UCL CHEMISTRY

FACULTY OF MATHEMATICAL AND

PHYSICAL SCIENCES (MAPS)

March 2013 DNA mutation by

particulate
radiation,
electromagnetic
radiation and free
radical attack
How plausible are X-men like
mutations?

Cosmina I. Dabu
P41 – LITERATURE REVIEW PROJECT

0
Abstract
The primary aim of the present review paper was to establish whether or not DNA damage of a potentially
mutagenic nature is induced by either particulate and electromagnetic radiation, or transition metals, specifically free
radicals produced by oxidations involving transition metals in Fenton-type reactions. We found that irradiation can
damage DNA by producing single strand (SSB), double strand (DSB) or multiple double strand breaks (MDSB),
due to both direct and indirect effects. We also found that the most important transition metals in mammalian cells
are Fe, Mn, Zn and Cu. The free radicals resulting from their oxidations induced DNA damage in the form of SSB,
DSB and MSB as well. The secondary aim of this paper, which was to create a description of the mechanisms and
chemical reaction pathways corresponding to the interaction between each of the above mentioned systems with
mammalian DNA was also addressed successfully. Finally, we determined that there is no record of potential
‘positive’, X-men like mutations as a result of these DNA damage systems.

Table of Contents

1. Introduction .......................................................................................................................................................................... 2
2. Brief overview of the biochemistry and mutation of DNA .......................................................................................... 4
2.1 Biochemistry ........................................................................................................................................................................ 4
2.2 Mutation.................................................................................................................................................................................... 5
3. DNA damage due to particulate and EM radiation ........................................................................................................ 6
3.1 Direct effects ....................................................................................................................................................................... 6
3.2 Indirect effects ........................................................................................................................................................... 11
3.3 Selected studies reflecting potential occurrence of human DNA damage in vivo ................................................... 13
4. DNA damage due to the attack of free radicals induced by transition metals .............................................................. 14
4.1 Brief overview of transition metals in mammalian cells ............................................................................................. 14
4.2 Fenton-type reactions and organic radicals .................................................................................................................. 15
4.3 The combined DNA damaging effect of radiation and transition metals ............................................................... 18
5. Conclusion ........................................................................................................................................................................... 19
Bibliography.................................................................................................................................................................................. 21

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 1. Introduction

Mutation. It is the key to our evolution. This process is slow, normally taking thousands and thousands of years, but
every few hundred millennia evolution leaps forward. Says Professor X from the widely known franchise X-men.
Fiction aside, is there any truth behind the baseline of the mutant story? What is mutation? What are the
mechanisms by which it occurs? Are there genetic factors, predispositions or external factors such as radiation or
certain molecules’ reaction with the DNA that could cause such mutations to occur in an individual?

Genetics has fascinated humans for many hundreds, possibly even thousands of years. An accurate, molecular level
description of it didn’t however come until the end of the 1800s. In 1869, Miescher isolated “nuclein” from
infected wounds’ pus, and renamed it “nucleic acid” several years later after he separated it into an acid and a
protein molecule. In the 1920’s, Muller observed the potential of spontaneous gene mutation as a result of a
cellular-level interaction of genetic material with certain physico-chemical agents. At that time, in the classical
concept of the gene, the gene was regarded as being ‘the atom’ of genetics; the smallest indivisible unit of genetic
transmission, recombination, mutation and function. Muller managed to induce similar changes in the genetic
material by irradiating it with X-rays, and noted that the number of mutations increased linearly with the amount of
radiation [677]. Around the same time, nucleic acids were found to be a major part of the chromosome [41]. Shortly
after, investigations of the nucleic acid lead to the conclusion that it was composed of four different units; of
adenylic, guanylic, thymidylic and cytidylic acids. In 1953, Watson and Crick had a breakthrough - they determined
the DNA’s main structure as it is known today [42].

The primary aim of the present review paper is to establish whether or not DNA damage of a potentially mutagenic
nature is induced by either particulate and electromagnetic radiation, or transition metals, specifically free radicals
produced by oxidations involving transition metals in Fenton-type reactions. This was done by presenting a
selection of studies which offer quantitative and qualitative data on DNA damage as a result to various specific
irradiation processes and contexts of transition metal oxidations. Secondly, we aim to create a description of the
mechanisms and chemical reaction pathways corresponding to the interaction between each of the above
mentioned systems with mammalian DNA. Half-jokingly, we set out to discover whether or not any of the
processes we explored had the potential of inducing ‘positive’, X-men like mutations. In more detail:

Chapter 2 briefly introduces the reader to the biochemistry of the DNA; its molecular structure and forms as well as
their relevance to chemical reactions and hence their importance in the context of this study. It also offers a very
simplified description of mutation.

In chapter 3, particulate and EM radiation are treated together, and within this chapter, the issues of direct effects
and indirect effects of the irradiation are treated separately. Firstly, in sections 3.1 and 3.2 we provide an overview
of several important aspects which need to be considered in order to understand and model the interaction between
DNA and radiation and its effects. Computational papers were used to present a description of radiation movement
through biological material and probability models for direct DNA damage based on total linear energy transfers
(LET) from incident particles. For indirect effects, the interaction between the products of water radiolysis and
DNA material was explored. The correlation between the type of LET of incident particles and the type of free
radical produced from water radiolysis was briefly discussed as well. Experimental papers were used to present
quantitative data on DNA damage under various types of radiation sources and energy intensities. To be noted is
that these subchapters served to establish and describe the chemical mechanism by which DNA damage occurs as a
result of EM and particulate irradiation. It did not however reflect plausible irradiation sources that people could
easily come in contact with. Section 3.3 looked for in vitro and in vivo studies which dealt with plausible sources of
radiation, such as cell phone radio frequency radiation, which a person could come in contact with and that could
produce DNA damage and hence induce mutations. We assumed that both direct and indirect effects contribute to
DNA damage and we also assumed that the damage leads to mutation.

Chapter 4 deals with Fenton-type reactions of various transition metals that are found within mammalian cells,
which produce DNA damaging free radicals, and their effects on the DNA. Section 4.1 introduces the selected
2
transition metals, their binding state and their concentrations within mammalian cells. Section 4.2 discusses the
general mechanism of Fenton and Fenton-type reactions, their resulting free radical products and their specific
effects on the cellular DNA. Section 4.3 briefly discusses the interesting repercussions of irradiation of the DNA
when it contains bound transition metals.

3
 2. Brief overview of the biochemistry and mutation of DNA

2.1 Biochemistry

Deoxyribonucleic acid (DNA) molecules are essential for living organisms as they contain the genetic information
for the formation and function of every cell. Ribonucleic acid (RNA) molecules and proteins are also involved in
the copying of the DNA and passing it on to new cells. The DNA contains two antiparallel strands and is found in
a double helix form. In eukaryotic cells, it is found in the nucleus (protected by the nuclear envelope's lipid bilayer)
and the mitochondria, which is the organelle responsible for generating the cell's adenosine triphosphate (ATP)
chemical energy supply. The DNA is a double helix consisting of two antiparallel strands that have a 5' end and a 3'
end respectively (ie. the phosphate at the end of the chain is either bonded to the C5 or the C3 of the sugar).

Its structure (Fig. 1) consists of four bases (purines

and pyrimidines) which pair up in a definite order,
adenine (A) with thymine (T) and cytosine with
guanine (G), and a deoxyribose sugar phosphate
backbone which is tied together through
phosphodiester bonds. Hydrogen bonds play an
essential role in keeping the double helix structure
of the DNA; the bases are connected together
through them, A-T containing two, and C-G three.
Computational studies have shown that the
hydrogen bonds are not purely electrostatic, but
exhibit a charge-transfer character (which carries a
comparable weight to the electrostatic one). This is
due to the O or N lone pairs donor interaction with
the acceptor N-H σ* orbitals. Additional
stabilization is given by the polarization within the
π- electron system, which reduces the bond length
by ~ 0.1Å [Fonseca Guerra].
Fig. 1 DNA basic structure [ref]
Due to its highly ordered nature, this molecular π-
stacked array allows charge transfer, which will later
become relevant for energy deposition and certain types of mutation [2]. To be noted is also the existence of the
minor and major grooves, which act as sites for metal-ion interaction. They have specific widths and are organized
charge centres with N-donors that can bind to metal ions.

There are three known forms of DNA, namely A, B and Z-DNA. B is the most prominent form, its discovery
(published in Nature in 1953) being one of the highlights of the 20th century science. Each form is important for
certain processes. Both the A and B forms have helices oriented to the right hand side, are right-handed forms.
The A form occurs in dehydrating solutions and is very similar to the B form, the difference being that B is more
stretched out. Due to its more compact form, the A type becomes important in DNA/RNA hybrids and in regions
of the RNA duplex form. The B form contains a minor and a major groove, which serve as binding sites for
proteins, such as transcription factors, which access the hydrogen bonds of the base pairs to read the existing
sequence. The Z form conforms to the same rules of base pairing as the other two forms, but is a left-handed form
and has more stretched out helices than the previous two forms.

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 a) b) c)

Fig. 2 Side views of :a) A-DNA b) B-DNA c) Z-DNA [ref]

The occurrence of different DNA forms is due to superhelical tension (usually forced by the process of
transcription) or the tendency of different base sequences to induce a certain shape (eg. alternating purines and
pyrimidines induce Z form) [2]. The biological role of the Z form is an area which continues to show new
discoveries. One of the most relevant for this review is that a very high number of genes from human
chromosomes contain sequences that induce Z-DNA near the sites which start transcription. This suggests that the
Z-DNA could have a functional role in starting the process of transcription. Another relevant role in connection
with mutations is its highly antigenic character, which has been linked several autoimmune diseases [3].

The base pair sequence, the number of base pairs per helix turn, the distance between the different components of
the DNA has a crucial effect on electron transfer mechanisms and radical chemical reactions. Of particular
importance is the redox characterization of the bases. G has been reported to be the best electron donor by P.
Jaruga hence with the lowest reduction potential (1.3V) [4]. G plays a key role in the mechanism of DNA free
radical induced damage, as the ·OH radical is a very good electrophile therefore it will add to sites with high
electron density. In terms of reduction potentials, G is followed by the other purine, A (1.4 V), and the pyrimidines
C (1.7V) and T (1.8V). This is directly related to the electron transfer mechanism. Due to similar reasons, C.R.
Treadway and colleagues report that electron transport occurs through an intrastrand pathway to a greater extent
than and interstrand one [5].

2.2 Mutation
Mutation is defined as “the changing of the structure of a gene, resulting in a variant form which may be
transmitted to subsequent generations, caused by the alteration of single base units in DNA, or the deletion,
insertion, or rearrangement of larger sections of genes or chromosomes” [5].

A simplified general description of mutation can be described using the example of cancer instauration and
development. Genetic instability (microsatellite or chromosomal) accelerates cancer progression by increasing the
likelihood of mutation at each step of progressive somatic mutations [6]. The cancer development process can be
described as taking place in three discrete steps: initiation, promotion and progression. In initiation, the cell’s genetic
material is mutated. The majority of the mentioned studies dealing with mutagenesis via the direct or indirect DNA
interaction with radiation or d-block metal induced radical attack on the DNA implicate this stage. Promotion occurs
after a cell has been mutated initially, it is vulnerable to the effects of promoters which stimulate cell proliferation.
In progression, a large number of daughter cells which contain the inherited mutation are created [7].

There are a number of factors that are known to cause damage to the DNA molecule. The body has ways of
repairing the damages in the DNA through certain proteins which correspond to various types of damage. For
instance, DNA ligase is used to fix broken side chains, DNA polymerase β tackles damaged bases within DNA,
endonuclease deals with damaged base pairs within DNA, and UV repair enzymes intervene in the case of dimers,
hydrations, breaks. If the radiation doses, the amount of d-block metals or other related parameters exceed certain
5
limits, the repair mechanisms cannot keep up with the damage anymore, thus mutations become permanent. We are
only concerned with permanent mutations.

We focused on two important categories of events which can lead to DNA damage: irradiation by particulates and
electromagnetic (EM) radiation and free radical attack induced by d-block metal oxidations. We also briefly
discussed the curious case of DNA irradiation while transition metals are bound to parts of the DNA molecule.

3. DNA damage due to particulate and EM radiation

3.1 Direct effects

Direct effects refer to the energy deposition of the fast particles on the DNA directly. In terms of the deposition
process, the excitation of a part of the DNA sugar-phosphate bond is followed by either deprotonation or direct
dissociation, both fates leading to DNA strand breaks.

A brilliant study published by A.Chatterjee and W.R. Holley (1991) [10] offers the theoretical background of
radiation movement through biological material and contains computational studies as well as some experimental
ones dealing with both the direct and indirect ways in which radiation induces DNA strand breaks. The process of
radiation-induced cell damage was studied from about 10-15 s when energy deposition occurs to 10-6 s. After the
latter time, the damage is said to be 'chemically stabilized'.

For this set of experiments, the authors used energetic heavy charged particles from a heavy- ion linear accelerator
coupled to a synchrotron. Various particles (from H+ to uranium) are accelerated, then injected into the
synchrotron to give out intense beams of charged particles with energies to a max of 1GeV/n (nucleon). In the
process of passing through tissues and cells, they lose energy and one of the physical quantities most associated
with this is -dE/dx, defined as the linear energy transfer (LET):

where z is the charge that the incident particle has, e is the charge of the electron, m is the electronic mass, v is the
velocity of the charged particle, NZ is the number of electrons per unit volume of measured cellular medium and I
is the mean excitation potential of the medium.

In the case of high LET charged particles, the energy distribution is showed by referring to the rc of the core. This is
the radius at which all of the energy loss due to glancing collisions is restricted.

It is given by:

with v the velocity of the charged particle, ε1 the lowest electronic transition energy and the
modified Planck constant.

The projectile's charge influences the energy density in the core, without changing its size. For reference, the radius
of the core corresponding to 600MeV/n is 82Å and the one for 10MeV/n is 15Å. This radius contains more than
50% of LET total, whilst the remaining energy is carried off to very large distances by energetic secondary
electrons. However, the average energy density which is deposited by these electrons is one or more orders of
magnitude less than the energy density from within the core region (Fig. 3). This outside-core region is referred to
as the 'penumbra', whose average radius is given by:

rp = 396(v) 2.7 (with v in units of 109 cm s-1 and the radius in Å)

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 Fig. 3 Cross-sectional view of a heavy particle trajectory. The dark
middle part presents 'the core', whilst the outside contour shows
'the penumbra' [10]

Differently charged particles that have the same LET values must have different velocities, hence different radii of
core and penumbra. Thus, they have different energy densities as a function of radial distance from the trajectory of
the incident particle. The average energy density inside the core (ρcore) is given by:

And inside the penumbra it is:

ρcore cannot be measured experimentally, however ρpen(r) was measured in previous studies. These expressions show
that the energy density inside the core is constant and the one in the penumbra changes proportional to 1/r2 (radial
distance from the trajectory of the incoming charged particle). A total LET can thus be determined by summing the
integrals of the above expressions within their respective radial regions. For the direct effect calculations, a 3D
DNA geometric model was used. The pathway of the fast particles was represented in three dimensions as well. Its
spatial orientation was given by: its impact parameter or the distance from the central axis of the DNA, the angle
that the trajectory makes with the central axis and the core radius rc. The coordinates of the particle pathway in
relation to the DNA can be utilized to calculate which sugar-phosphate groups are within the core (Fig. 4).

7
In order to calculate the average energy Eg lost to several sugar-phosphate molecules from within the core, the types
of molecular collisions were restricted to glancing collisions only, thus implying that only large impact parameter
collisions occur, so there is little deviation in the direction of the particles which thus leads to forward scattering.
Since the core radius was just defined in the context of glancing collisions, this is a sensible assumption to make and
for the purposes of observing DNA structure breaks, the direction of particle scattering within the core is not

Fig. 4 : Core of a 10 MeV/n Ne particle (rc=15Å) superimposed at a normal incidence both

to the plane of the figure and the axis of a structured DNA molecule that has a strand
separation distance of 20Å. The sketch is at a 1:1 scale of the core versus the DNA

essential considering that the tracks are equiprobable at any angle from the central axis. The average energy
calculation also makes use of Bragg's Rule – a molecule's stopping number is the sum of the stopping numbers of
all the atoms, which make the molecule [11]– and the stopping power formula (particle's average energy loss per
unit path length). A similar approach was taken to calculate the Eg from within the penumbra.

The average number of ionizations or excitations per sugar-phosphate that are at a radial distance r from the track
were given by:

Poisson statistics is used to give the probabilities describing the distribution of the numbers of excitations and
ionizations, as the energy deposition process is random:

Thus, for each incident particle pathway, the probability of ionization at each site of reaction within the penumbra
region radius, rp ,a random number, r#, is computed for each site, generating an “event”. If r# <P(>_1), the event is
noted to have produced an ionization, hence a break in the DNA strand. Breaks that are within 10 base pairs of
each other on opposite strands induce double breaks. So depending on its probability, an “event” produces no
strand breaks, one or more single breaks or double breaks. The yields of all the breaks can be calculated per unit
radiation dose, which is very useful and provides a good quantification method that can be compared to
experimental results. Thus, the yields for single strand breaks (SSB), YSSB, and double strand breaks (DSB), YDSB,
are given by:

where:

8
 with N the number of incident fast particles' pathways which are distributed uniformly over an area A inducing
either SSB or DSB on a DNA strip of molecular weight M and σ is the normalized cross section, ρ is the mass
density of the medium and LET is the total linear energy transfer of the fast particle.

The total Y can be expressed as :

where σ is the sum of the cross sections corresponding to the direct and indirect events which cause strand breaks.
For large values of LET, this assumption can induce overestimations of Y since it doesn't account for any spatial
correlation of direct and indirect causing damage. However, the error it gives was estimated at <10%, which still
provides a good approximate of the total strand breaks.

A direct comparison with experiments involving human DNA is difficult to do since a very high amount of
radiation exposure time is required in order for direct DNA damage to occur in the small SV40 virus DNA that was
used for the theoretical calculations (compare length of SV40 DNA molecule=1.8 x 10-6 m with mammalian DNA
length = 2-3 m). A way to do the comparison was by normalizing the results per unit base pair. Mammalian DNA
cell data was selected for cases where enzymatic repair of the DNA was absent, since this was the only way to
extract information on initial strand breaks, and was compared to the theoretical calculations (Fig. 5).

Fig. 5 Normalized yields of SSBs and DDBs against LET. The solid and open circles are various
reports’ published experimental data. The lines are from this paper’s theoretical calculations [10]

The experimental results (obtained from a variety of mammalian cell systems) compare reasonably well with the
theoretical calculations despite the differences in the experimental conditions which points to the conclusion that
these theoretical calculations can be used to produce theoretical predictions for an extensive range of experiments
dealing with DNA strand breakages and have a general character. Another notable observation is that the DNA
strand break pattern was different for different forms of the DNA. When the breaks were measured for DNA in a
chromatin form, their number was about six or seven times smaller than the number of DSB's seen for the linear
SV40 DNA. Another notable observation is that the DNA strand break pattern was different for different forms of
the DNA. When the breaks were measured for DNA in a chromatin form, their number was about six or seven
times smaller than the number of DSB's seen for the linear SV40 DNA (Fig. 5). There were several shortcomings to
this study, an important one being that it doesn't take into account the energy migration along the DNA chain
which implies that the actual site of damage in the DNA can be different from the location of the energy
deposition.

9
Several studies have been done which show that low energy electrons (<1 Kev Da-1), namely electrons with
energies lower than the DNA ionization potential (4.77-5.41 eV) are capable of damaging the DNA double strands;
low energy ions were shown to produce breakages in single and double stranded dried in vacuo DNA. C.A.
Hunniford et al. use the same property of LET of photon beams or light particles such as the electron passing
through tissue [12]. Additionally, they use the idea that ion beams deposit a very small amount of energy until they
reach a certain distance called the Bragg peak, where they deposit most of the energy. The ions can either directly
collide with DNA and break the strand or collide with secondary ions (with lower velocities) can take electrons
from the DNA, hence leaving 'holes' behind, which can induce breaks as a result of the change in the potential
energy surface. This is as slightly different approach to describing the direct effect than A. Chatterjee et al.’s,
nonetheless it is complementary and a step forward to describing the mechanism of action of these ions. Their
experimental approach dealt with dried plasmid pUC18 DNA in vacuo which was found to have been damaged upon
+
bombarding it with low-energy (<5KeV) Ar atoms. Unfortunately, these low energy ions have very low
penetration distances in air, hence the experiments were carried out under vacuum. The most extensive damage
form was SSBs, however DSBs were found to increase with ion kinetic energies, up to a plateau region at around
4KeV. The plasmid is usually isolated in supercoiled form, which makes it a more accurate system to compare with
human DNA in vivo. The notable effects of strand damages here were that a SSB converts the supercoiled form to
the open-circle form, whilst the DSB converts it into linear DNA (Fig. 6).

Fig. 6 Plasmid DNA converted from supercoiled to different forms

depending on the type of DNA lesion [12]

This is important as it offers a way to quantify the two different types of breakages. Additionally, MDSBs (multiple
DSBs) can occur randomly in the plasmid sequence, which are shown to produce multiple fragments of the DNA
(also shown in Fig. 6). This can be determined from the loss of the detected material from a gel electrophoresis as
compared to controls. 1-6 keV C+ and C2+ ions were used to test strand breakages on another plasmid, pBR322,
in vacuo as well. The magnitude of the breaks was found to be dependent on the number of ions incident on the
DNA piece, the ions’ kinetic energy and their charge state. Since LET is thought of as the energy transferred per
unit track, this does not affect the above conclusion on it. The MDSBs were the most extensive out of the three
potential types of breaks. An increase in the number of ions incident on the plasmid induced more damage, until
the saturation point of about 1000 ions per plasmid. An increase in the kinetic energy of the ions did not produce a
greatly increased proportion of DNA damage. However, it was observed that a higher proportion of molecules
underwent MDSBs as opposed to SSBs or DBSs, suggesting that ions with a higher kinetic energy have the ability
to cause more severe damage upon collision to the DNA.

Although the results did offer an important insight in the mechanism of radical attack on the DNA, they didn’t take
into account scavenging effects of hydrated DNA in vivo.

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 3.2 Indirect effects

In the context of DNA lesions, water radiation chemistry with various heavy charged particles is important, as the
cells, which contain the DNA in the nucleus, are made of up to 80% water and the radiolysis of water is induced by
energy deposition (thus yielding reactive species such as hydroxyl radicals). As a result, radiation exposure can also
indirectly cause changes in the DNA by the products of water radiolysis. Initiation is defined as the physical stage of
-15
energy deposition in the target medium. The primary process of ionization and excitation takes place in 10 s. This
+
produces mostly ·H, ·OH, H3O and solvated electrons. Next, the 'thermalization stage' occurs when these highly
reactive chemical species reach the temperature of the surrounding medium (~kT, with k-Boltzman's constant); that
takes about 10-12 s. Diffusion of radicals begins at the end of this process [10]. One of their fates leads to DNA
mutation or even cell death and it happens in about 10-6 s. This study has proposed modelling various types of
damage to DNA molecules on the basis of atomic and molecular physics laws and then cross-referencing them with
biochemical changes. It assumes that molecules such as proteins or enzymes that had deposed energy within the cell
are in a small enough concentration in comparison with water so they couldn't notably affect the DNA. However
they can have a scavenging effect, shielding DNA molecules from water-derived radicals, and this effect is significant
as the scavenger molecules and the radicals exist in comparable amounts. Experimentally this was reproduced quite
innovatively using an aqueous solution of Simian Virus DNA (SV40) – linear DNA - with 500mM Tris buffer1 to
simulate cellular conditions. Within a cell, the average migration distance of a water radical was found to be 30 Å, so
if a radical is produced at a larger distance from the DNA molecules the chances of it interacting with them are very
slim as it will be interfered with by scavengers.
-12
An ·OH was created with 17eV (UV region of EM spectrum) and the initial yields of radicals (10 s) were expressed
in terms of the number of a type of radical produced per 100 eV of energy which was deposited (G-values) and were:
GOH = 5.88, GH =0.88, Ge -solvate=5.00, GH3O+=5.00, having a Gaussian initial distribution, with a standard deviation
which slightly varies in the literature. Their formation with respect to each other's position is dependent on the
region they are formed in; within the core they form very close to each other, whereas in the penumbra they form
further away. The diffusion process they go through was simulated using Monte Carlo methods which allow the
reactants to “jump” in three dimensions on a distance I, in a time Δt, variables which are correlated through the
diffusion constant D, where with l the step size, chosen to be smaller or equal to 1Å in order to ensure the reactants
diffuse close enough to be within a critical distance characteristic of their species, thus insuring they have a good
chance of reacting with each other:

This model was put forward by Smoulchowksi [17]. The reaction radii RAB were calculated from experimental data
on the rate constants k using:

with DA and DB diffusion constants and k reaction rate constant.

·OH have D= 2x10-5 cm2s-1, ·H have D= 8x10-5 cm2s-1 and the k for water radicals reaction is of 109 L mol-1s-1
order. This considers DDNA =0, so keeps the DNA molecules fixed in space. Theoretical decay curves showing the
loss of water radicals from the system in pure water were derived based on an algorithm using these formulae,
though experimental results for comparison are hard to do at the moment because one would need to be able to fire
nanosecond or shorter time pulses. Data from a 20MeV pulsed electron beam was however successfully extracted
and produced a good comparison with the theoretical model. In these paper's experiments, the use of the Tris buffer
reduces the probability of radicals making a successful “jump”, thus changing the pure-water decay curves by a factor
of exp(-Δt/ 6 x 10-10 s). An assumption has been made concerning the production of DNA strand breaks – only
·OH radicals induce strand breaks by taking out hydrogen from the sugars (so on one of the five C positions). A

1Trisaminomethane-buffered saline is an isotonic, nontoxic, biocompatible buffer which is used to keep the pH stable in a
narrow range. Here, it keeps the structural integrity of the DNA and can act as a scavenger for water radicals
11
random number and the assumed Gaussian distribution give the initial radical position. Each radical could react
with a sugar or a base, based on its critical distance of approach, yielding radii for hydroxyl radicals as
follows:

•OH + ... Rxn radius/ Å

sugar 1.0
A 3.6
T 3.6
G 5.2
C 3.6

Fig. 7 shows an overview of all the DNA bases’ modification as a result of oxidative attack of the hydroxyl
radical.

Fig. 7 DNA base modification upon hydroxyl radical attack [13]

The authors also found that the DNA is damaged more by low- LET particles for the same dose of exposure; the
high-LET charged particles are thus less efficient in causing DNA damage. This is because an increase in LET
correlates to an increase in the energy density values from the core and the penumbra. This means that there will be a
higher density of radicals so a higher frequency of interactions between water radicals, hence there will be less of
them available to interact with the DNA and cause damage.

Water radiolysis is acknowledged to be essential in determining the mechanism of indirect radiation

damage to biological. A similar, more recent computational study focused on this was lead by M.P. Gaigeot
et al [14]. It followed the same principles as the study described above. It started from the irradiation of
biological tissues by fast heavy ions. Using Monte Carlo and Car-Parrinello (a type of ab initio molecular
dynamics which employ periodic boundary conditions and density functional theory) simulations, they have
shown that the HO2· is produced at a high rate by high LET particles as opposed to low LET ones, which
support the findings of the previous study.

Many studies also support the conclusion of water radiolysis having multiple products. In bimolecular
systems, two types of collisions are thought to be possible: electronic excitation, ionization and electron
capture by an ion and molecular fragmentation as a direct result of collision. These events produce several
reactive chemical species, out of which the most notable are: electrons, O• , O2•− , H• , OH• and HO2 • ,
along with OH- and H+.

12
3.3 Selected studies reflecting potential occurrence of human DNA damage in vivo

Even if direct and indirect DNA damaging effects were proven to happen as a result of particulate radiation, would
the human body be exposed to radiation that is of similar energy to these mutagenic radiations? For comparison, a
sketch of the electromagnetic radiation spectrum along with corresponding photon energies is described in Fig. 8.
Some literature suggests that any electromagnetic (EM) radiation with energy equal to or higher than UV light can
have mutagenic effects. Literature also suggests that radio-frequency (RF) EM radiation can induce oxidative stress
which can in turn increase the production of reactive radicals that induce DNA strand breaks. We selected a few
publications that dealt with high RF-EM fields, and RF used in mobile phone technology, as mobile phones are a
source of radiation which a significant number of people are exposed to daily.

EM fields increase free radical activity within cells by either increasing the production of reactive oxygen species
(ROS) or decreasing the activity of the antioxidant enzyme activity. DNA SSBs and DSBs breakages in rat brain cells
were reported by Lai and Singh [16], after exposure to a 2450 MHz RF field at 0.6-1.2 W/kg for a period of 2 hrs.
This also caused DNA-protein and DNA-DNA crosslinks. This damage could be counteracted by treating the rats
with free-radical scavengers.

Fig. 8 Electromagnetic spectrum: various types of EM radiations’ wavelengths and their corresponding
photon energies [15]

Paulaj et. al reported DNA single strand breakages in developing rat brain cells at a 35 days exposure to 2.45 and
16.5 GHz RF fields at 1 and 2.01 W/kg. The exposure of human lymphocytes and leucocytes at a specific absorption
rate (SAR) of 5-10 W/kg for 24 hrs was reported to produce chromosomal damage, by Tice et al; however this was
not observed for an exposure for 3 hrs at the same SAR or for an exposure of lower SAR [17]. In vitro studies have
shown to induce cellular signal transduction and stress response pathways, RF-EM waves have the potential of
-6
altering cell physiology [18]. The energy deposited in tissue by a 900 MHz GSM mobile phone is 4 x 10 eV and by a
1800 MHz one is 7 x 10-6 eV, both of which are significantly lower than the energy needed to break a chemical bond.

A very comprehensive study was done by D. Remondini et al,[19] where biological effects of mobile phone
microwaves (very high frequency radio waves) were studied in vitro. Six different types of human cells were exposed
to 900 and 1800 MHz waves; these included immortalized cell lines and primary cells. The later simulate in vivo
conditions very well, however they are very sensitive to cell isolation and extremely variable in their response, whilst
the former can be standardized, they genetically differ from primary cells and can be unstable, thus cell proliferation
changes or even cell death may occur. The results were analysed making use of bioinformatics statistics and were
inspected for biological relevance by cross-reference with various databases. While four of those cell types did not
show notable changes in the gene expression, two of them presented between 12 and 34 up- or down-regulated
genes. Analysis of the affected gene families doesn’t indicate a stress response, as stress usually up-regulates cellular
13
ribosomal RNA synthesis. However it has been indicated that some of the human cells might react with an increase
in expression of the genes that encode ribosomal proteins.

N.R. Desai et al [20] published an extensive and balanced review dealing with the in vitro effect of RF-EM waves on
biological material, focusing on cell phone radiation. As a short overview, pulse modulated RF radiation was shown
to induce morphological changes to exposed lymphocytes as well as increase their size. An increase in the exposure
time led to destruction in the organelle and the nucleus. For exposure times of 8h and 24h, chromatin and
mitochondrial alteration was observed. Additionally, a 48 hr RF radiation – GSM modulated at 1.8 GHz showed
cytoplasmic lysis and cell and nuclei membrane disruption. These results suggest that RF radiation played an
important role in inhibiting cell viability, and it did so in a time dependent fashion. A study done by Mashevich et al
confirms these findings; chromosomal instability was reported in human lymphocytes which were exposed to
continuous 830 MHz RF radiation at SAR level of 1.6-8.8 W/kg for 72 h [21]. Overall, the impact of RF on living
cells is still controversial; there are many papers that were unable to reproduce studies similar to the ones above,
observe no effects or contradict some of the findings. Examples would be studies by Kwee et. al [22]and Pacini et al
[23] who reported cell proliferation inhibition as a result of GSM basic RF exposure. The RF related study on
biological material is still at the beginning stage as it is very dependent on the types of cells and the experimental
conditions utilized.

4. DNA damage due to the attack of free radicals induced by transition metals
4.1 Brief overview of transition metals in mammalian cells

The body has protection mechanisms against mutagenesis, which reduce the exposure of the DNA to
damaging reagents such as ·O2-, H2O2, ·ROOH and ·OH. These radicals are the result of oxidations
involving metals. Human cells use a superoxide dismutase inside the cytoplasm, which can be equilibrated
using a small amount of metal ions, namely Cu and Zn. Additionally, within cells H2O2 reactions occur in a
special vesicle [24]. There is some literature which suggests Ca2+ could induce DNA strand damage through
a mechanism involving calcium activated nucleases as a result of an increase in calcium levels from the
cytoplasm and the nucleus induced by oxidative stress [26]. It is however not conclusive, as the only
indicator for this theory comes from the usage of the Ca2+ chelator quin 2 which when introduced
prevented oxidative stress induced DNA strand breaks. Hence, this paper will only involve oxidative stress
induced by transition metal ions.

In the human body, the most abundant transition metal ions which act as catalysts are iron (Fe), manganese
(Mn), zinc (Zn) and copper (Cu), along with traces of other metals which exist when no pathologies are
present or no toxicological exposures occurred. All of these can contribute to an increase in the production
of free radical.. As the DNA is found within the cells, the approximate concentration of the above
mentioned transition metals ions in the cell cytoplasm is relevant. Free Fe and Mn ions are present in
concentrations of about 10-16 M and the excess ions are carried out, after they have been oxidized to their
3+ oxidation state, by the blood-protein transferrin in an octahedral complex. Free Cu ions are found in
concentrations of 10-14 M and are kept in equilibrium through binding excess Cu2+in a tetrahedral
complexes by albumin carriers [24]. The average copper levels in normal tissues is reported to be around 20
x10-6 M and is found to increase in most tumours [27]. The ion distribution between the DNA and
proteins is not known, but is hypothesized to be determined by the degree of accessibility and binding
strength of the ions to the particular cellular components; different oxidation states induce different
binding strength [29]. Elementary analysis by energy loss spectrometry showed Fe bound to chromatin.
Iron containing proteins such as FUR or FNR found in the cytoplasm are linked to gene regulation and Cu
(bound in proteins) is localized on the external face of the mitochondrial membrane. Mn is a known
mutagenic agent and is found contained in a large number of vesicles such as: the lysosome (as protein acid
14
phosphase Mn(III) ), the mitochondria (superoxide dismutase enzymes), golgi apparatus (glycolysil
transferases), vacuoles (free Mn (II)).

4.2 Fenton-type reactions and organic radicals

Alkyl Hydroperoxides (ROOH) were reported by J. Termini [29] to induce mutations as a direct consequence of
their decomposition's production of radicals. Free radicals can also be generated when a hydroperoxide reacts with a
transition metal ion complex. Their distribution is influenced by the ligand structure and the nature of the transition
metal ion. Iron catalysed hydrogen peroxide is known as Fenton's reaction:

Fe2+ + H2O2 ----> Fe3+ + ·OH + OH-,

Fe3+ + H2O2 ----> Fe2+ + ·OOH + H+.

S. Goldstein, D. Meyerstein and G.Czapski reported that transition metals in their low oxidation states, LmMn+, tend
to behave like the Fenton reagent, hence are known as Fenton-type reactions. Here, the hydrogen peroxide is
replaced with an organic peroxide (ROOH), giving a generalized reaction:

ROOH + Mn++H+--> M(n+1)++ H2O + ·RO

In the case of a mixture of transition metal ions reacting with reducing agents in a context which contains oxygen, a
-
sequence of reaction takes place which eventually produces OH and ·OH [30]. If the Fenton-type reaction (Fig. 9)
proceeds via the above mentioned radical intermediates, it is considered to be radiomimetic – the mutation and
damaged sustained by the DNA mimics the one observed in the case of DNA damage done by ionizing radiation
(>10eV – approximately in the far UV region).

Fig. 9 Model of in situ Fenton reaction which produces DNA modifications

Several studies which involved substrate trapping and pulse radiolysis showed that the oxidation of various organic
substrates by Fe(II) EDTA (in large excess over the substrate) produces ·OH at rates directly comparable to the
independent generation of ·OH by ionizing radiation. This was generalized to different MLm complexes whose
natured altered the yields of oxidizing radicals, but in most cases the results were similar to the ones produced by
Fe(II) EDTA. To be noted is the production of protonated superoxide ( HO2·) by other transition metal complexes
such as Mn(III) and Co(II). This is normally not found in physiological pH conditions, having a pK of cca. 4.8, but it
is a strong oxidizing agent and has been linked to the formation of DNA strand breaks.

The ROOH's behave as the hydrogen peroxide in reactions with metal ions, and a special note should be made about
lipid hydroperoxides, whose structure influence the distribution of the produced radicals. This has repercussions on
mutagenesis. This has been studied in the context of the autooxidation of polyunsaturated lipids which is of interest
15
due to the correlation found between the high intake of polyunsaturated fats and the increase in the probability of
various cancers [29].

In the case of the ·OH reaction with DNA base T, the addition mostly takes place at the C-5 position
giving the corresponding 6-yl radical, in the case of G, the radical adds at the C-8 position giving the
corresponding 8-hydroxyguanine and formamidopyrimidine derivatives (Fig. 10). Similar adducts for A were
observed, in addition to adducts corresponding to ·OH addition at the C-4 position of the purines. These
direct reactions of the DNA bases with the hydroxyl radicals yield the unstable adducts which inflict DNA
damage.

Fig. 10 •OH interaction with guanine from the DNA and their reaction product

The mechanism of hydroxyl radicals reaction with purine and pyramidine bases is shown in the top Fig. 11.
Peroxyl radicals (ROO·) can also be generated in the aerobic Fenton reaction, from carbon radicals. These
can then oxidize other compounds, producing radical intermediates and alkyl hydroperoxides (Fig. 11,
bottom).

Fig. 11 Top: Hydroxyl radicals attack the methyl-substituted double bond, acting as the nucleophiles. The principal
pathway is double bond addition, resulting in products A and B, whilst the secondary pathway is abstraction of H
atoms, resulting in product C. Bottom: Peroxyl radicals oxidize the above substrates via H atom abstraction, producing
an oxidized radical intermediate and alkyl hydroperoxides

16
The ROOH's behave as the hydrogen peroxide in reactions with metal ions, and a special note should be made about
lipid hydroperoxides, whose structure influence the distribution of the produced radicals. This has repercussions on
mutagenesis. This has been studied in the context of the autooxidation of polyunsaturated lipids which is of interest due
to the correlation found between the high intake of polyunsaturated fats and the increase in the probability of various
cancers [29].

Akasaka and Yamamoto reported DNA oxidative base damage induced by Fe(III)/H2O2 in mammalian cells (which
show a greater damage than bacteria cells exposed to the same protocol); the mutation spectrum was primarily defined
by single base changes. The Fenton-induced mutation spectrum had 15% A/T base substitutions -compared to
spontaneous mutations which occur in ~ 2% of the time) – while the C/G base substitutions remained at a comparable
frequency and extent as with spontaneous mutations [32].

J.L. Sagripanti et al. have investigated the in vitro effects of low concentrations of Cu(II) and H2O2 on supercoiled
plasmid DNA. Plasmid DNA is a form of DNA packing found in bacteria and eukaryotic organisms and represents a
small DNA fragment that still keeps the property of a chromosome of autonomous replication. In vivo, plasmid DNA is
found tightly supercoiled so this provided a good comparison to oxidative stress reactions in a living organism. Even
though DNA in mammalian cells and proteins is found packed in a chromosome form, the results of the plasmid
experiment should still hold. Care should perhaps be taken on the quantity related parameters of the reactants, since the
extra bonding interactions in a chromosome may provide more stability to the molecule, so the DNA strands may be
harder to break. Whilst neither Cu(II) or H2O2 , introduced in concentrations up to 10-2 M, had any negative effects by
themselves, a mixture of 10-6 M Cu(II) and 10-5 M H2O2 was shown to induce strand breakages and inactivated the
transforming ability (ability to insert and express into another cell as a foreign DNA sequence). Metal chelators, catalase
and free radical scavengers inhibited the strand breaks. That indicates that it is indeed the Cu(II), H2O2 and resulting
·OH radicals whose reactions produce damages on both single strand and double strand samples. For the process of
enzymatic sequencing, the addition of a mixture of the above specified concentrations of copper and the hydrogen
peroxide was done in medium of pH 7-7.8. This is around what the pH of blood and many other human cells is. The
DNA sample was then treated with hot piperidine (1.0 M, 90°C, for 30 min) in order to cleave the DNA hence expose
the location of the damages [33]. For chemical sequencing, hence determination of oxidative base damage, an
electrophoresis method was used. The DNA sample went through a number of other reactions, which served to provide
a collection of bands on a gel (similar to the ones in Fig. 12). The original DNA sample (untreated) showed a major
band which points to the supercoiled form of DNA as well as a minor one which points to the circular form. The

Fig. 12 Gel electrophoresis of various potential forms of DNA [34]

treated sample showed DNA damage – the amount of the supercoiled form of DNA decreased and the circular and
linear form amounts increased. The rate of DNA single strand breaks was shown to be influenced by the incubation
period and incubation temperature; for 37ºC, and after an incubation period of roughly 30 min, around 10% of
supercoiled DNA remained. The results indicated that oxidation occurred preferentially in the 5' end of G in a
polyguanine. This is thought to be due to the π-stacking interaction between the G bases; thymine sites and isolated
guanines were also reported to be damaged by other sources, and adenine oxidation sites were rarely seen. However,
even though piperidine is the standard reagent used for biochemical degradation reactions such as DNA cleavage in the
case of modified nucleotides, it was shown to have an affinity for selectively cleaving G nucleotides if used by itself [35],
therefore the mechanism of hydroperoxide DNA bases oxidation might have not been properly investigated.
H.Rodriguez et.al [36] took a different approach, improving on the efficiency of determining DNA base oxidative

17
lesions; they used ligation mediated polymerase chain reaction (LMPCR) to detect rare DNA breaks. This technique
makes usage of small DNA linkers connected to the wanted piece of the DNA and amplifies several copies of that piece
over several orders of magnitude. This selective amplification technique is a trustworthy means to accurately study this
oxidative process without chemically interfering with any of the reaction elements. Additionally, instead of using
piperidine, DNA glycosylases/apurinic- apyramidinic (AP) lyases were used, thus determining the whole range of
oxidative damage to the DNA. The glycosylases create abasic residues (places in DNA which don't have a purine or a
pyramidine attached to the deoxyribose) at sites where oxidation occurs, which are then cleaved by AP lyases. A large
number of sites in isolated genomic DNA were probed after treating the DNA sample with Cu(II)/H2O2.. Rodriguez et.
al validated Sagripanti et al.'s work by determining the polyguanines as an oxidative hotspot. In addition to these
findings, oxidative damage to all the bases, in order of the damage extent G>C>T>>A has also been determined.
Similar findings were also reported, showing that DNA footprints obtained after oxidation by Fe(II) were virtually
identical with the ones obtained from Cu(II). Interestingly, Cr(VI) mediated Fenton reaction experiments showed a
different base damage pattern, which seems to suggest a difference in the DNA base metal ion binding preference.

4.3 The combined DNA damaging effect of radiation and transition metals

The interaction between radiation and DNA material that already had bound transition metals was found to result in
DNA conformational changes and mutagenesis. A thorough paper dealing with the changes in the DNA conformation
as a result of gamma irradiation in the presence of copper bound DNA molecules was written by C.N. Trumbore et al.
[28]. It was found that γ Irradiation of DNA solutions which contain copper induces DNA conformational changes.
Circular dichroism was used to diagnose these changes; the presence of a 187nm peak in the difference spectrum
appeared, which was predicted for a B-Z helical DNA conformation and similar observations were reported for the UV
spectra. It was proposed that, as a result of binding to the DNA, transition metals alter the DNA’s conformation in
their vicinity, which in turn causes increased vulnerability to radiation induced radical attack within or next to these
conformationally distorted DNA regions [37]. This could in itself be a source for potential mutation occurrence since
the conformation of the DNA and its associated proteins is essential to processes such as replication and transcription.
The presence of Z- DNA was correlated to the type and concentration of bound metals; their concentrations and
efficiency allows them to shift the conformational equilibrium from the B form to the Z form. As confirmed by studies
−
discussed in the above sections, ionizing radiation induced the oxidizing species •OH as well as the reducing one O2• .
Whilst the former one is known to produce SSBs and DSBs in the DNA, thus potentially causing conformational
changes, the latter one was reported to be unreactive with DNA, however it reduced Cu(II) to Cu(I), which in turn
caused similar conformational changes. In fact, it was shown to be twice as effective as the •OH radicals. Moreover, in
vitro experiments showed that DNA strand breaks and base damage due to ionizing radiation (which causes a number of
radical species) are insufficient to determine a change from B to Z conformations and they are in fact strongly
dependent on the metal type and concentration which are bound to the DNA at the time of irradiation. High metal
concentrations shifted the conformational equilibrium almost completely to the Z side for the alternating C-G base
sequence DNA parts. This is thought to have happened as a result of lowering the large activation energy barrier
between the two conformations; the •OH damages the DNA part which is Cu(II) complexed which induces strand
breaks as well as tighter binding of Cu(I) to the DNA [28]. Since the grooves of the DNA are known to accommodate
different transition metals, it is sensible to assume that similar effects may occur with metals other than copper.

This shines a new light on the understanding of the complex mechanisms of DNA alteration; it can be brought about in
distinct ways, with distinct repercussions and probably different end effects depending on the particular set of
conditions the DNA molecules are. So irradiating a DNA sample will have a different effect if the sample had
complexed transition metals or not.

18
5. Conclusion

Topics like mutagenesis or DNA alteration are much too complex to be thoroughly treated in an overview such
as this one. Their biochemical course of action cannot be studied by limiting it to the 'end product'. It is highly
sensible to initial conditions such as the existence of various compounds bound to the DNA in the moment of
radical attack, or the type radical production event.

The primary purpose of this literature review was answered. We set out to determine whether or not
mammalian DNA irradiation by particulate and EM radiation or DNA attack by free radicals induced by
transition metals’ oxidations induces mutagenic damage.

We presented that both direct effects of irradiation, and indirect effects can lead to DNA SSB’s, DSB’s or
MDSB’s. Important variables affecting the amount and type of breaks were found to be: the intensity of the
radiation and the total irradiation time, the type of DNA (A,B or Z-DNA) and its orientation, the amount of
linear energy transfer to the DNA (low-LET particles produced more damage). Direct effects involved energy
deposition of fast particles on DNA directly, whilst indirect effects were induced by the DNA interaction with
+
the products of water radiolysis, namely the highly reactive free radicals ·H, ·OH, H3O and solvated electrons.
Case studies dealing with mobile phones RF irradiation were inconclusive, though many seemed to suggest a
negative impact on cell organelles and potential damage to the cellular DNA.

We also wanted to see what kind of free radicals the Fenton-type reactions involving transition metals
produced, and what their interaction mechanism with the DNA was. The main free radicals produced were
found to be ·O2- , ·RO, ·ROOH, ·OH and ·OOH. The most abundant transition metals which can be
found in mammalian cells, and hence participate in Fenton-type reactions were found to be Fe, Mn,
Zn and Cu. If ·OH or ·OOH radicals are produced, the DNA damage occurs in the same way it does
in the case of irradiation with ionizing radiation. Studies involving oxidation of various organics
substrates by the studied transition metals showed extensive DNA damage, in the form of DNA SSB’s,
DSB’s or MDSB’s. Oxidative damage was shown to be preferential as follows G>C>T>>A. The presence of
transition metal ions, particularly of Fe and Cu were shown to have an essential effect on the type of outcome
radical attacks have on the DNA. These varied from strand breaking to DNA conformational changes, both
which have different repercussions and determine quite different mutations.

Unfortunately, none of the studies above suggested that any of the discussed mutagenic mechanism lead to
adaptive mutations. The situation seems to be quite grim – radiation energy deposition and oxidative stress was
mostly associated with cancers and tumours. Other literature suggests oxidative stress is linked to Parkinson’s
disease and Alzheimer’s disease, autism, heart failure [38]. There are very few examples were beneficial genetic
mutations were observed. An example would be a fairly new discovery reported by A.D. Sullivan et al [39]
where the 32-bp deletion in the host-cell chemokine receptor CCR5, CCR5Δ32 causes homozygous individuals
for this gene to be immune to HIV infection and heterozygous individuals to have a lower pre-AIDS viral load
and experience a hindered progression to AIDS.

The human body is a complex system and its self-regulation and feedback are hard to pinpoint and study in a
reductionist, segmented manner. As a result, many questions about mutagenesis in the context discussed in this
review remain to be answered: Bio-chemical mechanisms of the first steps of mutagenesis – DNA alteration
have been proposed, yet how can one track these all the way to their end result? How sensible to initial
conditions are these mutations? There is evidence that metal binding to the DNA changes the outcome of
radiation or radical attack on its molecules. Experiments should be designed to test or computationally simulate
the presence of other transition metals (or other potential metal traces) bound to the DNA. Interesting case
studies could include examining this in various pathologies dealing with abnormal metal levels in the body (eg.
Wilson’s disease, Hemochromatosis). Attention should also be drawn to organic molecules which can bind to
the DNA. Although this is a relatively new direction of research, studies have shown very interesting findings
19
and a lot of unmined potential. An example is methylene blue binding to alternating GC base sequences. There
are indications showing that it induces photosensitized reactions which can be utilized for selective DNA
backbone cleavage [40].

Over the years (particularly since the 1960’s) documented controversial inquires have been made in areas
dealing with what we can say come closest to the X-men type mutants such as: extrasensory perception,
telekinesis, psychokinetic-teleportation.

Whilst some studies dealing with the interaction between mind and matter continue, it is safe to say that
purposefully irradiating one’s self with γ rays or any other rays from the EM spectrum (such as by excessively
talking on the cell phone) or resorting to other drastic oxidative stress inducing methods will NOT bring
about superpowers (Fig. 13).

Fig. 13 Take-home message

20
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