The Topic is Radioprotective Efficacy of RK-IP-006 in mammalian system. Experiments performed were Antioxidant assay, SDS-PAGE, Western Blot to check the effect against radiation of 9Gy.

1. DISSERTATION REPORT ON THE TOPIC
RADIOPROTECTIVE EFFICACY OF RK-IP-006-IN MAMMALIAN SYSTEM
UNDER THE ELITE GUIDANCE OF
Dr. RAJ KUMAR (SCIENTIST ‘E’)
AT
Division of Radiation Biosciences
Institute of Nuclear Medicine and Allied Sciences
DRDO, Timarpur; Delhi-110054
SUBMITTED TO
DEPARTMENT OF BIOTECHNOLOGY
M.B.GOVT.PG.COLLEGE, HALDWANI
KUMAON UNIVERSITY
IN THE PARTIAL FULFILLMENT OF DEGREE
M.Sc. BIOTECHNLOGY
BY
NIKITA KHOLIYA
2017

3. -ACKNOWLEDGEMENT-
I would like to avail the opportunity to express my most sincere appreciation and deep gratitude to all those
people who in a way or other have contributed positively in completion of this project.
Firstly I express my sincere regards to Dr. Raj Kumar, Scientist‘E’ at the Division of Radiation Bioscience,
Institute of Nuclear Medicine and Allied Sciences, D.R.D.O, Delhi for allowing me to carry out this training
at this esteemed laboratory and gain valuable experience of recent technological advances. . His ideas and
suggestions during my training work inspired me to put in best of my efforts in my work. I have got his
encouragement and support at all levels. The knowledge, skills and thoughtfulness that he has imparted
will be cherished.
My college and my lecturers played one of the most important part of giving me such opportunity to carry
out this thesis and building up my self-confidence.
A great thanks to Dr. Shravan Kumar (Sci-C) for his wise and concrete suggestions and meticulous attention
which helped me to complete this work. Without her ideas, encouragement, guidance, advice and support
this project would have been impossible to complete.
I would further like to record my thanks to Ms.Darshana (JRF) Mr. Ashutosh Kumar Gupta (SRF), Mrs.
Neha Chhachhia (SRF),) Ms.Poonam Malhotra (SRF) for their unconditional help and support. There
enthusiastic support during my difficult times was most valuable and unforgettable, in fact I would like to
dedicate this and all I have done so far to them.
In my long list of people I cannot forget to express my heartfelt gratitude the cooperation of my parents
and their unwavering encouragement that always supported and assisted me in achieving my goals. I also
thank my co-trainees for their support and constructive discussions

4. DECLARATION
I hereby declare that project report entitled “RADIOPROTECTIVE EFFICACY OF RK-IP-006 IN
MAMMALIAN TISSUES” submitted by me to Department of Biotechnology in partial fulfilment of
the requirement for the award of the degree of M.sc in BIOTECHNOLOGY is a record of a bonafide
project work carried out by me under the guidance of Mr. RAJKUMAR. I further declare that the
work reported in this project has not been submitted and will not be submitted, either in part or in
full, for the award of any other degree or diploma in any other institute or university.
Signature of the candidate

5. CONTENT
Abstract
Introduction
Review of Literature
1. Types of Radiation
Non-ionizing
2. Ionizing Radiation
3. Ionizing Radiation Units
4. Radioactivity
5. Radiosensitivity
6. Radiation doses and Units
7. Determinants of Biological Effects of Radiation
8. Sequential Pattern of Radiation-Induced Biological Effects
9. Mechanisms of Radiation Damage
10. Free Radicals in Biology
11. Radiation Induced DNA Damage
12. Role of Antioxidants
13. Radioprotectors
Materials and Methods
Results
Discussion and Conclusion
References

6. ABSTRACT
Radiation exposure and its effect on health and surroundings has been a major concern now a days
all over world. Radiation absorbed in a cell have the potential to impact a variety of critical targets,
such as biomolecules. Radiation dose assessment is used to measure the probability of cancer
induction and genetic damage. Low levels of gamma rays cause a stochastic effects on health. High
doses produce multiple detrimental effects because of acute tissue damage, leading to multiple
organ dysfunctions and genetic changes.
Drug RK-IP-006 has been successful in reducing detrimental effects of gamma radiation in
Mammalian tissues of experimental mice, which is estimated by ANTIOXIDANT ASSAY (SOD,
Catalase, GSH, GST). The objective of our study was to perform In-vitro & In-vivo assay which
helped to show that how much biomolecules is damaged due to radiation and the phenomenon and
extent of its Radioprotection. Our study demonstrated that the drug has significant protective effects
against gamma irradiation induced DNA damage. Set of experimental mice treated with the drug
shows enhanced antioxidants activity in the sample as compared to the control group(R). Sample
treated by the drug is depicting less DNA Breakage. It was shown that the drug exerted the
maximum protective effects against gamma radiation induced DNA damage in lymphocytes when
incubated following irradiation at 9Gy, 80 SSD at a dose of 6mg/ml for 30 min. Besides, Results of
the present study indicated low level of p53 in the cytoplasmic fraction of the small intestine at Pre-
treated irradiated mice compared to only irradiated mice at 3rd day and 10 day respectively.
Expression of Hsp70 was higher in R group which shows that apoptosis has occurred in the cells
and DR group showed lower expression of Hsp70 indicating that drug protects the cells from
apoptosis to some extent. We concluded that RK-IP-006 is a potent radioprotector, in group treated
with drug prior to radiation anti apoptotic protein are well expressed, signifying protection of cells
against apoptosis.
Key words- SOD- Superoxide dismutase, GST- Glutathione-s-transferase, DNA- Deoxyribonuclease,
Gy-gray, SSD- sample to surface distance.

7. List of abbreviations
228 Ra Radium-228
60Co Cobalt-60
235 U Uranium-235
ATP Adenosine Triphosphate
BER Base excision repair
BM Bone marrow
BSA Bovine serum albumin
Bq Bequerel
CAT Catalase
C/kg Coulomb/kilogram
Ci Curie
CNSC Canadian Nuclear Safety Commission
DNA 2’- Deoxyribonucleic acid
DTT Dithiothreitol
DSB Double Strand Breaks
dTG Ditolyorthoguanidine
EDTA Ethylenediaminetetraacetic acid
EGTA Ethylene glycol-bis aminoethyl ether
ELF Extremely low frequency
GST Glutathipne-s-transferase
Gy Gray
H2O2 Hydrogen Peroxide
HOCl Hypochlorous acid
IM Intramuscularly
IR Ionising radiations
LO alkoxylradical
LET Linear energy transfer
LD Lethal Dose
LMDSs Locally multiply damaged sites
Nacl Sodium chloride
NER Nuleotide excision repair
NaoH Sodium hydroxide
NO Nitric oxide
O.D Optical density
OH• Hydroxyl radical

8. PHGPX Phospholipid hydroperoxide glutathione
peroxidase
PI Propidium Iodide
ONOO- Peroxynitrite
Rad Radiation
S Spleen
SOD Superoxide dismutase
SSB Single Strand Breaks
SSD Source to Sample Distance
Sv Sievert
UV Ultraviolet

9. INTRODUCTION
Radioactive substances give out radiation all of the time. There are three types of nuclear radiation:
alpha, beta and gamma. Alpha is the least penetrating, while gamma is the most penetrating. Radiation
can be harmful, but it can also be useful.
Radiation is a form of energy that is emitted during nuclear transitions from unstable nuclei of an
atom (e.g.235 U228 Ra, etc) and are transmitted in the form of electromagnetic waves, and/or
particles. There are various isotopes which are synthetic radio-isotopes like 60 Co, as they are
artificially generated in the nuclear reactors. Life has evolved in a world containing significant levels
of ionizing radiations in the background (Eric J Hall and Amato J Gaacia, 2006)
The discovery of X-rays by Roentgen has revolutionized the understanding and application of
ionizing radiation. Technology often caused misery to mankind when used with wrong intention.
World War II has witnessed devastating power of nuclear explosion and in last five decades, the
occurrence of several nuclear accidents have caused tremendous loss of humans, economy and even
retarded growth of the affected regions. Concerns arising from exposure to radiation have always
drawn attention whenever such accidents had occurred. A large number of working environments are
known to have more background radiations and the health concerns regarding physiological effects
(short term & long term), injuries and even the probability of fatality could be expected if the exposure
level exceeds accidently. Genetic damages in a surviving population may give rise to mutations and
genetic disorders which could be passed on to their subsequent generations. In fact, radiation exposed
individuals are more likely to have compromised immune responses thereby living with higher
chances of infection related diseases.(Dale,W. M ,1992 )
WBI of low-dose exposure is likely to be the scenario for radiation workers in hospitals and industries,
radiation emergency members, military forces in radiation combat zones and in routine cancer
radiotherapy. Gamma rays and X-rays are the two most common types of radiations encountered in
the above environments. These radiations can travel long distances with low attenuation in the
biological and physical media. Cellular responses to radiation are directed by the Law of Bergonie
and Tribondeau which states that the undifferentiated cells such as stem cells or less developed tissues
with high metabolic rates are highly radiosensitive. Therefore hematopoietic system is more sensitive
to radiation. Gastrointestinal system is next to hematopoietic in terms of 1radiosensitivity. Thus whole
body exposure affects hematopoietic system, causing depletion of blood parameters resulting in

10. lymphocytopenia, thrombocytopenia, or increased number of granulocytes, neutrophils are also
reported. Bone marrow, spleen and lymphocytes are some of the key constituents of the
haematopoietic system. When these are affected, their systemic repair failure may become life
threatening; hence their protection and recovery are necessary for survival and quality of life. Other
organs are also not spared in case of whole body exposure at increasing radiation doses, leading to
multiple organ dysfunctions and hence the chances of survival become critical(Hall E.J 1998)
Radiation damage initiates as it starts heating up the cell. Radiations absorbed in a cell have the
potential to impact a variety of critical targets, such as biomolecules. However damage to DNA is
considered critical for cell survival. Radiations may impact the DNA directly or indirectly, i.e. it can
either cause ionization of the atoms in the DNA molecules, visualized as a “direct hit” or they may
induce radiolysis of the water molecules in the cell and can generate reactive species, which may
attack the DNA molecule and abrupt the gene functioning.
Thus, free radical scavengers in the form of antioxidants that are present inside cells are the first
responders. Various antioxidant enzymes (catalase, glutathione, glutathione peroxidase) and non-
enzymatic molecules (vitamin C & E, polyphenols, cofactors such as copper, zinc, manganese, iron
and many others) act by neutralizing the free radicals.
However, their collective capacity is limited as this system maintains a redox balance, therefore
excess burden of free radicals generated during radiation exposure in cells increases probability of
oxidative damages. Various free radical scavengers are known to be effective in reducing the free
radical burden. Therefore, among various approaches for development of radiation countermeasures
(radioprotectors, mitigators and therapeutic agents) antioxidant as potential approach is under
investigations in various research laboratories.
In view of these scenarios, we have conducted this study to investigate the radioprotective efficacy
of the given antioxidant molecule in WBI mice against gamma radiation mainly in the hematopoietic
cells and tissues.

11. REVIEW OF LITERATURE
Radiation can cause immediate effects (radiation sickness), but also long term effects which may
occur even after many years (cancer) or several generation later (genetic effects). Biological effects
of radiation result from both direct and indirect action of radiation. Direct action is based on direct
interaction between radiation particles and complex body cell molecules, (for example direct breakup
of DNA molecule). Indirect action is more complex and depends heavily on the energy loss effects of
radiation in the body tissue and the subsequent chemistry. Humans have been exposed to natural
background radiation since the dawn of time. Scientific and technological advancements have further
increased radiation burden in humans, since exposure to low level of radiation frequently has become
common during medical diagnostic procedures, space or air travel, cosmic radiation and use of certain
electronic gadgets. Ionizing radiation produces cancer, loss of neural function and death in humans
and animals. It also induces killing, mutation, and chromosomal aberrations in cells. Ultraviolet
radiation (UVR) has many positive effects, but overexposure of organisms can generate harmful
effect on tissue. Most of the radiation-induced damage to bio molecules occurs in aqueous media,
such as those prevailing in living system, caused by the formation of free radicals resulting from the
radiolysis of water. Reactive oxygen species and lipid peroxides have been implicated in pathogenesis
of a number of diseases, including cancer, diabetes mellitus, rheumatoid arthritis, infectious diseases,
atherosclerosis and ageing.
Radiation induced damage mediated by free radical is an important mechanism of its action. Various
plant and microbial products have free radical scavenging property in imparting protection against
radiation exposure and its further negative consequences. Synthetic compounds and herbal products
that have antioxidant potential may serve as a preliminary source of investigation for radioprotection.
Radiation is a process in which energetic particles or energy or waves travel through a medium or
space. It occurs naturally in sunlight and sound waves. Man-made radiation is used in x-rays, nuclear
weapons, nuclear power plants and cancer treatment.
Radiation is energy in the form of rays or high-speed particles. Atoms are composed of nucleus
contains minute particles called protons and neutrons, and the atom's outer shell contains other
particles called electrons. There are more than 100 different kinds of atoms present on earth. The
lightest of these is the hydrogen atom and the heaviest is the uranium atom. Generally, the heavier
atoms are not as stable as the lighter atom. These unstable atoms are known as radioactive atoms.
They become stable only by emitting some radiation. The emission of radiation by them is referred
to as radioactivity. (Haque R, Saha S,Bera T 2011)

12. 1. TYPES OF RADIATIONS
Radiation is energy in the form of waves of particles. There are two forms of radiation – non-ionizing
and ionizing –
2.1 Non-ionizing radiation
Non-ionizing radiation is a type of electromagnetic radiation that does not carry enough energy per
quantum to ionize atoms or molecules (Podgorsak, et al. 2006) that is, to completely remove an
electron from an atom.
Examples of non-ionizing radiation include:
 Microwaves
 Visible light
 Radio waves
 TV waves
 Ultraviolet radiation (except for the very shortest wavelengths)
2.2 Ionizing radiation

13. Ionizing radiation is capable of knocking electrons out of their orbits around atoms, upsetting the
electron/proton balance and giving the atom a positive charge. Electrically charged molecules and
atoms are called ions. Ionizing radiation includes the radiation that comes from both natural and man-
made radioactive materials (Etter, L.E and Thomas C.C, 1965)
There are several types of ionizing radiation:
2.2.1 PARTICULATE RADIATION
Alpha radiation (α)
Alpha radiation consists of alpha particles that are made up of two protons and two neutrons each and
that carry a double positive charge. Due to their relatively large mass and charge, they have an
extremely limited ability to penetrate matter. Alpha radiation can be stopped by a piece of paper or
the dead outer layer of the skin (Pattison 2001). Consequently, alpha radiation from nuclear
substances outside the body does not present a radiation hazard. However, when alpha-radiation-
emitting nuclear substances are taken into the body (for example, by breathing them in or by ingesting
them), the energy of the alpha radiation is completely absorbed into bodily tissues. For this reason,
alpha radiation is only an internal hazard. An example of a nuclear substance that undergoes alpha
decay is radon-222, which decays to polonium-218.

14. Beta radiation (β)
Beta radiation consists of charged particles that are ejected from an atom’s nucleus and that are
physically identical to electrons. Beta particles generally have a negative charge, are very small and
can penetrate more deeply than alpha particles. However, most beta radiation can be stopped by small
amounts of shielding, such as sheets of plastic, glass or metal. When the source of radiation is outside
the body, beta radiation with sufficient energy can penetrate the body’s dead outer layer of skin and
deposit its energy within active skin cells. However, beta radiation is very limited in its ability to
penetrate to deeper tissues and organs in the body. Beta-radiation-emitting nuclear substances can
also be hazardous if taken into the body. An example of a nuclear substance that undergoes beta
emission is tritium (hydrogen-3), which decays to helium-3.
Neutron radiation (n)
Apart from cosmic radiation, spontaneous fission is the only natural source of neutrons (n). A
common source of neutrons is the nuclear reactor, in which the splitting of a uranium or plutonium
nucleus is accompanied by the emission of neutrons. The neutrons emitted from one fission event can
strike the nucleus of an adjacent atom and cause another fission event, inducing a chain reaction. The
production of nuclear power is based upon this principle. All other sources of neutrons depend on
reactions where a nucleus is bombarded with a certain type of radiation (such as photon radiation or
alpha radiation), and where the resulting effect on the nucleus is the emission of a neutron. Neutrons
are able to penetrate tissues and organs of the human body when the radiation source is outside the
body. Neutrons can also be hazardous if neutron-emitting nuclear substances are deposited inside the
body. Neutron radiation is best shielded or absorbed by materials that contain hydrogen atoms, such
as paraffin wax and plastics. This is because neutrons and hydrogen atoms have similar atomic
weights and readily undergo collisions between each other.
Figure summarizes the types of radiation discussed in this document, from higher-energy ionizing
radiation to lower-energy non-ionizing radiation. Each radiation source differs in its ability to
penetrate various materials, such as paper, skin, lead and water.

15. 2.2.2 ELECTROMAGNETIC RADIATION
Photon radiation (gamma [γ] and X-ray)
Photon radiation is electromagnetic radiation. There are two types of photon radiation of interest for
the purpose of this document: gamma (γ) and X-ray. Gamma radiation consists of photons that
originate from within the nucleus, and X-ray radiation consists of photons that originate from outside
the nucleus, and are typically lower in energy than gamma radiation.
Photon radiation can penetrate very deeply and sometimes can only be reduced in intensity by
materials that are quite dense, such as lead or steel. In general, photon radiation can travel much
greater distances than alpha or beta radiation, and it can penetrate bodily tissues and organs when the
radiation source is outside the body. Photon radiation can also be hazardous if photon-emitting nuclear
substances are taken into the body. An example of a nuclear substance that undergoes photon emission
is cobalt-60, which decays to nickel-60.(Fedorowski A, Steciwko A.1988)
Figure 1: Penetration abilities of different types of ionizing radiation (Canadian Nuclear
Safety Commission's (CNSC's))
2.3 IONIZING RADIATION UNITS
The amount of radiation delivered needs to be known in order to determine possible harmful
biological effects and to reach definite conclusions in studies that use ionizing radiation. Specific
units are required for radiation measurements. Units of radiation measurements have changed
dramatically over the years, and some units have been completely abandoned (e.g., the pastille), while
other units have been introduced (Hutchison, et al. 1985)
2.4 RADIOACTIVITY
This is the transition of an unstable nucleus to a steady state through the emission of particulate or
electromagnetic radiation from the nucleus (Beyzadeoglu, et al. 2010).

16. Rad: This is the amount of radiation that causes one hundred erg (of energy) to be absorbed per gram
of irradiated material (rad = radiation absorbed dose).
1 rad = 100erg/g.
Gray (Gy): This is the amount of radiation amount that cause one joule to be absorbed per kilogram
of irradiated material.
1 Gy = 1 J/kg.
1 Gy = 100cGy = 100 Rad.
Fig 2: Radiosensitivity of various cells and tissues from least to highly sensitive
(Cyberphysics_Radiation risks)
2.5 RADIOSENSITIVITY
It is the radio susceptibility of cell, tissues, organs, organisms or other substances to the injurious
action of radiation. Cells are least sensitive when in the S phase, then the G 1 phases, then G 2phases
and the most sensitive in M phases of the cell cycle. In general, it has been found that cell radio

17. sensitivity is directly proportional to the rate of cell division and inversely proportional to the degree
of cell differentiation. (Khan, et al. 1999).
1.5.1 Relative radio sensitivities of various tissues/ organs are as follows:
 High radio sensitivity: Bone marrow, lymphoid organs, blood, testes, ovaries, intestines.
 Fairly High radio sensitivity: Skin and other organs with epithelial cell lining (such as oral
cavity, oesophagus, rectum, bladder, cornea, vagina, uterine cervix etc).
 Moderate radio sensitivity: Stomach, optic lens, growing cartilage, fine vasculature,
growing bone.
 Fairly Low radio sensitivity: Salivary glands, mature cartilage or bones, kidneys, liver,
pancreas, respiratory organs, thyroid, adrenal and pituitary glands
 Low radio sensitivity: Spinal cord, muscle, brain.
2.6 RADIATION DOSES AND UNITS:
There are mainly three types of doses to measure radiations, which include [CNSC, Dec
2012]:
a) Absorbed dose- It is the dose of energy which is deposited in an object or a tissue when exposed
to ionizing radiations. The absorbed dose is measured in a unit called Gray (Gy). One gray is
equivalent to a unit of energy (joule) deposited in a kilogram of a substance. (Jung H R)
b) Equivalent dose- It is the dose which is required to equate different types of radiation with
different biological effects, by the virtue of a radiation weighting factor (wR). For instance, 1 Gy
of alpha radiation is more harmful to a given tissue than 1 Gy of beta radiation. To obtain the
equivalent dose, the absorbed dose is multiplied by a specified radiation weighting factor (wR). The
unit of equivalent dose is expressed in Sievert (Sv) i.e., 1 Sv of alpha radiation will have the same
biological effect as 1 Sv of beta radiation. In other words, the equivalent dose provides a single unit
that accounts for the degree of harm that different types of radiation would cause to the same tissue
(Hwang SY)

18. c) Effective dose-
Different tissues and organs have different radiosensitivities. For example, bone marrow is much
more radiosensitive than muscle or nerve tissue. To obtain an indication of how exposure can affect
overall health, the equivalent dose is multiplied by a tissue weighting factor (wT) related to the risk
for a particular tissue or organ. This multiplication provides 6the effective dose absorbed by the body.
The unit used for effective dose is also the Sievert.
Table 1: Relationship between units and doses of radiation
Radioactivity Absorbed dose Dose Equivalent Exposure
Common units Ci Rad Rem R
SI units Bq Gy Sv C/Kg
Ref: [ORISE_ORAU]
2.7EFFECTS OF RADIATION EXPOSURE ON PUBLIC HEALTH
In general, the amount and duration of radiation exposure affects the grievous or type of health
effect. There are two broad categories of health effects: chronic (long-term) and acute (short-term).
a) Chronic exposure
Chronic exposure in which energy is absorbed over longer period and leads to chronic effects which
occur several year after exposure. (Yehezkelli et. al. 2002; Shafi, et al.2016)
b) Acute Exposure
Acute exposure in which energy from radiation is absorbed over few hour or days and leads to acute
effects which occur within several hours to months after exposure. (Emilien, et al. 2017; Strom, 2013)
2.8 EFFECTS OF RADIATION ON BIOLOGICAL MACROMOLECULES
Ionizing radiation can produce different types of damage on DNA, RNA, Lipids, Proteins and other
bio-molecules.
a) Effect of radiation on DNA: Ionizing radiation causes the formation of strand breaks in cellular
DNA, as well as other types of lesions in the chromatin of cells. The amount of DNA damage induced
is determined by the type of radiation as well as the presence of other molecular components in close
proximity to DNA, in particular the presence of proteins because it is well known that most molecular
interactions between proteins and DNA occur via amino acids. It is estimated that each Gray (Gy) of
radiation leads to about 100,000 ionizations within a cell, damage to over 1,000 bases, about 1,000
SSBs and about 20 – 40 DSBs. Despite this, 1 Gy kills only 30% of mammalian cells due to the

19. effectiveness of DNA repair - particularly for non-DSB (double strand break) lesions (Roots, et al.
1985).
b) Effect of radiation on lipid: Lipid per oxidation has been found as the main type of damage to
membrane lipids and lipoproteins. Ionizing radiation induced lipid oxidative modifications of poly
unsaturated fatty acids (PUFAs) appears as a dynamic process initiated by hydroxyl free radicals
generated by water radiolysis, amplified by a propagating-chain mechanism involving alkyl and
peroxyl free radicals, and leading not only to hydro peroxides but also to a lot of other lipid oxidized
end-products, lipid hydro peroxides and conjugated dienes which are early products of lipid
peroxidation (Cuttler, et al.2009)
2.9 MECHANISMS OF RADIATION DAMAGE
Radiation damage starts at the cellular level. Radiation which is absorbed in a cell has the potential
to impact a variety of critical targets in the cell, the most important of which is the DNA. Nuclear
DNA is the main target of ionizing radiation, exposure of which is followed by many types of DNA
damages such as double strand breaks (DSB) are considered the most relevant lesion for mutations
and carcinogenesis, and unrepaired or misrepaired DSBs are a serious threat to genomic integrity
(Roots R , Kraft G& Gosschalk, E1985).
More recent studies have shown that radiation-induced cell membrane damage triggers a cascade of
events which could also result in cell death. It is now accepted that radiation- 12induced cytotoxicity
results from damage to these structures, although the integration of signals from both targets, at the
molecular level remains an open question (Bourguignon et al., 2005).
The mechanism by which the damage occurs can happen via one of the two scenarios (Bodansky
B.2007):
2.9.1 DIRECT ACTION
In the first scenario, radiation may impact the DNA directly, causing ionization of the atoms in the
DNA molecule. This can be visualized as a “direct hit” by the radiation on the DNA, and thus is a
fairly uncommon occurrence due to the small size of the target; the diameter of the DNA helix is only
about 2 nm. It is estimated that the radiation must produce ionization within a few nanometres of the
DNA molecule in order for this action to occur. (Ward, J.E)

20. 2.9.2 INDIRECT ACTION
Gamma radiations and X-rays interact with water molecules within the cells causing radiolysis. The
products of radiolysis are mostly oxygen centered reactive species (ROS), which are called free
radicals (for e.g. OH*, peroxinitrite, superoxide, hydroxide ion, proton, peroxy radicals and others).
These free radicals are able to diffuse over a distance to interact with the critical biological targets
such as the DNA. Because they are able to diffuse some distance in the cell, the initial ionization
event does not have to occur so close to the DNA in order to cause damage. Thus, damage from
indirect action is much more common than damage from direct action, especially for radiation that
has a low specific ionization. An ionizing radiation can also interact with molecules in a cell
particularly with water (radiolysis) to produce free radicals, which then induces damages.(Wei, H.
And Yu, K.N 2010)
Figure 5: Mechanisms of radiation damage- Direct or Indirect [Hall, 7 edition]
When the DNA is attacked, either via direct or indirect action, damage is caused to the strands of
molecules that make up the double-helix structure. Most of this damage consists of breaks in only
one of the two strands and is easily repaired by the cell, using the opposing strand as a template. If,
however, a double-strand break occurs, the cell has much more difficulty repairing the damage either

21. by NHEJ or HRR and may make mistakes. This can result in mutations, or changes to the DNA code,
which can result in consequences such as cancer or cell death. Double-strand breaks occur at a rate
of about one double-stand break to 25 single-strand breaks. Thus, most radiation damage to DNA is
reparable. (Freidberg E.)
2.10 FREE RADICALS IN BIOLOGY
A free radical can be defined as any molecular species capable of independent existence that contains
an unpaired electron in an atomic orbital. The presence of an unpaired electron makes the molecule
or atom very reactive and thus unstable. Many radicals are unstable and highly reactive. It can attain
stability by either donating an electron to or accept an electron from other molecules, therefore
behaving as oxidants or reductants (Valko M et al.)
Figure 6: Presence of an unpaired electron in the outer shell of a free radical molecule (The art
of medicine, Evelyn Schwagger lab)
Oxygen is an element indispensable for life. When cells use oxygen to generate energy, free radicals
are created as a consequence of ATP (adenosine triphosphate) production by the mitochondria. These
by-products are generally reactive oxygen species (ROS) as well as reactive nitrogen species (RNS)
that result from the cellular redox process and are commonly referred to as Free radicals. These
molecules e,g., superoxide (O -2 ), hydroxyl radicals (OH ), protons (H + ), electrons (e - ),
perhydroxyl radical (HO 2 ), nitric oxide (NO ), peroxynitrite (ONOO - ) are unstable and highly

22. reactive with other molecules in their quest to attain molecular stability. The chemical reactions
involving manufacture of energy for our cells, called mitochondrial respiration, these free radicals are
produced as by-products (Bagchi and Puri, 1998).
Figure 7: Major ROS and RNS generation systems. Generation mechanisms of ROS or RNS are
depicted in blue boxes, while reactive species are shown in stars (Nava Bashan, 2009)
These species play a dual role as both toxic and beneficial compounds. The delicate balance between
their two antagonistic effects is clearly an important aspect of life. At low or moderate levels, ROS
and RNS exert beneficial effects on cellular responses and 20immune function. At high
concentrations, they generate oxidative stress, a deleterious process that can damage all cell structures
(Lien Ai Pham-Huy et al., 2008)

23. 2.10.1 SOURCES OF FREE RADICALS
Free radicals and other ROS are derived either from normal essential metabolic processes in the
human body or from external sources (exogenous) such as exposure to radiations, ozone, cigarette
smoking, environmental pollutants, certain drugs, pesticides and Industrial solvents.
Figure 8: Summary of endogenous (blue) and exogenous (pink) sources of free radicals
Free radical formation occurs continuously in the cells as a consequence of both enzymatic and
nonenzymatic reactions (endogeneous sources). Enzymatic reactions, which serve as the source of
free radicals, include those involved in the respiratory chain, in phagocytosis, in prostaglandin
synthesis, and in the cytochrome P-450 system. Free radicals can also be formed in non-enzymatic
reactions of oxygen with organic compounds as well as those initiated by ionizing reactions.
2.10.2 ROLE OF FREE RADICALS IN PHYSIOLOGICAL FUNCTIONS

24. Free radical reactions produce progressive adverse changes that accumulate with age throughout the
body. Such “normal” changes with age are relatively common to all. However, superimposed on this
common pattern are patterns influenced by genetics and environmental differences that modulate free
radical damage (V. Lobo, 2010).
Thus ROS/RNS are recognised to play a dual role as both deleterious and beneficial species. The
beneficial effects of ROS/RNS occur at low/moderate concentrations and involve physiological roles
in cellular responses to noxia, as for example in defence against infectious agents, in the function of
a number of cellular signalling pathways, and the induction of a mitogenic response. Ironically,
various ROS-mediated actions in fact protect cells against ROS-induced oxidative stress and re-
establish or maintain "redox balance" also termed as "redox homeostasis". The "two-faced" character
of ROS is clearly substantiated. For example, ROS within cells may act as secondary messengers in
intracellular signalling cascades which induce and maintain the oncogenic phenotype of cancer cells;
however ROS can also induce cellular senescence and apoptosis and can therefore function as anti-
tumourigenic species (Berlett, B.S. Stadtman, E.R. (1997)
Overproduction of ROS results in oxidative stress, a deleterious process that can be an important
mediator of damage to various biomolecules. Impairments to these vital molecules are manifested as
diseases such as atherosclerosis, inflammatory conditions, certain cancers, and the process of aging,
determined by genetic and environmental factors. Oxidative damage to the biomolecules (proteins,
lipids and DNA) can be characterized as follows: (Evans C., Burdon R. 1994)
2.10.3 Damage to proteins
Proteins can be oxidatively modified in three ways: oxidative modification of specific amino acid,
free radical-mediated peptide cleavage, and formation of protein cross- linkage due to reaction with
lipid peroxidation products. Protein containing amino acids such as methionine, cysteine, arginine,
and histidine seem to be the most vulnerable to oxidation. Free radical mediated protein modification
increases susceptibility to enzyme proteolysis. Oxidative damage to protein products may affect the
activity of enzymes, receptors, and membrane transport. (Martin weik)
Oxidatively damaged protein products may contain very reactive groups that may contribute to
damage to membrane and many cellular functions. Peroxyl radical is usually considered to be free
radical species for the oxidation of proteins. ROS can damage proteins and produce carbonyls and

25. other amino acids modification including formation of methionine sulfoxide and protein carbonyls
and other amino acids modification including formation of methionine sulfoxide and protein
peroxide. Protein oxidation affects the alteration of signal transduction mechanism, enzyme activity,
heat stability, and proteolysis susceptibility, which leads to aging.
2.10.4 Lipid peroxidation
Lipid peroxidation is a free radical process involving a source of secondary free radical, which further
can act as second messenger or can directly react with other biomolecule, enhancing biochemical
lesions. Lipid peroxidation occurs on polyunsaturated fatty acid (PUFA) located on the cell
membranes and it further proceeds with radical chain reaction. Hydroxyl radical (OH -) is thought to
initiate ROS and remove hydrogen atom, thus producing lipid radical and further converted into diene
conjugate. Further, by addition of oxygen it forms peroxyl radical; this highly reactive radical attacks
another fatty acid forming lipid hydroperoxide (LOOH) and a new radical. Thus lipid peroxidation is
propagated.(Yin, H.; Xu, L. & Porter, N.A. (2011)

26. Figure 9: Lipid peroxidation reaction (http://www.google.co.in/)
Due to lipid peroxidation, a number of compounds are formed, for example, alkanes, malonaldehyde,
etc. These are used as markers in lipid peroxidation assay and have been verified in many diseases
such as neurogenerative diseases, ischemic reperfusion injury, and diabetes. These compounds can

27. damage cell membranes by disrupting fluidity and permeability and they can also adversely affect the
function of membrane bound proteins such as enzymes and receptors. (Cuttler, et al.2009)
1.10.5 Oxidative damage to DNA
It has been described in the above section that nucleic acids are susceptible to oxidative damage
induced by free radicals. It has been reported that especially in aging and cancer that, DNA is
considered as a major target. Oxidative nucleotide as glycol, dTG, and 8-hydroxy-2 deoxyguanosine
is found to be increased during oxidative damage to DNA under UV radiation or free radical damage.
It has been reported that mitochondrial DNA are more susceptible to oxidative damage that have role
in many diseases including cancer. It has been suggested that 8-hydroxy-2-deoxyguanosine can be
used as biological marker for oxidative stress (Hattori Y et al., 1996).
1.10.6 Types of free radical
 Reactive oxygen species - The most important free radicals in the body are the radical
derivatives of oxygen better known as reactive oxygen species. These include oxygen in its
triplet state (3O2) or singlet state (1O2), superoxide anion (O2
⁻), hydroxyl radical (OH), nitric
oxide (NO), peroxynitrite (ONOO⁻), hypochlorous acid (HOCl), hydrogen peroxide (H2O2)
alkoxyl radical (LO).
 Carbon centred radical – Carbon centred free radical (CCl3) arises from the attack of an
oxidizing radical on an organic molecule.
 Hydrogen centred radical - Hydrogen centred radicals result from attack of the hydrogen
atom (H).
 Sulphur centred radical – Sulfur centred radical produced in the oxidation of glutathione
resulting in the thiyl radical (R-S).
 Nitrogen centred radical - A nitrogen centred radical also exists for example the phenyl
diazine radical.
Production of free radicals in the human body

28. Free radicals and other reactive oxygen species are derived either from normal essential metabolic
processes in the human body or from external sources such as exposure to X-rays, ozone, cigarette
smoking, air pollutants and industrial chemicals. Free radical formation occurs continuously in the
cells as a consequence of both enzymatic and non-enzymatic reactions. Enzymatic reactions which
serve as sources of free radicals include those involved in the respiratory chain, in phagocytises, in
prostaglandin synthesis and in the cytochrome P450 system. Free radicals also arise in non-enzymatic
reactions of oxygen with organic compounds as well as those initiated by ionizing radiations. Some
internally generated sources of free radicals are: (Lien Ai Pham-Huy et al.2008)
 Mitochondria
 Phagocytes
 Xanthine oxidase
 reactions involving iron and other transition metals
 arachidonate pathways
 peroxisomes
 inflammation
 Ischemia/reperfusion.
Some externally generated sources of free radicals are:
 cigarette smoke
 environmental pollutants
 radiation
 ultraviolet light
2.11 RADIATION INDUCED DNA DAMAGES

29. DNA damages generated by ionizing radiation are very complex and fundamental for studies related
to cellular radiation response and effects such as cellular necrosis, cell transformations and later the
possibility of tumorigenecity. DNA damage onset repair processes may give rise to gene mutations
and cytogenetic end points such as micronuclei and chromosomal aberrations (Wei Han and K. N.
Yu, 2010). Majorly DNA strand breaks include SSB& DSB. The main forms of DNAdamage induced
by high or low LET radiation include single-strand breaks (SSBs) and double-strand breaks (DSBs),
sugar and base modifications, oxidative damage of bases, interstrand cross-links, DNA- protein cross-
links and locally multiply damaged sites (LMDSs) [Averbeck D, 2000].
Among all DSBs and LMDSs are very dangerous and lethal to cell leading to mutagenesis, genomic
instability and carcinogenesis.
Ionizing-radiation-induced base damages have been extensively studied in vitro by irradiation of free
bases, nucleosides, oligonucleotides or DNA in the solid state or in aqueous solutions (von Sonntag
1987; Te ́oule 1987; Nicoloff and Hoekstra 1996).
Although certain types of DNA base damages such as 8-hydroxydeoxyguanosine have significant
biological significance in some studies, available data indicate that such isolated base damages
probably play a minor role in radiation mutagenesis (Ward 1998).
Figure 10: Radiation induced DNA lesions [University of Oxford, Biochemistry Dept.]
SSB is caused by the reaction of any of the deoxyribose hydrogens (Ward 1998). In the presence of
oxygen, radiation will increase the production of alkali-labile sites (Hutchison 1985). Most of the
SSBs induced by ionizing radiation can be repaired via DNA ligation (von Sonntag 1987).
In contrast, DSBs caused by ionizing radiation or other carcinogenic chemicals are considered the
most relevant lesion for mutations and carcinogenesis. Unrepaired and misrepaired DSBs are serious
threats to the genomic integrity (Hoeijmakers 2001). DSBs lead to chromosomal aberrations, which

30. simultaneously affect many genes to cause malfunction and death in cells (Rich et al. 2000). It is
noted that DSBs can also be generated in a number of natural processes including oxidative
metabolisms, replication, meiosis, and production or formation of antibodies (Chaudhry et al. 1997;
Dahm-Daphi et al. 2000).
2.11.1 DNA REPAIR AND RADIOSENSITIVITY
The immediate response to IR induced DNA damage is the stimulation of the DNA repair machinery
and the activation of cell cycle checkpoints, followed by down-stream cellular responses such as
apoptosis that removes damaged cells (Helen Budworth et al., 2010).
All living organisms have a variety of repair mechanisms to respond to DNA damage. Considering
that mammalian cells, on average, undergo about 10,000 measurable DNA modification events per
cell per hour [Moustacchi E., 2000], maintenance of DNA integrity and gene function requires
machinery of high efficiency and fidelity. The mechanisms of DNA repair, the signalling pathways
involved in radiation sensitivity and non-targeted effects are key aspects essential to understanding
radiation effects at the level of genetic information, the crosstalking cell to cell system, and different
and unexpected mechanisms that may amplify the response (Alapetite C et al., 1999, Gatti RA et al.,
1988, Angele S et al., 2003).
The predominant repair pathway that activates due to IR-induced DNA damage is the base excision
repair (BER), which is responsible for the removal of damaged bases and DNA single-strand breaks
through gap-filling by DNA polymerase and ligation of DNA ends. Nucleotide excision repair (NER)
is the major pathway for the repair of bulky DNA damages that cause DNA helical distortion (Batty
DP, Wood RD, 2000)
The diversity of lesions in DNA has three main causes:
a) Environmental agents, such as the UV component of sunlight, IR and genotoxic chemicals.
b) Products of normal cellular metabolism that include reactive oxygen species derived from
oxidative respiration and products of lipid peroxidation.

31. c) The spontaneous hydrolysis of nucleotide residues that leaves non-instructive abasic sites
(Hoeijmakers JH, 2001).
Damage Tolerance
One damage tolerance mechanism, called translesion DNA synthesis (TLS), involves the replication
machinery bypassing sites of base damage, allowing normal DNA replication and gene expression to
proceed downstream of the damage. Recently, various DNA polymerases with more flexible base-
pairing properties permitting translesion synthesis, has been discovered. As a result of these low
fidelity enzymes, mutations to the DNA sequence are incorporated. In fact, this process is responsible
for most damage-induced point mutations and is particularly relevant for oncogenesis (Friedberg E.,
2003).
Figure 11: Special DNA polymerases both in prokaryotes and eukaryotes copying damaged
DNA; Translesion DNA synthesis (Microbial molecular genetics, NAIST_GSBS)
2.11.2 Effect of radiation on protein
During ionizing radiation induced damage to protein, the type of reactions and consequences are quite
similar to those of DNA; abstraction of H atoms and binding to aromatic rings, leading to backbone

32. breakage and modification of side chains. All these events lead to peptide chain fragmentation and
modification of amino acid side chain. (Maurya, et al. 2011).
2.12 ROLE OF ANTIOXIDANTS
Antioxidants are stable molecules capable of neutralizing rampaging free radicals by donating an
electron or hydrogen atom, thereby reducing their capacity to damage. A large number of antioxidants
are present intracellularly and these are antioxidant enzymes such as SOD, catalase, glutathione
peroxidise whereas non enzymatic molecules include glutathione, seasamol, albumin, melatonin, etc.
Many antioxidants can be supplemented at cellular level through foods in the diet. Antioxidants delay
or inhibit cellular damage mainly through their free radical scavenging property (Halliwell B., 1995).
These low- molecular-weight antioxidants can safely interact with free radicals and terminate the
chain reaction before vital molecules are damaged. (K. Bagchi and S. Puri, 1998)
Figure 12: Antioxidants stabilizing a free radical (Roy Health
consultant)(https://www.google.co.in/search=free+radicals)
2.12.1 MECHANISM OF ACTION
Two principle mechanisms of action have been proposed for antioxidants (Rice-Evans CA and
Diplock AT., 1993). The first is a chain- breaking mechanism by which the primary antioxidant
donates an electron to the free radical present in the systems. The second mechanism involves removal
of ROS/reactive nitrogen species initiators (secondary antioxidants) by quenching chain-initiating
catalyst. Antioxidants may exert their effect on biological systems by different mechanisms including

33. electron donation, metal ion chelation, co-antioxidants, or by gene expression regulation (Krinsky
NI., 1992).
2.12.2 LEVELS OF ANTIOXIDANT ACTION
The antioxidants acting in the defense systems act at different levels such as preventive, radical
scavenging, repair and de novo, and the fourth line of defense, i.e., the adaptation (Niki E., 1993).
1. The first line of defense is the preventive antioxidants, which suppress the formation of free
radicals. Although the precise mechanism and site of radical formation in vivo are not well elucidated
yet, the metal-induced decompositions of hydroperoxides and hydrogen peroxide must be one of the
important sources. To suppress such reactions, some antioxidants reduce hydroperoxides and
hydrogen peroxide beforehand to alcohols and water, respectively, without generation of free radicals
and some proteins sequester metal ions. Glutathione peroxidase, glutathione-s-transferase,
phospholipid hydroperoxide glutathione peroxidase (PHGPX), and peroxidase are known to
decompose lipid hydroperoxides to corresponding alcohols. PHGPX is unique in that it can reduce
hydroperoxides of phospholipids integrated into biomembranes. Glutathione peroxidase and catalase
reduce hydrogen peroxide to water (Chitra, K.P, and Pillai K.S 2002)
2. The second line of defense is the antioxidants that scavenge the active radicals to suppress chain
initiation and/or break the chain propagation reactions. Various endogenous radical-scavenging
antioxidants are known: some are hydrophilic and others are lipophilic. Vitamin C, uric acid, bilirubin,
albumin, and thiols are hydrophilic radical- scavenging antioxidants, while vitamin E and ubiquinol
are lipophilic radical-scavenging antioxidants. Vitamin E is accepted as the most potent radical-
scavenging lipophilic antioxidant (Puri S 1998)
3. The third line of defense is the repair and de novo antioxidants. The proteolytic enzymes,
proteinases, proteases, and peptidases, present in the cytosol and in the mitochondria of mammalian
cells, recognize, degrade, and remove oxidatively modified proteins and prevent the accumulation of
oxidized proteins. The DNA repair systems also play an important role in the total defense system
against oxidative damage. Various kinds of enzymes such as glycosylases and nucleases, which repair
the damaged DNA, are known. (Shinde A, Ganu J)

34. 4. There is another important function called adaptation where the signal for the production and
reactions of free radicals induces formation and transport of the appropriate antioxidant to the right
site (Leibfritz,D,Moncol, Crocin P, Telser, 2007)
2.13 MECHANISM OF RADIOPROTECTION
People can protect themselves against IR by using compounds that prevent indirect action, and repair
direct or indirect damages in the cells after IR exposure. In general, radioprotective agents suppress
reactive compound formation (free-radical scavengers), detoxify radiation-induced species, target
stabilization of vital bio-molecules, and enhance the repair and recovery processes. Radioprotectors
can also act as immunomodulators, i.e., they stimulate the proliferation of hematopoietic and
immunopoietic stem cells (Nair, et al. 2001). In relation to the IR exposure radioprotective agents can
be classified as chemical radioprotectors/ prophylactic preparations, mitigators, and therapeutic
preparations (Maurya, et al. 2011; Parida, et al. 2001). Prophy-lactic formulations, also known as
classical radioprotectors, are used before IR exposure to prevent tissue damage. This class of
radioprotectors belongs to compounds with thiol (sulfhydryl) groups and/or compounds with
antioxidant properties (Stone, H. B., et al. 2004) (Devasagaya, et al. 2011) Mitigators are applied
during or after IR exposure, but before manifestation of radiation symptoms, with the aim to minimize
toxicity, and prevent or reduce the negative effects of radiation on cells/tissues (Maurya, et al. 2011).
This class of compounds includes chelating agents (chelators) and blocking agents, such as KI, which
protects the thyroid gland from radioactive isotope iodine-131, by preventing its accumulation in the
thyroid (Dayem, et al. 2006).Therapeutic preparations are applied after radiation exposure, in the cure
treatment and healing from the acute radiation syndrome and delayed effects of radiation exposure,
such as fibrosis, vascular damages, and numerous damages of organs.

35. Figure 13: Mechanism of Radiation (http://www.google.com= radiation+mechanism)
2.14 RADIOPROTECTOR
Radioprotectors are compounds that are designed to reduce the damage in normal tissues by radiation
and to be effective must be present before or at the time radiation while mitigators may be used to
minimize toxicity when applied even after radiation has been delivered (Citrin, et al. 2010)
Radioprotective treatments that have been proposed over the past decades include thio compounds,
growth factors, cytokines and natural antioxidants. (Yazlovitskaya, 2013)
2.14.1 Radioprotector can be classified the basis of origin.
1. Synthetic Radioprotector
2. Natural Radioprotector
A) SYNTHETIC RADIOPROTECTOR
Cysteine is a radioprotector it is also toxic and induce nausea and vomiting at the dose level for radio-
protection. A development programme was initiated in 1959 by the United States army in studies

36. conducted at the Water Reed Institute of Research to identify and synthesis drugs capable of
conferring protection to individuals in a radiation environment, but without the debilitating toxicity
of cysteine or cystamine. The first compound, WR-438, called cystophos, was said to be administered
orally, although in fact these sulfhydryl compound break down in stomach acid and are effective only
if administered indigenously or intraperitonealy. The utility is how ever constrained due to their
toxicity effects (Nikjoo, et al.1998).
The second compound, WR-2721, now known as amifostine, is perhaps the most effective of those
synthesized in the Walter Reed series. It gives good protection to the blood forming organs, as can be
seen by the dose-reduced factor for 30 day’s death in mice, which approach the theoretically
maximum value of 3. In 1948, Patt discovered that cystein protects mice against X- rays if the drug
was injected ingested in large amounts before the radiation exposure. At about the same time, Bacq
and his colleagues in the Europe independently discovered that cysteamine could also protect animal
from total body irritation. This compound has a structure represented by
SH – CH2 – CH2 – NH2
Animal injection with cysteamine to concentrations about 150 mg/kg require dose of X- rays 1.8
times larger than control animals to produce the same mortality rate.(Kumar D , Paul T.2003)
B) HERBAL RADIOPROTECTOR
The molecular drugs since are toxic, herbal drugs have been tested as an alternative due to their multi-
mode action, less toxicity and consequently lesser side effect. The results obtained with herbal radio
protector have been very promising.several plants have been screened for radioprotection. The studies
were performed on high-altitude plants viz., “podophylum” known to the ancient Hindu physicians
since ancient times of its characteristic anti-helminthic properties and used it extensively as cathartic,
emetic and cholagogue. The Herbal Radio protector products are being the current interest of many
workers due to their antioxidant activities (Agora, et al., 2003).
2.15 RADIOPROTECTIVE PATHWAYS
The mechanistic/biological basis for development of a radioprotective strategy necessitates an
understanding of the molecular biology underlying the mechanism of the cellular, tissue and organ
specific radiation damage response. Examples of the pathways for focus include: nuclear DNA strand
breaks, communication of nuclear stress responses through the cell cytoplasm to mitochondria,
mitochondrial response to nuclear signalling, and mitochondrial initiation of apoptosis (targeting
multiple arms of the apoptotic regularity machinery. Dai Y, Grant S)
Finally other cells respond to the inflammatory cytokine cascade that follows cell killing in a second
wave of cell death.

37. This second wave may slowly persist or may occur in a delayed but severe fashion leading to the
rapid onset of what is called chronic effects described above (recognition ligands on apoptotic cells:
a perspective. Gardai j, Bratton DL, Ogden CA, Henson PM)
2.15.1Blocking nuclear DNA damage and its communication to the mitochondria
Overlapping pathways of cellular protection from ionizing irradiation, ultraviolet irradiation and heat
have been revealed in the discovery of damage repair genes, genes for induction of antioxidant
proteins (Genes required for ionizing radiation resistance in yeast. (Bennett CB , Lewis LK ,
Kathikeyan G , Lobachev KS , Jin YH , Sterling JF , Snipe JR , Resnick MA), free radical scavengers,
and by study of the evolution of heat shock proteins. A common pathway in defense against ionizing
irradiation involves protection of single and double strand nuclear DNA breaks, which lead to
induction of the self-destructive pathways of apoptosis, autophagy and mitotic arrest as well as
delayed mutations. There is evidence that all phyla in both the plant and animal Kingdoms maintain
common genetic functions for adjusting to conditions of low level ionizing irradiation.A
radioprotector could well be one that protects against DNA strand breaks.
2.15.2 Mitochondrial Stabilization
Development of radioprotectors has also followed on knowledge of the intrinsic radiation resistance
of specific transgenic mice that display overproduction of a mitochondrial localized antioxidant
protein. Also of importance was the observed relative radiosensitivity of a knockout strain of mice
deficient in production of an antioxidant radioprotective protein such as MnSOD Agents which
increase the cellular antioxidant pool anticipating large quantities of irradiation induced ROS, thus
anticipate the need to neutralize these molecules.
Such radioprotective agents include MnSOD transgene therapy and small molecules MnSOD mimics.
Other strategies to elevate cellular antioxidant stores, would be to deliver the immunostimulant
TLR5-Flagellinor another biological agent or derived product that elicits a stress response in cells
including upregulation of MnSOD gene transcription and its protein production to achieve the goal
of increasing the cellular antioxidant response capacity.
Yet other relevant approaches would include small molecules that could act as ROS scavengers
(radioprotection in-vitri & in-vivo by minicircle plasmid carrying the human mangnese superoxide
dismutase transgene.Zhang X, Epperly MW, Kay MA, Chen ZY, Dixon T, Franicola D, Greenberger
BA, Komanduri P, Greenberger JS).Other examples of therapeutic agents which have been developed

38. along the lines of protecting the mitochondria in cells from initiating apoptosis do so by elevating
antioxidant levels in response to irradiation such as WR2721 (Amifostine) which was designed as a
ROS scavenger molecule.
2.16 RADIOPROTECTION OF SPLEEN AND BONE MARROW
Different substances, of natural and chemical nature have been tested for their radioprotective
potentials. The oral administration of Mentha extract (ME) before exposure to gamma radiation was
found to be effective in increasing the frequency of radiation-induced endogenous spleen colonies,
weight of the spleen in animals and a significant increase in the body weight of animals in the Mentha
and radiation combined group at different radiation doses (Radioprotection of swiss albino mice by
plant extract Mentha Piperita(Linn.), Samarth RM, Kumar A) . Significant increases in total
erythrocyte and leucocyte counts, hemoglobin concentration, and hematocrit values in the animals of
the Mentha and radiation combined group was also seen.
3,3'-Diselenodipropionic acid (DSePA), a diselenide and a derivative of selenocystine, was also
evaluated for it’s in vivo radioprotective effects in Swiss albino mice, at an intraperitoneal dose of 2
mg/kg body wt, for 5 days before whole-body exposure to gamma-radiation. DSePA was found to
attenuate radiation-induced DNA damage. It inhibited p21 in both spleen and liver tissues. DSePA
also inhibited radiation-induced apoptosis in the spleen and reversed radiation-induced alterations in
the expression of the proapoptotic BAX and the antiapoptotic Bcl-2 genes.

39. MATERIALS AND METHODS
3.1 DRUG: RK-IP-006
Calculation:
Av. wt. of mice x No. of mice x Dose of drug (150 mg/kg)
1000
3.2 MATERIALS
Table 3.1- Lists of Materials Used
Sr. No. Material Name Manufacturing/Supplier
1 Injection Syringes Dispovan, New Delhi
2 Whatman filter paper Whatman filter paper
3 Eppendorf Tarsons Ltd.
4 Micropipette tips Tarsons Ltd.
5 Gloves Surgicare Ltd.
6 Glassware Borosil, India
7 Magnetic beats Rama Scientific workers, New
Delhi
8 Aluminium foil Home foil
9 Parafilm Cole-Parmer India Pvt Ltd,
Mumbai
10 Nitrocellulose membrane Thermo Fisher Ltd.
11 Conical centrifuge tubes Tarson Ltd., BD.
12 Pipettes Tarson Ltd.
13 Mask Surgicare Ltd.

40. Table 3.2 - list of instrument used
Sr. No. Instrument name Manufacturer/supplier
1 Homogenizer Remi udyog , Bombay
2 Sonicator Star Micronic Device
3 Centrifuge Habil
4 Microplate Scanner Bio-teck
5 Digital weight balance Sartorius
6 U.V transilluminator AAB
7 Transblote Bio-Rad
8 Water bath Ignos
9 Hot plate Khera
10 Vortex Spinix
11 Shaker Tarson
12 Ice machine Blue star
13 Autoclave YORCO Scientific instrument

41. 14 pH- meter Hanna
15 Gel Scanner Emerson
16 DNA isolation instrument Chemagic prepito
18 Vertical gel system Bio- Rad
19 Horizontal gel system Bio-Rad
20 DNA quantify
spectrophotometer
Bio-teck
3.3 EXPERIMENTAL ANIMAL
 preparation of animal- Female C57BL/20 mice were obtained from the Experimental Animal
facility of the Institute of Nuclear Medicine & Allied Sciences (INMAS), Delhi, India.Mice
were housed in cages under optimum conditions of temperature (25°C ± 2°C), humidity (50–
60%) and light (14 h of light and 10 h of dark), and provided with standard food and water ad
libitum. After acclimatisation (5–6 days), mice were weighed and the average body weight
was 25.0 ± 2 g. All study protocols were approved by the institutional animal ethics
committee.
 Treatment with drug- Animals were treated with 150mg/kg whole body weight
intramuscularly prior to 2 hours before radiation.
 Y-Irradiation- Animals were exposed to whole-body γ- radiation at 9-Gy radiation dose using
the (60Co) Cobalt Tele-therapy Unit, Bhabhatron-II (Panacea Biotech India) at a dose rate of1
Gy/min, source to sample distance (SSD) 80 cm and field size 35 × 35 cm 2 . The dose rate
was routinely calibrated as a part of quality assurance by the radiation safety officer of the
institute and the system was operated by a trained operator.

42. 3.4 EXPERIMENTAL DESIGN
Group 1(Control) Without any treatment
Group 2(Drug) Mice were administered with RK-IP006
Group 3(Radiation-9Gy) Mice were exposed to 9Gy gamma
radiation
Group 4(D+R) Mice were administered with RK-IP006
2hrs prior to radiation
Sample preparation procedure for In-Vivo:
1. Animal were grouped as C, D, R, D+R and were examined daily.
2. Treatment was given to animals according to their group at a concentration of 150mg/kg body
weight.
3. Animal were dissected according to respected time point by giving cervical dislocation.
4. Bone Marrow cells were flushed out from humerus and femur bones using PBS
5. Spleen was messed using foster slides and passes through 0.75µ filter.
6. Single cell suspension of bone marrow and spleen cells was made.
7. Single suspended cells were sonicate 2 cycles for 30 sec.
8. The samples were centrifuged at 13000g for 10min.
9. Supernatant was discarded and 500µl RBC lysis buffer was added , Sample were again
centrifuges at 13000g for 10 min.
10. Supernatant was discarded and 1ml PBS was added.
11. Supernatant was collected and PI was added to protect degradation of protein in sample.
12. Samples were stored at -80 ͦ C for further experiments.

43. Figure 13- Isolation of bone from C57BL
Figure 14- preparation of bone marrow cells

44. METHODS
3.5.1. PROTEIN ESTIMATION BY BRADFORD METHOD
The Bradford assay is used to measure the concentration of total protein in a sample
Principle
The principle of this assay is that the binding of protein molecules to coomassie dye under acidic
conditions results in a color change from brown to blue. This method actually measures the presence
of the basic amino acids residue arginine, lysine and histidine, which contributes to formation of the
protein-dye complex. Unlike the BCA assay, reducing agents (i.e DTT and beta -mercaptoethanol)
and metal chelators (i.e EDTA, EGTA) at low concentration do not cause interference. However, the
presence of SDS even at low concentration can interfere with protein-dye binding.
Materials and Reagents
 Distilled Water
 Bovine Serum Albumin (BSA) (Sigma-Aldrich)
 Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number : 27815)
EQUIPMENTS
 Microplate scanner
 Pipetts
 Eppendorfs (1.5 ml )
PROCEDURE
A. Standard assay procedure
1. prepare 7 tubes and label them as 0 ,10 , 20 , 40 ,60 ,80 , 100 .
2. Respective concentration of BSA was transfer in eppendorfs.
3. Distilled water was added to make up the volume upto 100µl and 900µl Bradford reagent were
added in each eppendorfs.
4. 200µl sample were transferred in 96 well plate in triplet form

45. 5. O.D was taken at 595nm.
Table3.3: standard of BSA
Standard 0.1%mg/ml BSA(µl) Distilled water
(µl)
Bradford Reagent
(µl)
0 0 100 900
10 10 90 900
20 20 80 900
40 40 60 900
60 60 40 900
80 80 20 900
100 100 0 900
Figure 15 - bradford protein assay in microvolume
B. Sample assay procedure.
1. Eppendorfs were labelled as C, C1, D, D1, R, R1, DR, DR1.
2. In each eppendorfs 10µl sample, 90µl distilled water and 900µl Bradford reagent were
transferred in each eppendorfs.
3. 200µl sample were transferred in 96 well plate in triplet manner.
4. O.D was taken at 595nm.
Table 3.4 - Protein estimation of sample by Bradford Assay
Sample Protein conc.(µl) Distilled water (µl) Bradford Dye (µl)
Control (C) 10 90 900
Drug (D) 10 90 900
Radiation (R) 10 90 900
Drug+Radiatn (DR) 10 90 900

46. Precautions
1. Bradford reagent is light sensitive therefore it should be done in dark.
2. Pipetting should be done properly to limit the variation in readings.
3.5.2. ANTI OXIDANTS ASSAY
A number of methods are available for determination of antioxidant activity. These assays differ from
each other in terms of reagents, substrates, experimental condition, reaction medium and standard
analytical evaluation methods. Evaluation of natural and synthetic antioxidants requires antioxidant
assay.
Figure 16 - Microplate scanner for antioxidant activity

47. 3.5.2.1CATALASE ASSAY
Catalase (CAT) is an enzyme responsible for the degradation of hydrogen peroxide. It is a protective
enzyme present in nearly all animal cells. It is an ubiquitous antioxidant enzyme that is present in
most aerobic cells. Catalase (CAT) is involved in the detoxification of hydrogen peroxide (H2O2), a
reactive oxygen species (ROS) which is a toxic product of both normal aerobic metabolism and
pathogenic ROS production. This test demonstrate the presence of catalase, an enzyme that catalyse
the release of oxygen from hydrogen peroxide
Specificity
The reaction of CAT occurs in two steps. A molecule of hydrogen peroxide oxidizes the heme to an
oxyferryl species. A porphyrin cation radical is generated when one oxidation equivalent is removed
from iron and one from the porphyrin ring. A second hydrogen peroxide molecule acts as a reducing
agent to regenerate the resting state enzyme, producing a molecule of oxygen and water.
2H2O2  2H2O + O2
ROOH + AH2  H2O + ROH + A
CAT is a tetrameric enzyme consisting of four identical tetrahedrally arranged subunits of 60 kDa
that contains a single ferriprotoporphyrin group per subunit, and has a molecular mass of about 240
kDa (Buschfort, et al. 1997). CAT reacts very efficiently with H2O2 to form water and molecular
oxygen; and with H donors (methanol, ethanol, formic acid, or phenols) with peroxidase activity.
In animals, hydrogen peroxide is detoxified by CAT and by GPX. CAT protects cells from hydrogen
peroxide generated within them. Even though CAT is not essential for some cell types under normal
conditions, it plays an important role in the acquisition of tolerance to oxidative stress in the adaptive
response of cells. Survival of rats exposed to 100% oxygen was increased when liposome’s containing
SOD and CAT were injected intravenously before and during the exposure (Aebi, et al. 1980) The
increased sensitivity of transfected CAT-enriched cells to some drugs and oxidants is attributed to the
property of CAT in cells to prevent the drug-induced consumption of O2 either for destroying H2O2
to oxygen or for direct interaction with the drug. (Turrens, et al. 1984)
Application:
 It is used in the food industry for removing hydrogen peroxide from milk prior to cheese
production.
 It is use in food wrappers where it prevents food from oxidizing.
 It is valuable in differentiating aerobic and obligate anaerobic bacteria.

48.  It is also used in the textile industry, removing hydrogen peroxide from fabrics to make sure
the material is peroxide-free.
 A minor use is in contact lens hygiene - a few lens-cleaning products disinfect the lens using
a hydrogen peroxide solution; a solution containing CAT is then used to decompose the
hydrogen peroxide before the lens is used again.
 Recently, CAT has also begun to be used in the aesthetics industry. Several mask treatments
combine the enzyme with hydrogen peroxide on the face with the intent of increasing cellular
oxygenation in the upper layers of the epidermis.
PROTOCOL
Principle: Catalase enzyme acts on the H2O2 and leaves it into H20 & O2 creating.
Reagent Required:
1. 50 mM Phosphate Buffer (pH – 7.0) foe 100ml.
 NaH2PO4 - 780mg/ml
 Na2HPO4 - 710mg/ml
2. 30 mM H2O2 (25ml P.B & 15µL H2O2).
Note:
Before preparing H2O2, take the absorbance at 240nm, the value should be 1.5 for 20ml, add 33.3ul
of H2O2 (Prepare freshly in 50 mM of Phosphate Buffer)
Reaction Mixture:
Blank : Only Phosphate buffer (1ml)
Sample: 700µl Phosphate buffer
+
20µl sample
+
250µl H2O2
Reading:

49. Read the sample at 240nm for 1min. at as interval of 15sec.
Calculation:
µmol. /min = ▲O.D/ min. x Volume of reaction sample
0.71 x Volume of sample
3.5.2.2 Superoxide dismutase Assay (SOD)
Superoxide dismutase is an enzyme that alternately catalyzes the dismutation of the superoxide
radical into either ordinary molecular oxygen or hydrogen peroxide.
In 1967 biochemist Irwin Fridovitch of Duke University and Joe McCord discovered the antioxidant
enzyme SOD, which provides an important means of cellular defence against free radical damage.
This breakthrough caused medical scientists to begin to look seriously at free radicals. In most cases
the process is automatically controlled and the number of free radicals does not become dangerously
high. Fortunately, the body has, throughout the course of millions of years of evoluation become
accustomed to coping with free radicals and has evolved various schemes for doing this (Chitra K.P.,
et al. 2002). SOD is the antioxidant enzyme that catalysed the dismutation of the highly reactive
superoxide anion to O2 and to the less reactive species H2O2. Peroxide can be destroyed by CAT or
GPX reactions (Fridovich I., 1995) ( Sandalio, L.M, et al. 1997).
O2
⁻ + O2⁻ + 2H2
+  H2O2 + O2
In humans, there are three forms of SOD: cytosolic Cu/Zn-SOD, mitochondrial Mn-SOD, and
extracellular SOD (EC-SOD) (Sandstro´m, J., et al. 1994)( Sun, E., et al.1995). SOD destroys O2-
by successive oxidation and 10 Antioxidant Enzyme reduction of the transition metal ion at the active
site in a Ping Pong type mechanism with remarkably high reaction rates (Meier, B., et al. 1998). All
types of SOD bind single charged anions such as azide and fluoride, but distinct differences have
been noted in the susceptibilities of Fe-, Mn- or Cu/Zn-SODs. Cu/Zn-SOD is competitively inhibited
by N3- , CN- and by F-(Leone, M., et al. 1998)( Vance, C.K., et al.1998). Mn-SOD is a homotetramer
(96 kDa) containing one manganese atom per subunit those cycles from Mn (III) to Mn (II) and back
to Mn (III) during the two step dismutation of superoxide (MacMillan-Crow, L.A., et al. 1998).
Application:
 This enzyme has been known to promote the rejuvenation and repair of cells, while reducing
the damages caused by free radicals.

50.  This enzyme is also used for treatment of inflammatory diseases, burn injuries, prostate
problems, arthritis, corneal ulcer, and reversing the long term effects of radiation and smoke
exposure.
 If superoxide dismutase is made into a lotion and applied to the skin, it will prevent the
formation of wrinkles. It will also heal wounds, reduce the appearance of scars, and lighten
skin pigmentation that has been caused by UV rays.
 SOD is also known to help carry nitric oxide into our hair follicles. This is beneficial for
people who are experiencing premature hair loss due to a genetic predisposition or free
radicals. Because this enzyme is a very potent antioxidant, SOD combats the effects of free
radicals that are causing hair follicles to die. Since nitric oxide relaxes the blood vessels and
allows more blood to circulate to the hair follicles and SOD helps to remove the free radicals,
hair loss can be prevented and even reversed. Taking dietary supplements that provide an
adequate supply of Superoxide dismutase will be helpful in maintaining overall well being
and health because it protects our entire body from the harmful effects of free radicals.
Protocol
Principle:
Pyragallol in contact with air converts into a quizolic compound forming colour SOD, prevent the
oxidation of pyragallol and thereby the colour formation.
Reagent required:
1. 50 mM Tris HCL (pH – 8.2), store at 4⁻ C.
2. 30mM EDTA (stock) the morality of EDTA in reaction of pyragallol in reaction mixture
should be 0.2mM.
 50mM Tris HCL buffer : 302.75 mg in 50ml D.W
 EDTA : 3.8mg/10ml
 Pyragallol : 111.7mg/10ml

51. Figure17 pH meter
Reaction mixture:
Blank : 930ul buffer
+
70ul EDTA
Control : 840ul buffer
+
65ul EDTA
+
65ul pyragallol.
Sample : 840u l buffer
+
65ul EDTA
+
20ul Sample
+
65ul pyragallol.
Reading: Take O.D at 420 nm for 3min. at 30 sec. intervals.
Calculation:
umol / min = ▲O.D blank – O.D treated sample / ▲ O.D Blank X 100

52. 3.5.3. SDS PAGE ASSAY
Aim:
SDS-PAGE was performed to separate and observe the protein pattern of the sample by the method
of Lammeli (1970)
Principle:
SDS-PAGE was performed to accomplish the following:
a) To observe the protein pattern of the enzyme mixture.
b) To determine the homogeneity of the purified enzyme mixture.
c) To determine the molecular weight of the purified enzyme.
4.2 REAGENTS REQUIRED:
1) 30% Acrylamide / bis stock solution
 Acrylamide = 29.2 gm/ml
 N´-N bis methylene – acrylamide = 0.8 gm/ml
 Distilled water = 100ml
Filter & stored in dark bottle at below 4⁻ C.
2) 0.5 M tris HCL buffer (pH – 6.8) for stacking gel.
 Tris base = 6.05 gm
 Distilled water = 100ml
Adjust pH 6.8 with HCL
3) 1.5 M tris HCL buffer (pH – 8.8) for Resolving gel
 Tris base = 6.05gm
 Distilled water = 100ml
Adjust pH 8.8 with HCL
4) 10% (w/v) SDS stock solution
 SDS = 10g
 Distilled water = 100ml
5) 10% APS stock solution (Fresh)
 APS = 100mg

53.  Distilled water = 1ml
6) TEMED stored at 4⁻C in the dark bottle
7) 10 X Running buffer (Electrophoresis) buffer pH – 3
 Tris base = 30.0 gm
 Glycine = 14.4 gm
 10% SDS = 10ml
 Distilled water = 1000ml
Stored at room temp.
8) Sample buffer or loading buffer (SDS reducing buffer 2X)
 0.5 M tris HCL (pH – 6.8) = 1.25 ml
 Glycerol (5%) = 2.5 ml
 Bromo – phenol blue (0.5%) = 0.2 ml
 10% SDS = 2.0 ml
 Distilled water = 10 ml
Stored at room temp and add 5µl ß – mercaptoethanol/ml of sample buffer prior to use. Dilute the
sample at least 1:2 with sample buffer and heated at 95 ͦ C for 4 min.
9) Staining solution (500 ml)
 0.1% Coomassie brilliant blue – R250 = 0.5 gm
 40% Methanol = 250 ml
 10% GAA = 50 ml
 Distilled water = 500 ml
10) Destaining solution (500 ml)
 10% Methanol = 50 ml
 7.0% GAA = 35 ml
 Distilled water = 415 ml

54. PROCEDURE
A) Preparation of Resolving gel:
 Set the casting frames (clamp two glass plates in the casting frames) on the casting stands.
 Prepare the gel solution (as described above) in a separate small falcon tube.
 Swirl the solution gently but thoroughly.
 Pipette appropriate amount of separating gel solution (listed above) into the gap between the
glass plates.
 To make the top of the separating gel be horizontal, fill in methanol (either isopropanol) into
the gap until it overflow.
Wait for 20-30min to let it gelate.
Table 3.5 - Resolving Gel for Trisglycine SDS PAGE
Reagents 10% 12% 15%
Distilled water 1.9ml 1.6ml 1.1ml
30% Acrylamide 1.7ml 2.0ml 2.5ml
Tris HCL (8.8) 1.3ml 1.3ml 1.3ml
10% SDS 0.05ml 0.05ml 0.05ml
10% APS 0.05ml 0.05ml 0.05ml
TEMED 0.002ml 0.002ml 0.002ml
B) Preparation of stacking gel:
 Discard the methanol and see separating gel left.
 Pipet in stacking gel until a overflow.
 Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let it
solidify.
 Make sure a complete solidify of the stacking gel and take out the comb. Take the glass plates
out of the casting frame and set them in the cell buffer dam. Pour the running buffer

55. (electrophoresis buffer) into the inner chamber and keep pouring after overflow until the
buffer surface reaches the required level in the outer chamber.
Table 3.6: 5% Stacking Gels for Tris glycine SDS PAGE
Reagents 2ml 3ml
Distilled water 1.4 2.1
30% Acrylamide 0.33 0.5
0.5 M tris HCL (6.8) 0.25 0.38
10% SDS 0.02 0.03
10%APS 0.02 0.03
TEMED 0.002 0.003
C) Prepare the samples:
 Samples were mixed with 5x dye.
 Heat them in boiling water for 5-10 min.
 Samples were centrifuged 3000rpm for 1min.
 20µl sample were loaded into wells and make sure not to overflow.
 Don't forget loading protein marker into the first lane or in middle lane.
 Then cover the top and connect the anodes.
 Set an appropriate volt and run the electrophoresis when everything's done.
 As for the total running time, stop SDS-PAGE running when the down most sign of the protein
marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass
plate. Generally, about 1 hour for a 100V voltage and a 12% separating gel. For a separating
gel possessing higher percentage of acrylamide, the time will be longer.
 Set an appropriate volt and run the electrophoresis when everything's done.
 As for the total running time, stop SDS-PAGE running when the down most sign of the protein
marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass
plate. Generally, about 1 hour for a 100V voltage and a 12% separating gel. For a separating
gel possessing higher percentage of acrylamide, the time will be longer.

57. Figure 18 - Vertical gel system for SDS PAGE gel electrophoresis (http://www.google.co.in=
sds+page+electrophoresis)
Figure 19 - SDS PAGE gel electrophoresis ((http://www.google.co.in))

58. 3.5.4. WESTERN BLOT ASSAY
AIM:
Western Blotting was performed by the rapid method of Towbin et al., (1979) to detect the expression
pattern of a protein. To detect the antigens blotted on a nitrocellulose membrane with the use of an
antibody.
PRINCIPLE:
Western blotting (also known as protein blotting or immune blotting) is a rapid and sensitive assay
for detective and characterization of proteins. Western blotting technique exploits the inherent
specificity by polyclonal or monoclonal antibodies.
It is an analytical method wherein a protein sample is electrophoreses on an SDS-PAGE and electro
transferred onto nitrocellulose membrane. The transferred protein is detected using specific primary
antibody and secondary enzyme labelled antibody and substrate. A protein sample is subjected to
polyacrylamide gel electrophoresis. After this the gel is placed over a sheet of nitrocellulose and the
protein in the gel is electrophoretically transferred to the nitrocellulose. The nitrocellulose is then
soaked in blocking buffer (3% skimmed milk solution) to "block" the nonspecific binding of proteins.
The nitrocellulose is then incubated with the specific antibody for the protein of interest. The
nitrocellulose is then incubated with a second antibody, which is specific for the first antibody. For
example, if the first antibody was raised in mouse, the second antibody might be termed "goat anti-
mouse immunoglobulin". This means is that mouse immunoglobulin were used to elicit an antibody
response in goats. The second antibody will typically have a covalently attached enzyme which, when
provided with a chromogenic substrate, will cause a colour reaction. Thus the molecular weight and
amount of the desired protein can be characterized from a complex mixture (e.g. crude cell extract)
of other proteins by western blotting.
REAGENTS AND MATERIALS:
1. Nitrocellulose membrane
2. Plastic staining box
3. Electro blotting apparatus
4. Whatmann No.1 filter paper
5. Transfer buffer (500 ml, pH 8.3) Tris–HCl -25 mM Glycine -192 mM Methanol-20%
6. 10X Tris buffered saline (TBS) (100 ml, pH 7.6) Tris -2.4 g NaCl -8 g they were dissolved in low
amount of double distilled water, the pH was adjusted to
7. And the total was made upto 100 ml with double distilled water.
8. Blocking solution (50mL) 5% Non-dry fat milk powder - 0.25g 1X TBS (pH 7.6) - 50mL 0.1%
Tween- 20 - 0.05mL

59. 9. Washing buffer (100mL) (TBS) 1X TBS (pH 7.6) -100 ml 0.1% Tween - 20 - 0.1 ml
10. Preparation of primary antibodies
11. Preparation of secondary anti bodies
12. Colour indicator solution 0.05% 0f BCIP/NBT premix solution and 0.01% of H2O2 were dissolved
in 1X PBS (pH 7.6). This chromogen substrate was prepared just prior to the treatment.
13. Ponceau S red solution (100mL) Ponceau S red - 0.5 g Glacial acetic acid - 5%
PROCEDURE:
1. After SDS-PAGE, the gel was equilibrated in blotting buffer for 20 min at room temperature. While
the gel was equilibrating, a piece of nitrocellulose membrane was cut into the same dimension as the
gel it was wet slowly by sliding it at 45o angle into transfer buffer and was soaked for 20 min.
2. The pieces of Whatmann No.1 filter paper, four pads were also soaked in transfer buffer for 20
min.
3. Then, the pads, filter paper, nitrocellulose membrane and gel were assembled in the semi-dry blot
apparatus in the following order: The two pre-soaked pads were placed at the bottom and a glass
pipette was rolled over the surface of the pad to remove air bubbles. Then, the Whatmann No.1 filter
paper was placed followed the nitrocellulose membrane. Carefully, the equilibrated gel was placed
on top of the nitrocellulose membrane. The second Whatmann No.1 filter paper and followed it, the
second set of pad were placed on top of the gel. (After each step care was taken to remove the
bubbles). The transfer cell and plug was assembled and the gel transferred though transferblot. After
the transfer, protein were visualized by staining in ponceau S solution for 5 min, destained in the
distilled water and the molecular marker was marked with in delible ink and destained for 10 min.
The membrane was blocked in blocking buffer for 1h at room temperature. Then, the membrane was
washed again with washing buffer and incubated with primary antibody overnight at 4oC. The next
day, the membrane was washed again with washing buffer and incubated with AP-conjugated
secondary antibody for 2 h at room temperature. The membrane was washed and TTBS solution was
added and incubated at room temperature and watched for colour development, which is usually
completed within 5–10 min. The membrane was rinsed with distilled water to stop the reaction of
BCIP/NBT. It was then placed on filter paper to air dry. Dilutions of the primary and secondary
antibody were standardized after several trials. The specific protein was detected as a band in the
nitrocellulose membrane.

60. Figure 20- Transblot instrument for gel transfer (http://www.google.com=
western+blot+instrument)
The presence of specific protein or the presence of antigen or specific antibody was visualised as a
bluish grey coloured band.
SAMPLE PREPARATION FOR IN-VITRO
1. Human blood was collected in vacutainer tubes.
2. In D group only drug (RK-IP-006) was given. In R group only radiation was given and In DR
group drug (RK-IP-006) was given 1hr. prior to radiation.
3. 250ml blood was fed in96 well plates and set the required protocol of Chemagic prepito
instrument.
4. After 60min. the DNA sample was collected from the machine.
5. Samples were stored at -20 ͦ C for further experiments.

61. 3.5.5 LADDER ASSAY
Aim: To study the DNA damage cause by radiation and its protection through the drug (RK-IP-006).
Principle:
Techniques which permit the sensitive detection of DNA damage have been useful in studies of
environmental toxicology, carcinogenesis, and aging. Since the effects of environmental toxicants,
cancer, and aging are often tissue and cell-type specific it is important to develop techniques which
can detect DNA damage.
Quantization of nucleic acids is commonly performed to determine the average concentrations
of DNA or RNA present in a mixture, as well as their purity. DNA can also be quantified by
measuring the UV-Vis spectrophotometer. A spectrophotometer to measure the amount of ultraviolet
radiation absorbed by the bases. The OD260/OD280 ratio is an indication of nucleic acid purity. Pure
DNA has an OD260/OD280 ratio of ~1.8; pure RNA has an OD260/OD280 ratio of ~2.0. Low ratios could
be caused by protein contamination.
To determine the concentration of DNA in the original sample, perform the following calculation:
 dsDNA concentration = 50 μg/mL × OD260 × dilution factor
Figure 21 Spectrophotometer for DNA Quantify
Requirement:
Human blood, Agarose, 10mg/ml EtBr, RK-IP-006, Radiation source, chemagic prepita instrument,
pipette, vacutainer tubes, 96 well plates, eppendorf , spectrophotometer, U.V transilluminator

62. 1. Eppendrof were marked C, D, R, DR. Drug (RK-IP-006) were prepared (6mg/ml) and 100µl
were transferred in D and DR marked eppendrof and 600Gy radiation were given by GC-5000
instrument to R, DR.
2. 250 ml blood was fed into 96 wellplate & set the required protocol of Chemagic prepito(R)
instrument.
3. After 60min. the DNA sample was collected in Eppendorf from the machine (keep the DNA
sample in -20 refrigerator).and the DNA samples were quantified by using spectrophotometer
4. Meanwhile 1% Agarose gel was prepared (500 agarose was dissolved in 50ml TAE buffer
put 400µl EtBr in the agarose gel as the gel comes to room temperature).
5. As the gel was solidified the DNA sample & Dye were loaded into the wells (10µl DNA & 10
µl Dye)
6. Agarose gel Electrophoresis was run at 60V
7. After 2 hr.the gel was examined in U.V Transilluminator.
Precaution.
 Gel should be prepared and run in dark.
 Pipetting should be done properly.

63. Figure 22 - Chemagic prepita instrument for DNA isolation (http://www.google.com=
DNA+isolation)
Figure 23 - Barcode of DNA isolation Instrument

64. RESULTS
4.1 Ladder Assay:
To evaluate the effect of drug (RK-IP-006) and gamma radiation in human blood, DNA ladder assay
was performed. The result of the study indicated that the drug confers radioprotection to the DNA as
in figure 25 (lane 3-4) there is less smearing in DR group as compared to R group, indicating less
damage in DR group than R. The C and D group shows same amount of smearing and the intensity
of bands are also similar. The result suggests that the drug confers protection to the DNA against
gamma radiation .Result also indicates that there is no effect of drug on DNA in normal conditions.
Figure 24- effect of drug and radiation induced DNA damage. The agarose gel electrophoresis of
DNA isolated from the human blood of gamma ray-irradiated. Lane 1: Control (non-irradiated); lane
2: Radiation alone; lane 3: drug (3mg/ml) + radiation; lane 4: drug (4mg/ml) + radiation; lane 5: drug
(5mg/ml) + radiation; lane 6: drug(6mg/ml) + radiation.

65. 4.2. Antioxidant enzyme Assays
4.2.1 Catalase:
To evaluate the effect of radioprotective drug RK-IP-006 and gamma radiation on catalase activity,
in bone marrow and spleen samples, colorimetric assay was performed. The results of the study
indicated significant inhibition in catalase activity after post irradiated intervals of 6h and 24h in bone
marrow (Fig 26 a, b) and in spleen (Fig 27). However, significant increase in catalase activity was
observed in the bone marrow and spleen samples of irradiated mice which were also pre-treated with
RK-IP-006.
This result suggests that the activity of catalase enzyme is enhanced in the presence of the drug RK-
IP-006. Thus, the drug seems to confer radioprotective effect through modulation of catalase activity
in samples against gamma radiation.
(a) (b)
Figure 25- Effect of gamma radiation and RK-IP-006 treatments on catalase activity in
irradiated mice bone marrow.
Catalase (Spleen):
To evaluate the effect of radioprotective drug RK-IP-006 and gamma radiation on catalase activity in
irradiated mice spleen, catalase assay was performed. The results of the study indicated significant
inhibition in catalase activity at 6h and 24h after radiation treatment (Fig 27 a, b) However, significant
induction in catalase activity was observed in the spleen of irradiated mice were pre-treated with RK-
C D R DR
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
µmoles/min./mgofprotein
6 Hrs BM (Catalase)
C D R DR
0
2000
4000
6000
8000
10000
12000
µMoles/min./mgofprotein
24 Hrs BM (Catalase)

66. IP-006. These results suggest catalase activity protection efficacy of radioprotective drug against
gamma radiation.
(a) (b)
Figure 26 -Effect of gamma radiation and RK-IP-006 treatments on catalase activity in
irradiated mice spleen.
4.2.2 Superoxide dismutase:
To evaluate the effect of radioprotective drug RK-IP-006 and gamma radiation on superoxide
dismutase activity in bone marrow and spleen samples, SOD assay was performed.
The results of the study indicates significant inhibition in SOD activity at 6h and 24h after radiation
treatment in bone marrow (Fig28 a , b) and spleen (Fig29) However, significant induction in SOD
activity was observed in the samples of irradiated mice which were pre-treated with RK-IP-006 (2hrs
pre-irradiation). These results suggest protection of SOD activity by radioprotective drug against
gamma radiation.
C D R DR
0
200
400
600
800
1000
SODunits/mgofprotein
6Hrs BM(SOD)
C D R DR
0
5
10
15
20
25
30
35
40
45
50
SODunits/mgofprotein
24 Hrs BM SOD
C D R DR
0
500
1000
1500
2000
2500
3000
3500
4000
µMoles/min./mgofprotein
6 Hrs Spleen(Catalase)

67. (a) (b)
Figure 27 -Effect of gamma radiation and RK-IP-006 treatments on superoxide dismutase
activity in irradiated mice Bone marrow.
Superoxide dismutase (Spleen)
(a) (b)
Figure 28 -Effect of gamma radiation and RK-IP-006 treatments on superoxide dismutase
activity in irradiated mice spleen.
C D R DR
0
100
200
300
400
500
600
700
800
900
1000
SODunits/mgofprotein
6 Hrs Spleen(SOD)
C D R DR
0
200
400
600
800
1000
1200
1400
1600
1800
SODunits/mgofprotein
24 Hrs Spleen(SOD)

68. D RC DR
4.3. SDS PAGE Assay
C D R DR C D R DR
Figure – (29a, b) Protein profiling of 1, 3 and 7days of intestine sample through SDS PAGE.

69. 4.4. Western Blot analysis
In order to correlate the cellular changes with molecular alterations which appeared in small intestine
of the irradiated and RK-IP-006.treated mice, time-dependent protein expression analysis was carried
out
Figure 30- Western blot analysis of p53, Hsp70, Bax/Bcl2, ß acting expression.
Hsp70 expression:
The result of study indicated that the expression of Hsp70 was higher in R group which shows that
apoptosis has occurred in the cells and DR group show the lower expression of Hsp70 which indicates
that drug protects the cells from apoptosis to some extent. As in Fig 32, on the 3rdday a slight decrease
in the expression of Hsp70 in drug pre-treated and irradiated group was observed in comparison to
irradiated only group. At the end of 10 day, significant reduction in the expression of Hsp70 was
observed.
Figure 31-Effect of radiation and drug treatment on HSP-70 expression in irradiated mice intestine
P53 expression:

70. The results of study indicate low level of p53 in the cytoplasmic fraction of the small intestine of RK-
IP-006. However, 1 days post- irradiation expression of p53 is same in all group as shown in fig. 33
that DNA damage does not occur after 1 days irradiation where else after 3 and 10 days irradiation
expression of p53 increased in R group which shows that after irradiation of 3 and 10 days DNA
damage occurred. In result pre-treated irradiated mice compared to only irradiated mice at 3rd day and
10 day respectively.
Figure 32- Effect of radiation and drug treatment on p53 expressionin irradiated mice intestine
Bax/Bcl-2 ratio:
In fig34 It was observed that in R group Bax/Bcl2 level is higher which shows that apoptosis has
occurred in the cells. Bax/Bcl2 ratio was calculated for the three time periods and it was found to be
promoting the anti-apoptotic property of the drug RK-IP-006.by maintaining the higher expression
of Bcl-2 as compared to bax from day 3rd itself and significant results at 10 day of the study.
Figure 33- Effect of radiation and drug treatment on Bax/Bcl-2 ratio expression in irradiated
mice intestine

71. Discussion and Conclusion
An acute full-body equivalent single exposure dose of 1 Sv (1000 mSv) causes slight blood changes,
but 2.0–3.5 Sv (2.0–3.5 Gy) causes very severe syndrome of nausea, hair loss, and haemorrhaging,
and will cause death in a sizable number of cases about 10% to 35% without medical treatment. A
dose of 5 Sv (5 Gy) is considered approximately the LD50 (lethal dose for 50% of exposed population)
for an acute exposure to radiation even with standard medical treatment. A dose higher than 5 Sv (5
Gy) brings an increasing chance of death above 50%. Above 7.5–10 Sv (7.5–10 Gy) to the entire
body, even extraordinary treatment, such as bone-marrow transplants, will not prevent the death of
the individual exposed.
In this study, we demonstrate that DNA strand breaks and oxidative damage occur at high doses of
gamma radiation and can be detected by the ladder assay and significantly increased levels of DNA
strand breaks were observed at 600 Gy in naked DNA of whole blood. This is likely due to the fact
that gamma radiation caused damage to DNA directly or indirectly as a result of reactive oxygen
species (ROS) generation.[ CITATION Wan061 l 1033 ].
The present study demonstrated that the drug has significant protective effects against gamma
irradiation induced total DNA damage.
On examining gamma irradiation induced total DNA damage; drug showed protective effects at all
concentrations used in the present study.
In the present study, the radioprotective effect of drug was also investigated, to determine whether
drug treatment for 30 min prior to or following gamma irradiation was more effective.
Drug exerted the maximum protective effects against gamma radiation induced DNA damage in
lymphocytes when incubated following irradiation at a dose of 6mg/ml for 30 min.
Radiation induced damage mediated by free radical is an important mechanism of its action. Free
radicals are Reactive Oxygen Species (ROS), which include all highly reactive, oxygen‐containing
molecules. Types of ROS include the hydroxyl radical, the super oxide anion radical, hydrogen
peroxide, singlet oxygen, nitric oxide radical, hypochlorite radical, and various lipid peroxides. These
free radicals may either be produced by physiological or biochemical processes or by pollution and

72. other endogenous sources. All these free radicals are capable of reacting with membrane lipids,
nucleic acids, proteins and enzymes and other small molecules, resulting in cellular damage.
The aim of the present study was to evaluate the radioprotective effect of drug in mice exposed whole-
body to different doses of gamma radiation. The exposure of animals to gamma radiation resulted in
radiation-induced sickness and mortality; the higher doses killed all the animals within 10 days. The
bone marrow stem cells are more sensitive to radiation damage than the intestinal crypt cells, but the
peripheral blood cells have a longer transit time than the intestinal cells. Hence the gastrointestinal
syndrome appears earlier than the bone marrow syndrome. In mice, death from 11 to 30 days post
irradiation is due to the hemopoietic damage.
In this study, we demonstrate that the drug had significant protective effects against gamma radiation
attack on various macromolecules present in the cell and its extra-cellular environment.
On examine gamma irradiation induce damage the protein of BM, Spleen sample, drug showed
protective effects at all protein used in present study.
In the present study, the radioprotective effect of drug was also investigated, to determine whether
drug treatment for 2 hr. prior to or following gamma irradiation was more effective.
Drug exert the maximum protective effects against gamma irradiation induce cells damage when
incubated following irradiation at 9Gy, 80 SSD.
In order to correlate the cellular changes with molecular alterations which appeared in small intestine
of the irradiated and RK-IP-006 treated mice, time-dependent protein expression analysis was carried
out. p53 is responsible for DNA damage induced apoptosis. Radiation stress induces p53 expression.
Results of the present study indicated low level of p53 in the cytoplasmic fraction of the small
intestine of RK-IP-006. Pre-treated irradiated mice compared to only irradiated mice at 3rd day and
10 day respectively.
Similarly another stress marker protein Hsp70 was observed to be elevated in irradiated alone group
of mice. On the 3rd a slight decrease in the expression of Hsp70 in drug pre-treated and irradiated

73. group was observed in comparison to irradiated only group. At the end of 10 day, significant reduction
in the expression of Hsp70 was observed.
Bax/Bcl2 ratio was calculated for the three time periods and it was found to be promoting the anti-
apoptotic property of the drug RK-IP-006by maintaining the higher expression of Bcl-2 as compared
to bax from day 3rd itself and significant results at 10 day of the study.
In the light of the above results we can conclude that RK-IP-006 is a potent radioprotector .The
expression of the apoptotic and antiapoptotic proteins indicates the fate of the cells after gamma
irradiation. In the radiation alone group apoptotic proteins are highly expressed, indicating the onset
of programmed cell death. But at the same time, in the group which is treated with drug prior to
radiation anti apoptotic protein are well expressed, signifying protection of cell against apoptosis.
Moreover, the ladder assay also indicates that the drug confers radioprotection to the DNA against
the gamma radiation. The radiation alone group shows greater smearing and less intense DNA bands
indicating DNA damage .But in ‘DR’ group there is less smearing and more intense DNA bands .The
antioxidant assays also indicates that the activity of antioxidant enzymes is enhanced in the presence
of the drug.