structural Changes Within the Nucleus
1. Restitution, the breaks rejoin in their original configuration with no visible damage (Fig. 7.11). In this case,
no injury to the cell occurs because the chromatid
has been restored to its original condition. The process of healing by restitution is believed to be how
95% of single-chromosome breaks mend.
2. Deletion, a part of the chromosome or chromatid is
lost at the next cell division, thus creating an aberration known as an acentric fragment (Fig. 7.12), which
results in a cell mutation.
3. Broken-end rearrangement, a grossly misshapen
chromosome may be produced. Ring chromatids,
dicentric chromosomes, and anaphase bridges are
examples of such distorted chromosomes and chromatids (Fig. 7.13). This results in a cell mutation.
4. Broken-end rearrangement without visible damage
to the chromatids, whereby the chromatid’s genetic
material has been rearranged, yet the chromatid
appears normal. Translocations are examples of
such rearrangements (Fig. 7.14). This results in a
cell mutation.
Changes such as those outlined in items 2, 3, and 4
inevitably result in mutation because the positions of
the genes on the chromatids have been rearranged, thus
altering the heritable characteristics of the cell.
4
Target Theory
The biologic effects of exposure to radiation stem
primarily from the ionizations occurring at sensitive
CHAPTER 7 Molecular and Cellular Radiation Biology
123
A
Fig. 7.11 The process of restitution, whereby the breaks rejoin in the original configuration with no visible
damage. (A) The chromatid (single-strand chromosome) break occurs because of a photon interaction. (B) The
fragment is fully separated from the rest of the chromatid. This same type of damage could occur to a metaphase or X-shaped chromosome if S phase had already occurred. (C) The broken fragment has reattached in
its original location through the action of repair enzymes.
B C
S
S
A B C
Fig. 7.12 The process of deletion, in which part of a chromosome is lost at the next cell division, thus creat-
ing an acentric fragment. (A) The chromatid or single-strand chromosome break results from a photon interaction. (B) The fragment is fully separated from the rest of the chromatid. (C) After the next DNA synthesis
phase of the cell cycle (labeled S), the remainder of the single-strand chromosome has been replicated
normally, but with fragments missing from the two arms of the metaphase chromosome. The replicated
fragment is acentric, a section of genetic material without a centromere.
124
CHAPTER 7 Molecular and Cellular Radiation Biology
A B C
D
S
S
F
Fig. 7.13 The process of broken-end rearrangement may result in grossly misshapen chromatids. (A) Two
chromatid breaks occur in a single chromatid as a result of the interactions of two photons. (B) The fragments
from opposite ends unite before the DNA synthesis phase. (C) The ends of the chromatid that are still attached to the centromere also unite and form a “ring” chromatid. (D) Chromatid breaks occur in two different
chromatids. (E) The fragments are fully separated from the rest of their respective chromatids. (F) The ends
of the chromatids and the ends of the fragments have joined before DNA synthesis, thus forming a dicentric
(two centromeres) and an acentric (no centromere) fragment. (G) After DNA synthesis (labeled S), the chromatid is elongated but cannot split in two. The two centromeres are “bridged.” This type of chromatid damage
leads to reproductive death of the cell (i.e., it cannot replicate or divide into two cells).
G
E
CHAPTER 7 Molecular and Cellular Radiation Biology
A B
Fig. 7.14 If radiation breaks off parts of two different chromatids that are near each other (A) then the
broken parts may reattach to the wrong chromatids (B) resulting in no visible damage. However, this
rearrangement of genetic material may drastically alter a cell’s function and lead to cell death or failure to
replicate. This same type of damage could occur to a chromosome if S phase had already occurred. In this
case, the cell may divide, but the genetic material in the daughter cells is compromised and those cells may
not function properly.
125
cellular points secondary to energy transfers from radiation. These affected sites in a cell or, more specifically,
on a vital molecule within the cell are known as targets.
Whether or not such sites are struck by radiation is a
random process. From all existing evidence, it appears
that producing a serious effect typically requires more
than one radiation “hit” on a specific target. The damage from a single hit ordinarily is not conclusive because
of repair mechanisms. This concept of radiation damage to specific sensitive locations resulting from discrete
and random events is known as target theory.
To summarize the importance of target theory,
among the many different types of molecules that lie
within the cell, a master, or key, molecule that maintains
normal cell function, and thereby ensures cell survival,
is considered to be present (Fig. 7.15). Because this molecule is unique in any given cell, no similar molecules in
the cell are available to replace it; if a critical location on
the master molecule is a target receiving multiple hits
from ionizing radiation, the master molecule may be
inactivated. Healthy cell function will then cease, and
the cell will die (Fig. 7.16). If, conversely, it receives only
a single hit, then the master molecule most likely will
still be operational. Experimental data strongly support
this concept and confirm that DNA is the irreplaceable
master, or key, molecule that exists as the preeminent
vital target. Destruction of other important large-scale
molecules that are present in the cell does not typically
result in cell death. The reason is that cells have a number of similar molecules to take control and perform
necessary functions in the event of the destruction of
one or more of the molecules. Consequently, if only a
few non-DNA cell molecules are made dysfunctional by
radiation exposure, the cell will probably not display
any evidence of injury after irradiation.
As radiation passes through the molecular structure of
living systems, it does not preferentially seek out master
molecules in cells to destroy them; it interacts with these
key molecules only by chance. The target theory concept
is useful for understanding both cell death and nonfatal
cell abnormalities caused by exposure to radiation.
Interactions between ionizing radiation and molecular targets such as DNA occur through both direct and
indirect action. However, discerning which of the two
types of effects or actions has been at work in any given
case of cell death is virtually impossible.
126
CHAPTER 7 Molecular and Cellular Radiation Biology
TARGET THEORY MASTER MOLECULE
Master
molecule, or key
Fig. 7.15 The yellow circle depicts a target that represents a potential location for either direct hits from
external incident radiation or indirect hits caused by the formation of very interactive free radicals from incident radiation interactions with the numerous water molecules surrounding the target. (From Radiobiology
and radiation protection: Mosby’s radiographic instructional series, St. Louis, 1999, Mosby.)
X-ray photon
A
Cell
X-ray photon
B
Fig. 7.16 The target theory holds that the cell will die after
exposure to ionizing radiation only if the master, or key, molecule (DNA) is inactivated in the process. (A) An x-ray photon
passes through the cell without interacting with the master
molecule, which is located in the cell’s nucleus, no measurable
effect results. (B) An x-ray photon enters the nucleus and interacts with and inactivates the master molecule; the cell dies as
a result.
Nucleus No effect
Cell death
Master (key)
molecule
EFFECTS OF IRRADIATION ON THE ENTIRE CELL
For the cell as a whole, damage to the cell’s nucleus
reveals itself in one of the following conditions:
1. Instant death
2. Reproductive death
3. Apoptosis, or programmed cell death (interphase
death)
4. Mitotic, or genetic, death
5. Mitotic delay
6. Interference with function
Instant Death
Instant death of large numbers of cells occurs when a
volume is irradiated with an x-ray or gamma ray dose
of approximately 1000 Gyt in seconds or a few minutes.
This massive influx of energy causes gross disruption
of cellular form and structure and severe changes in
chemical machinery. As a result of receiving such an
enormous dose of ionizing radiation, the cell’s DNA
macromolecule breaks up and cellular proteins coagulate. Radiation doses high enough to cause this type of
damage are vastly more significant than those used for
diagnostic examinations or even standard therapeutic
treatments.