What is Radiobiology:

Radiation biology (also known as radiobiology) is a medical science that

involves the study of the action of ionizing radiation on biological tissues and
living organisms. We are all living in a background of radiation which could be
natural (cosmic radiation, terestrial radiation from plants, rocks and water,
inhalation from the air we breath and even from our bodies, ingestion from the
food we eat; or man made (atmospheric testing, medical sources, industrial and
nuclea fuel). However, no matter how big or small, radiation is harmful and
potentially lethal to living beings but could be beneficial in medicine such as in
the radiation therapy (radiotherapy) for the tratment of cancers and
thyrotoxicosis.
All living organisms are made up of protoplasm which consists of inorganic
(proteins, carbohydrates, nucleic acids, lipids) and organic (water and minerals)
compounds dissolved or suspended in water. The smallest unit of protoplasm
capable of independent existence is the cell which is the basic microscopic unit
of all living organism.
Group of cells that together perform one or more functions is referred to as
tissue. Group of tissues that together perform one or more functions is called an
organ. Group of organs that perform one or more functions is an organ system
or an organism.

Radiolysis
Water is a major constituent of all living matter, representing 70-85% of the
weight of cells. For this reason water plays a major role in radiobiology because
its radiolysis produces reactive species which are responsible for direct and/or
quasi-direct effects. Its absorption properties being similar to those of the
human body, water is widely used for clinical dosimetry. Radiolysis is a series
of phenomena by which molecules are destabilized by ionizing radiation such
as x-rays, gamma rays, photons, electrons or heavy ions leading to new
chemical species. Radiolysis occurs in any environment under irradiation. The
deposition of these ionising radiation in cells causes exitation and ionization of
water molecules with production of electrons in the irradiated medium.
Radiolysis of water leads to the production of oxidizing (H2O2, O2, OH. , O2. ,
O2 ion) and reducing (H2, H. , e-aqu) species among which some species are non-
stable (solvated electron, e-aqu) and others are stable molecules (H2O2, O2, H2)
Exposure of cells to ionizing radiation induces high energy radiolysis of H2O
water molecules into H+ and OH-.. These radicals are themselves chemically
reactive and in turn recombine to produce a series of highly reactive
combinations such as superoxide (HO2) and peroxide (H2O2) that produce
oxidative damage to molecules within the cell.
The initial event is the transfer of ~ 7 - 100 eV, an amount of energy sufficient
to cause (multiple) ionizations or excitations in water molecules.
Transfer of energy to the medium in biological systems usually involves
ionization of a water molecule, but can also involve the cellular macromolecules
(e.g., DNA) directly
Through ionizations and excitations the passage of a charged particle through
biological medium creates three species in the local vicinity of the particle
track:
INITIAL PHYSICAL EVENTS.
The initial event is the transfer of about 7-100 eV, an amount of energy
sufficient to cause ionizations or excitations in water molecules.
a

S Some of these reactions produce radicals.

.Radicals refers to an atom or molecle that contains an unpaired electron
.Radicals are highly reactive
.Radicals can be neutral or charged

H2O*─> HO. + H. ( It takes about 5 eV to break the O-H bond)

The water radical cation (H2O.+) is very strong acid and can lose a proton at about 10-4 sec to
neighboring water molecules to form hydroxyl radical

H2O.+ + H2O → .OH + H3O+

 e-aq + H. +H2O → H2 +OH-

H. + H . → H 2

Most of these reactions remove chemically reactive species from the system.

With time (by about 10-6 sec ) all the reactive species have diffused sufficiently
far that further reactions are unlikely. T he chemical development of the track
is over by 10-6 second.

CELL BIOLOGY AND GENETIC NAPPARATUS

The cell (from Latin cella, meaning "small room") is the basic structural,
functional, and biological unit of all known organisms. A cell is the smallest
unit of life. Cells are often called the "building blocks of life". The study
of cells is called cell biology or cellular biology.
Genetics and cell biology is the science of heredity. It is studied at the level of whole
organisms (classical or transmission genetics), the DNA itself (molecular genetics),
or whole populations (population and evolutionary genetics).
Genetics use molecular approaches to investigate how genes determine physical
traits including succeptibility to diseases and inherited disorders. Cell biology focuses
on the structure and function of the components of living cells (such as the cell
membrane, flagella responsible for cell movements, cell differentiation in developing
organisms, abnormal cell division and metabolic interactions between cell
compartments).

CELL THEORY: Cells are the basic unit of life. The theory states that:
1) All living organisms are made up of one or more cells and the products of
those cells
2) All cells carry out life activities (require energy, grow, have limited size)
3) New cells arise only from other living cells by process of cell division.

Three main components:

1) Plasma membrane/ cell membrane
2) Cytoplasm
3) Nucleus: this is the largest organelle in the cell. It contains the the genetic
information (DNA) on special strands called chromosomes

GENETIC APPARATUS:

Genetic material, also known as deoxyribonucleic acid (DNA) and ribonucleic

acid (RNA), plays a fundamental role in the composition of living organisms.
DNA and RNA are called genetic material because, Genetic material is that
substance which controls the formation and expression of traits in an organism
and can replicate and pass on from a cell to its daughter cell and from one
generation to the next.

We look like our mom or dad because of our genetic material known as
deoxyribonuleic acid (DNA). DNA is the hereditary material found in the
nucleus of eukaryotic cells (animal and plant) and the cytoplasm of prokaryotic
cells (bacteria) that determines the composition of the organism. DNA is found
in the nucleus of every cell, and it is exactly the same in each cell. There is
another type of genetic material found in cells and viruses known as ribonucleic
acid (RNA). DNA contains all the secrets that make one awesome, so it
important that the cell keeps the DNA safe. DNA is comprised of four chemical
(nitrogen) bases also known as nucleotides: adenine (A), guanine (G), cytosine
(C), and thymine (T). The order of these bases is what determines DNA’s
instructions or genetic code. These bases pair with one another, A with T and C
with G. Each base is attached to a sugar (ribose) and a phosphate molecule.
STRUCTURE:
DNA is double stranded, and each strand is held together by the pairing of the
nucleotides; A with T and C with G. The double stranded DNA is spirally
coiled to form a helix and thats why it is referred to in many textbooks as a
double helix. This double helix looks much like a ladder with the nucleotides
representing the rungs on the ladder and the sugar and phosphate are the sides.
The double helixes are then wrapped around proteins called histones and
packaged into chromosomes
The other type of genetic material called ribonucleic acid (RNA) is structurally
similar to DNA, but there are a few differences--primarily in their chemical
bases. Both have a sugar, phosphate group, and four chemical bases. Unlike
DNA which is double stranded, RNA is single stranded. The other major
difference is the bases that are found in RNA. RNA contains adenine (A),
guanine (G), cytosine (C), and uracil (U). So, in RNA the base thymine is
replaced with uracil. In RNA the base pairs are A loves U and C loves G.
.
EFFECT OF RADIATION ON DNA, AMINO ACIDS AND PROTEINS
When cells are exposed to ionizing radiation, radiochemical damage can occur
either by direct action or indirect action. Direct action occurs when alpha
particles, beta particles or x-rays create ions which physically break one or both
of the sugar phosphate backbones or break the base pairs of the DNA.
Ionizing radiation affects living things on an atomic level, by ionizing
molecules inside the microscopic cells that make up your body. When ionizing
radiation comes in contact with a cell any or all of the following may happen

1 It may pass directly through the cell without causing any damage.

2 It may damage the cell but the cell will repair itself.

3 It may affect the cell’s ability to reproduce itself correctly, possibly causing a
mutation

4 It may kill the cell. The death of one cell is of no concern but if too many cells
in one organ such as the liver die at once, the organism will die.

The nucleus of each cell contains microscopic bodies called chromosomes. The
chromosomes are organised in pairs and are responsible for the funtion and
reproduction of each cell in an organism’s body.

Types of DNA Damage: Radiation can produce a variety of lesions in DNA

• Rupture of the strand

• Alteration to bases
• Destruction of sugars
• Crosslinks and formation of dimers

Radiation damage to DNA: DNA Strand Breaks

Single strand breaks.

● Can take place at the phosphdiester bond, or at the bond between the base and
the sugar. ● A large proportion of the single strand breaks are caused by
hydroxyl radicals (OH●) ● Single strand breaks are readily repaired using
the opposit strand as a template. However base pair sustitution and frame shift
mutations can occur

Double strand breaks:

● Involves breakage of both strands at points less than 3 neucleotides apart.
●Production by single particle crossing both strands
●Production by two independent events
●Can be measured by various techniques such as sucrose gradient
centrifugation ● Double strand breaks have shown a
direct proportionality to radiation dose

Double-strand breaks are believed to be the most detrimental lesions produced

in chromosomes by ionizing radiation. Because such breaks are difficult to
repair, they can cause mutations and cell death. Unrejoined double strand breaks
are cytotoxic (they kill cells). Double strand breaks can also result in the loss of
DNA fragments which, during the repair process, can cause the joining of non-
homologous chromosomes (chromosomes not of the same pair) leading to the
loss or amplification of chromosomal material.

X-ray dose of ~1 Gy produces about 1000 single strand breaks and about 50-
100 double strand breaks in a typical mammalian cell. This dose causes about
50% cell death. DSBs are not necessarily lethal.

Base changes:
Bases can be damaged or destroyed or chemically modified by radiation.
Hydroxyl radical and byproducts can add to bases.
Pyrimidines (T, C) more sensitive than purines.
The biological significance of base damage is less than that of strand damage

EFFECTS OF RADIATION ON PROTEINS

Protein irradiation will produce many free radicals and ion pairs which will
react with adjacent (within 2-3 nm proximity) molecule, in this case protein.
While reacting with proteins, free radicals and ion pairs can modify amino acids
and break peptide bonds; thus changes the conformation of protein. This is
called indirect effect of ionizing radiation which is predominant in gamma
radiation or X-ray which possess low Linear Energy Transfer (LET) property.
In the direct effect of ionizing radiation, predominant in case of alpha and beta
radiation containing high Linear Energy Transfer (LET) property, protein
denaturation/deformation will be caused directly by these particles
through modification of amino acids and breakdown of peptide bonds.

Ionizing radiations cause fragmentation and aggregation of protein molecules.

When protein molecules are degraded there is a variety of products and also
many amino-acids are degraded.

Interaction of ionizing radiation with lipids

The lipid component of cell membranes is generally estimated to be ∼5 nm in
thickness with significant exposure to the aqueous cellular environment.
Though radiation is capable of directly damaging lipids, lipid bilayer mimetics
have indicated that indirect damage induced by water radiolysis products is a
larger contributor toward overall lipid modification by ionizing radiation.
Radiation induces lipid peroxidation, particularly the peroxidation of
polyunsaturated fatty acids (PUFAs), leading to an increase in membrane
permeability, disruption of ion gradients and other transmembrane processes,
and altered activity of membrane-associated proteins). Studies of ionizing
radiation targeted to the cell membrane revealed the induction of apoptosis at 5–
10 Gy via increased ceramide levels. This outcome was observed even in cells
without a nucleus, revealing another pathway for cellular ionising radiation
damage independent of nucleic acid damage.

SCELL SURVIVAL CURVES

A cell survival curve describes the relationship between the surviving fraction
of cells (i.e. the fraction of irradiated cells that maintain their reproductive
integrity (clonogenic cells)) and the absorbed dose. It describes the relationship
between radiation dose and the fraction of cells that “survive” that dose. This is
mainly used to assess biological effectiveness of radiation
A cell survival curve is a plot of the number of cells that survive to form
colonies as a function of radiation dose. Thus cell survival curves measure
reproductive cell death. Some damaged cells may continue to function for a
time but if they do not reproduce they are not counted as survivors. Cell
survival as a function of radiation dose is graphically represented by plotting the
surviving fraction on a logarithmic scale on the ordinate against dose on a linear
scale on the abscissa.

The type of radiation influences the shape of the cell survival curve. Densely
ionizing radiations exhibit a cell survival curve that is almost an exponential
function of dose, shown by an almost straight line on the log–linear plot. For
sparsely ionizing radiation, however, the curves show an initial slope followed
by a shoulder region and then become nearly straight at higher doses. Factors
that make cells less radiosensitive are: removal of oxygen to create a hypoxic
state, the addition of chemical radical scavengers, the use of low dose rates or
multifractionated irradiation, and cells synchronized in the late S phase of the
cell cycle.

Cell death : this can represent (i) loss of specific function eg in differentiated
cells of nerve, muscle and secretory cells. 100 Gy destroys cell function in non-
proliferating systems of nerves and muscle cells.

ii) loss of ability to divide eg in proliferating cell such as stem cells in

hematopoietic system or interstinal epithelium. 2 Gy is the mean lethal dose for
loss of proliferative capacity for proliferating cells.

iii) loss of reproductive intergrity eg reproductive death.

Survival means retention of reproductive intergrity, ie the capacity for sustained

proliferation in cells that proliferate. Proof of reproductive intergrity is the
capacity of a single cell to grow into large colony , visible to the the naked eye.
A surviving cell that has retained its reproductive intergrity and is able to
proliferate indefinitely into a large number of progeny is said to be clonogenic.
 Cell survival curve for high LET (densely ionizing) radiation and low LET
(sparsely ionizing) radiation (a) Model: multitarget/single hit, (b) Model:
linear quardratic

Type of radiation influences the shape of the survival curve:

i) For densely ionizing radiation (high LET) the cell survival curve is
almost an exponential function of dose (shown by an almost straight
line on a log-linear plot
ii) For sparsely ionizing radiation (low LET) the survival curves show an
initial slope followed by a shoulder region and then become nearly
straight at high doses.

Theories of biological effects of radiation.

The theories are based on 1) direct or target action theory and 2) indirect action
or Poison Chemical Theory.

Direct or Target action theory: this suggests that cell damage results when
ionising radiation directly hits critical areas or targets within the cell. Eg if x-
ray photons directly strike the DNA of a cell, critical damage occurs, causing
injury to the irradiated organism. Direct injuries from exposure to ionising
radiation occur infrquently. Most x-ray photons pass through the cell and cause
little or no damage.
Indirect action or Poison Chemical Theory: x-ray photons are absorbed by the
water compnent of a cell forming free radicals.these radicals combine to form
toxins such as H2O2 which cause cellular dysfunction and biologic damage.

Radiation can cause biological damages on cells either indirect or direct action.
Radiation produces moving electron in human body. The electrons cause
further ionisation and excitation resulting in chemical and molecular changes.
Radiations produce free radicals which are unpaired electrons that are
chemically reactive. Most of the radiation damage is caused by OH radicals
from the water composition of the human body. These radicals can further
interact with DNA, RNA, or protein molecules and cause damage to tissues and
affect its normal function. Eg chromosome break and aberration.

Biological effects of radiation can be a result of the direct or the indirect

mechanisms. These effect can be acute or delayed. Acute effects are results of
high level radiation exposure causing immediate effect such as radiation
sickness. Delayed effects occure after a very long period of time and they
include cancers, cataract and hypothjyroidism and genetic (hereditory)
disorders.

Types of bilogical effects of radiation:

1) Stochastic effects: a stochastic effect is one in which the probability of

occurence increases with increasing absorbed dose rather than the its
severity. Stochastic means random and the severity of this effect is
independent of the radiation dose. Stochastic effect is classified into two:
i) somatic and ii) genetic effects.

i)Somatic: radiation effect occurs on an exposed individual during his

lifetime. Eg radiation induced cancer.

ii)Genetic: this is not seen in the person irradiated but it is passed on to

future generations. Radiation induced mutation that affect offspring.
Genetic damage cannot be repaired.

Properties of stochastic effects: the main mechanism of this effect is cell

modification. It no threshold dose; it can occur at even low doses; it
cannot be completely avoided.
2) Determistic (Non-stochastic) effects: deterministic effect is one in one in
which severity increases with increasing absorbrd dose. They have a
threshold below which the effect does not occur. The threhold may be
very low and may vary from person to person.however , once the
threshold has been exceeded, the severity of an effect increases with dose.

Properties of deterministic effect: the main mechanism of this effect is

cell killing. It has threshold dose; the effect occurs only at high dose; it
can be completely avoided.

RADIOSENSITIVITY AND MODIFIERS

Radiosensitivity: this the relative susceptibility of cells, tissues or

organisms to the harmful effect of ionizing radiation. Generally, it has
been noted that cell radiosensitivity is directly proportional to the rate of
cell division and inversely proportional to the degree of cell
differentiation. Bergonie and Tribondaeu law stated that tissues will be
more radiosensitive if (I) the cells are undifferentiated (II) They have
greater proliferative capacity (III) they divide more rapidly.

Radiosensitivity depends on several factors such as: 1) ability to repair

damage, 2) hypoxia, 3) cell cycle position and 4) growth fraction.

Radiosensitization is a physical, chemical or pharmacological

intervention that increases the lethal effect of radiation when
administered in conjunction with it during radiotherapy. Tumor cells are
more sensitive to radiotherapy. However clinical benefit can be expected
only if there is differential effect demonstrated between tumors and
normal tissues.

Radiosensitizers are agents that increase the lethal effects of radiation

when administered in conjunction to radiotherapy. They are compounds
that when combined with radiation achieve greater tumor inactivation
than would have been expected from the additive effect of each modality.
To be clinically effective, they should improve the therapeutic ratio
TCP/NTCP ( where TCP is the tumour control probability and NTCP is
the normal tissue complication probability), because if an intervention
 equally increases the effect and side effect, then it is not useful. A
radiosensitizer may or may not have lethal effects against tumour cells
when administered alone without radiation.

CHARACTERISTICS OF RADIOSENSITIZERS: i)Lack of toxity, ii)

potent radiosensitizing effect, iii) non-cell cycle specificity, iv)
amenable to dose intense or prolonged infusion schedules, v) adaptable
to convenient out-patient admnistration.

Radiosensitive phase of cell cycle: M> G2> G1> early S> late S.

TYPES OF RADIOSENSITIZERS: physical and chemical:

hyperthermia, hyperbaric oxygen, carbogen +/- nicotinamide, arcon,
modifiers of haemoglobin, non-hypoxic cell sensitisers, hypoxic cell
sensitisers, hypoxic cytotoxins, biological modifiers, chemotherapeutic
drugs.

RADIATION EFFECT MODIFIERS

The effect of radiation will be modified by choosing some parameters such

as Linear energy transfer, oxygen effect, radiosensitizers, and radio
protecters,etc.

Linear Energy Transfer (LET):

The linear energy transfer (L) of charged particles in medium is the quotient
of dE/dl, where dE is the average energy locally imparted to the medium by
a charged particle of specified energy in traversing a distance of dl.
L = dE/dl

OxygenEffect:

The ratio of doses administered under hypoxic to aerated conditions

needed to achieve the same biological effect is called the oxygen
enhancement ratio (OER). For sparsely ionizing radiations, such as x- and
γ-rays, the OER at high doses has a value of between 2.5 and 3.5. The
OER has been determined for a wide variety of chemical and biologic
systems with different endpoints, and its value for x-rays and γ-rays always
tends to fall in this range. There is some evidence that for rapidly growing
cells cultured in vitro, the OER has a smaller value of about 2.5 at lower
doses, on the order of the daily dose per fraction generally used in
radiotherapy. This is believed to result from the variation of OER with the
phase of the cell cycle: Cells in G1 phase have a lower OER than those in
S, and because G1 cells are more radiosensitive, they dominate the low-
dose region of the survival curve. For this reason, the OER of an
asynchronous population is slightly smaller at low doses than at high
doses. This result has been demonstrated for fast-growing cells cultured in
vitro, for which precise survival measurements are possible, but would be
difficult to show in a tissue. There is some evidence also that for cells in
culture, the survival curve has a complex shape for doses below 1 Gy (100
rad). What effect, if any, this has on the OER is not yet clear.

RadioSensitizers:

Radiosensitizers are chemical or pharmacologic agents that increase the

lethal effects of radiation if administered in conjunction with it. Many
compounds that modify the radiation response of mammalian cells have
been discovered over the years, but most offer no practical gain in
radiotherapy because they do not show a differential effect between tumors
and normal tissues. There is no point in employing a drug that increases
the sensitivity of tumor and normal cells to the same extent. There are only
two types of sensitizers that have found practical use in clinical
radiotherapy:

Halogenated pyrimidines sensitize cells to a degree dependent on the

amount of the analogue incorporated. In this case, a differential effect is
based on the premise that tumor cells cycle faster and therefore
incorporate more of the drug than the surrounding normal tissues

Hypoxic-cell sensitizers increase the radiosensitivity of cells deficient in

molecular oxygen but have no effect on normally aerated cells. In this
case, a differential effect is based on the premise that hypoxic cells
occur only in tumors and not in normal tissues.

These two classes of sensitizers are discussed in turn. The basic

strategy of all radiosensitizers . The aim is to move the tumor control
curve to lower doses by sensitizing tumor cells but not affecting the
normal-tissue complication curve, or at least not altering it as much. The
outcome would be to increase the tumor control probability for a given
level of normal-tissue complications.

.
RADIOPROTECTORS
Radioprotectors are agents that protect cells (organs, organisms) from
the damaging effects of ionizing radiation. These agents reduce the
effective dose of the radiation, measured in terms of the dose reduction
factor (DRF)

DRF is the ratio of the radiation dose in the presence of drug to produce
a given biological effect to the dose in absence of drug to obtain the
same biological effect. Sulfhydryl compounds (e.g., amifostine) are
radioprotectors that contain free SH groups, which interrupt the chain of
events that utilizes free radicals to indirectly damage target molecules
(i.e., free radical scavengers).
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