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Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser JRole of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem 266: 37-56

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Abstract and Figures

The development of cancer in humans and animals is a multistep process. The complex series of cellular and molecular changes participating in cancer development are mediated by a diversity of endogenous and exogenous stimuli. One type of endogenous damage is that arising from intermediates of oxygen (dioxygen) reduction - oxygen-free radicals (OFR), which attacks not only the bases but also the deoxyribosyl backbone of DNA. Thanks to improvements in analytical techniques, a major achievement in the understanding of carcinogenesis in the past two decades has been the identification and quantification of various adducts of OFR with DNA. OFR are also known to attack other cellular components such as lipids, leaving behind reactive species that in turn can couple to DNA bases. Endogenous DNA lesions are genotoxic and induce mutations. The most extensively studied lesion is the formation of 8-OH-dG. This lesion is important because it is relatively easily formed and is mutagenic and therefore is a potential biomarker of carcinogenesis. Mutations that may arise from formation of 8-OH-dG involve GC --> TA transversions. In view of these findings, OFR are considered as an important class of carcinogens. The effect of OFR is balanced by the antioxidant action of non-enzymatic antioxidants as well as antioxidant enzymes. Non-enzymatic antioxidants involve vitamin C, vitamin E, carotenoids (CAR), selenium and others. However, under certain conditions, some antioxidants can also exhibit a pro-oxidant mechanism of action. For example, beta-carotene at high concentration and with increased partial pressure of dioxygen is known to behave as a pro-oxidant. Some concerns have also been raised over the potentially deleterious transition metal ion-mediated (iron, copper) pro-oxidant effect of vitamin C. Clinical studies mapping the effect of preventive antioxidants have shown surprisingly little or no effect on cancer incidence. The epidemiological trials together with in vitro experiments suggest that the optimal approach is to reduce endogenous and exogenous sources of oxidative stress, rather than increase intake of anti-oxidants. In this review, we highlight some major achievements in the study of DNA damage caused by OFR and the role in carcinogenesis played by oxidatively damaged DNA. The protective effect of antioxidants against free radicals is also discussed.
Content may be subject to copyright.
Molecular and Cellular Biochemistry 266: 37–56, 2004.
c
2004 Kluwer Academic Publishers. Printed in the Netherlands.
Role of oxygen radicals in DNA damage and
cancer incidence
Marian Valko,
1
Mario Izakovic,
1
Milan Mazur,
1
Christopher J.
Rhodes
2
and Joshua Telser
3
1
Faculty of Chemical and Food Technology, Slovak Technical University, SK-812 37 Bratislava, Slovakia;
2
School of
Chemistry, University of Reading, Reading, UK;
3
Chemistry Program, Roosevelt University, Chicago, IL USA
Received 21 January 2004; accepted 11 March 2004
Abstract
The development of cancer in humans and animals is a multistep process. The complex series of cellular and molecular changes
participating in cancer development are mediated by a diversity of endogenous and exogenous stimuli. One type of endogenous
damage is that arising from intermediates of oxygen (dioxygen) reduction oxygen-free radicals (OFR), which attacks not only
the bases but also the deoxyribosyl backbone of DNA. Thanks to improvements in analytical techniques, a major achievement
in the understanding of carcinogenesis in the past two decades has been the identification and quantification of various adducts
of OFR with DNA. OFR are also known to attack other cellular components such as lipids, leaving behind reactive species
that in turn can couple to DNA bases. Endogenous DNA lesions are genotoxic and induce mutations. The most extensively
studied lesion is the formation of 8-OH-dG. This lesion is important because it is relatively easily formed and is mutagenic
and therefore is a potential biomarker of carcinogenesis. Mutations that may arise from formation of 8-OH-dG involve GC
TA transversions. In view of these findings, OFR are considered as an important class of carcinogens. The effect of OFR is
balanced by the antioxidant action of non-enzymatic antioxidants as well as antioxidant enzymes. Non-enzymatic antioxidants
involve vitamin C, vitamin E, carotenoids (CAR), selenium and others. However, under certain conditions, some antioxidants
can also exhibit a pro-oxidant mechanism of action. For example, β-carotene at high concentration and with increased partial
pressure of dioxygen is known to behave as a pro-oxidant. Some concerns have also been raised over the potentially deleterious
transition metal ion-mediated (iron, copper) pro-oxidant effect of vitamin C. Clinical studies mapping the effect of preventive
antioxidants have shown surprisingly little or no effect on cancer incidence. The epidemiological trials together with in vitro
experiments suggest that the optimal approach is to reduce endogenous and exogenous sources of oxidative stress, rather than
increase intake of anti-oxidants. In this review, we highlight some major achievements in the study of DNA damage caused
by OFR and the role in carcinogenesis played by oxidatively damaged DNA. The protective effect of antioxidants against free
radicals is also discussed. (Mol Cell Biochem 266: 37–56, 2004)
Key words: radicals, DNA damage, cancer incidence, antioxidants, pro-oxidants, carotenoids, oxidative stress, review
Abbreviations: OFR oxygen-free radicals; ROS reactive oxygen species; XO xanthine oxidase; EPR Electron Paramagnetic
Resonance; MDA – malondialdehyde; GSH – glutathione; NADH – nicotineamide adenine dinucleotide; AFR ascorbate-free
radical; LDL – low-density lipoprotein; 8-oxoG – 8-oxoguanine; CAR – carotenoids; FAPy-G – 2,6-diamino-5-formamido-4-
hydroxypyrimidine
Address for offprints:M.Valko, Faculty of Chemical and Food Technology, Slovak Technical University, SK-812 37 Bratislava, Slovakia (E-mail:
marian.valko@stuba.sk)
38
Introduction
Oxygen, while indisputably essential for life, can also par-
ticipate in the destruction of tissue and/or impair its ability
to function normally [1, 2]. Oxygen-free radicals (OFR), or
more generally, reactive oxygen species (ROS) are products
of normal cellular metabolism. It has been estimated that the
average person has around 10,000–20,000 free radicals at-
tacking each body cell each day. For an over-trained athlete,
this figure can be increased by roughly 50%.
In some cases, ROS are produced specifically to serve
essential biological functions, whereas in other cases, they
represent byproducts of metabolic processes [3]. Despite
the cell’s antioxidant defence system to counteract oxida-
tive damage from ORF, radical-related damage of DNA and
proteins have been proposed to play a key role in the develop-
ment of age-dependent diseases such as cancer, arterioscle-
rosis, arthritis, neurodegenerative disorders and others [4, 5].
All ROS have the potential to interact with cellular compo-
nents including DNA bases or the deoxyribosyl backbone of
DNA to produce damaged bases or strand breaks [6]. Oxy-
gen radicals can also oxidize lipids or proteins thus generating
intermediates that react with DNA by forming adducts [7].
Some oxidative DNA lesions are promutagenic and oxida-
tive damage is proposed to play a role in the development of
certain cancers [8].
Electronic structure of oxygen radicals
Free radicals can be defined as molecules or molecular frag-
ments containing one or more unpaired electrons in atomic
or molecular orbitals [9]. This unpaired electron(s) usually
gives a considerable degree of reactivity to the free radical.
Radicals derived from oxygen represent the most important
class of radical species generated in living systems [10].
Molecular oxygen (dioxygen) has a unique electronic con-
figuration and is itself a radical. In the ground state, it is a
biradical with two parallel unpaired electrons in antibonding
π
orbitals thus forming a triplet state molecule [11]. These
two unpaired electrons give oxygen a spin quantum number
(S)ofone (S = 1, each unpaired electron contributing 1/2)
and a spin multiplicity of three (2S + 1 = 3), that is, dioxy-
gen is a triplet molecule [12]. Valence bond theory describes
dioxygen as a double bond species (O=O), however molec-
ular orbital (MO) theory predicts that dioxygen is a biradical
(
OO
), a structure which better accounts for the reactivity
of dioxygen with other radical molecules [12].
In addition to the triplet state of dioxygen (denoted
3
g
)it
also exists in two singlet states, denoted as
1
g
and
1
+
g
,
higher in energy than the ground state by 23.4 and 37.5
kcal/mol, respectively (see Fig. 1). The lifetime of
1
+
g
is
extremely short and it rapidly interconverts to
1
g
, which
is probably the most relevant source of singlet dioxygen in
biological systems [11].
The addition of one electron to dioxygen forms the super-
oxide anion radical (O
2
) (Fig. 1). This electron fills one of
the π
orbitals. The addition of another electron to the sec-
ond π
orbital forms peroxide dianion (O
2
2
), which has all
electrons paired and therefore is not an oxygen radical [11].
The kinetic stability of dioxygen is explained by its elec-
tronic configuration. As mentioned above, in the ground state
the π
electrons are parallel. Therefore, when oxygen reacts
with an atom or a molecule, the substrate also must possess
parallel spins. However, electron pairs in most atoms and
molecular bonds contain antiparallel electrons, therefore, the
oxidizing ability of molecular oxygen is reduced. A second
reason for the reduced oxidative ability of dioxygen is the
thermodynamic disadvantage of the one-electron reduction
of oxygen (O
2
+ e
O
2
)versus the two electron reduc-
tion (O
2
+ 2e
O
2
2
), which fills both π
orbitals [13, 14].
The thermodynamic parameters for the reduction of dioxy-
gen molecule are summarized in Table 1 and the four single-
electron reduction steps from molecular oxygen to water are
shown in Fig. 2.
Sources and properties of oxygen
radicals
Superoxide radical
With the exception of unusual circumstances such as ionis-
ing radiation, ultraviolet light, and other forms of high en-
ergy exposure, free radicals are produced in cells generally
by electron transfer reactions, which can be enzymatically
mediated or non-enzymatically mediated. Cellular sites of
superoxide generation and its compartmentalization is shown
in Fig. 3. The major source of free radicals under normal cir-
cumstances is the electron leakage that occurs from electron
Table 1. Standard reduction potentials for dioxygen and
related species (the values of E
φ
(V) are for aqueous
solutions with O
2
at 1 atm, pH = 7)
E
φ
(V)
One electron reduction
O
2
+ e
O
2
0.33
O
2
+ e
+ 2H
+
H
2
O
2
+0.94
H
2
O
2
+ H
+
+ e
OH + H
2
O +0.38
OH +H
+
+ e
H
2
O +2.33
Two electron reduction
O
2
+ 2H
+
+ 2e
H
2
O
2
+0.30
H
2
O
2
+ 2H
+
+ 2e
H
2
O +1.35
Four electron reduction
O
2
+ 4H
+
+ 4e
2H
2
O +0.82
39
Fig. 1.Various forms of dioxygen.
Fig. 2. The active oxygen system: four single-electron reduction steps from
molecular oxygen to water.
transport chains, such as those in the mitochondria and en-
doplasmic reticulum, to molecular oxygen, which generates
superoxide [15].
Intracellular sources of superoxide/hydrogen peroxide
Mitochondria
The production of superoxide, the most common radical in
biological systems, occurs mostly within the mitochondria of
a cell. The mitochondrium is a small intracelullar organelle
which is responsible for energy production and cellular res-
piration. Mitochondria accomplish this task through a mech-
anism called the “electron transport chain. In this mecha-
nism, electrons are passed between different molecules, with
each pass producing useful chemical energy. Oxygen occu-
pies the final position in the electron transport chain. Even
under ideal conditions, some electrons “leak” from the elec-
tron transport chain [16, 17]. These leaking electrons in-
teract with oxygen to produce superoxide radicals, so that
under physiological conditions, about 1–3% of the oxygen
molecules in the mitochondria are converted into superoxide
[18–21].
The primary site of radical oxygen damage from superox-
ide so produced is mitochondrial DNA (mtDNA). The cell
repairs much of the damage done to nuclear DNA, but mtDNA
cannot be readily fixed. Therefore, extensive mtDNA damage
Fig. 3. Cellular sites of superoxide generation and its compartmentalization.
accumulates over time and shuts down mitochondria, causing
cells to die and the organism to age.
Cytochrome P-450
The phase I cytochrome P-450 is the terminal component
of the monoxygenase system found within the endoplas-
matic reticulum of most mammalian cells [22]. The main
role of cytochrome P-450 is that of detoxification of for-
eign compounds into less toxic products. In order to perform
this detoxification function, this enzyme uses oxygen to oxi-
dise the foreign compounds. This enzyme is also involved in
hydroxylation reactions, which also remove/inactivate toxic
compounds in the body and are heavily involved in steroido-
genesis. During these oxidation and hydroxylation reactions
electrons may be ‘leaked’ onto oxygen molecules, forming
superoxide radicals – O
2
[22].
40
Fig. 4. Superoxide generation within enzymatic oxidations as, e.g. in the
biological degradation of purines.
Cytoplasmatic oxidases
Cytochrome oxidase is found at the end of the elec-
tron transport chain in the mitochondrion [23]. The elec-
tron transport chain uses oxygen to oxidise nicotineamide
adenine dinucleotide (NADH) and FADH
2
during aero-
bic respiration to generate energy. Cytochrome oxidase
adds four electrons onto a molecule of dioxygen in a
series of reduction reactions (see also Fig. 2). Each
of these reduction reactions may potentially have su-
peroxide radicals as a byproduct, which are potentially
damaging.
Xanthine oxidase
Xanthine oxidase (XO) is a highly versatile enzyme that is
widely distributed among species (from bacteria to man) and
within the various tissues of mammals [24]. XO is an im-
portant source of OFR. It is a member of a group of en-
zymes known as molybdenum iron–sulfur flavin hydroxy-
lases and catalyses the hydroxylation of purines [25, 26].
In particular, XO catalyses the reaction of hypoxanthine
to xanthine and xanthine to uric acid (see Fig. 4). In both
steps, molecular oxygen is reduced, forming the superox-
ide anion in the first instance and hydrogen peroxide in the
second.
Microsomes and peroxisomes
Microsomes are responsible for 80% of the H
2
O
2
produced in
vivo at 100% hyperoxia sites [27]. Peroxisomes are known to
produce H
2
O
2
,butnot O
2
, under physiological conditions
[28]. Although the liver is the primary organ where peroxi-
somal contribution to the overall H
2
O
2
production is signifi-
cant, other organs that contain peroxisomes are also exposed
to these H
2
O
2
-generating mechanisms. Peroxisomal oxida-
tion of fatty acids has recently been recognized as a poten-
tially important source of H
2
O
2
production with prolonged
starvation.
Extracellular sources of superoxide
Membrane NADPH oxidases(s)
The NADPH oxidases are a group of plasma membrane-
associated enzymes found in a variety of cells of mesodermal
origin. The most thoroughly studied of these is the leukocyte
NADPH oxidase (E.C.1.23.45.3), which is found in phago-
cytes and B-lymphocytes. If a phagocytic cell such as the
neutrophil is exposed to a stimulus, it has the ability of rec-
ognizing the foreign particle and undergoing a series of reac-
tions called the respiratory burst [29]. The respiratory burst
enables the cell to provide oxidising agents for the destruc-
tion of the target cells. When NAD(P)H oxidase is activated,
it takes NAD(P)H from the cytoplasm and passes electrons
to O
2
-producing superoxide within the plasma membrane or
on its outer surface [30, 31]
2O
2
+ NAD(P)H 2O
•−
2
+ NADP
1
+ H
+
(1)
In chronic granulomatous disease, there is a hereditary defect
of NAD(P)H oxidase enzyme resulting in decreased produc-
tion of superoxide and the patient’s leukocytes cannot kill the
pathogens [32].
As summarized in Fig. 5, granulocytes and other phago-
cytic cells possess a membrane NADPH oxidase, which takes
reducing equivalents from the hexose monophosphate shunt
and transfers these to molecular oxygen to produce super-
oxide and other active oxygen species. A further myeloper-
oxidase converts peroxide produced in this system to mi-
crobiocidal products, probably including hypochlorite [33].
Production of activated products by this system probably
plays a key role in cell-mediated immunity and microbio-
cidal activity. There is evidence for similar systems in T-
lymphocytes, platelets, and mucus. An NADPH oxidase of
non-inflammatory cells may have a role in mediating cyclic
nucleotide metabolism [33].
Properties of superoxide
Despite the moderate in vitro chemical reactivity of superox-
ide in aqueous solution, it has been proven to be able to do a
considerable degree of in vivo damage. However, superoxide
can undergo a dismutation reaction [34]
2O
•−
2
+ 2H
+
SOD
−→ H
2
O
2
+ O
2
(2)
This reaction is accelerated in biological systems by the
SOD enzymes by about 4 orders of magnitude. It should be
noted that SOD enzymes work in conjunction with H
2
O
2
removing enzymes, such as catalases and glutathione (GSH)
peroxidases [35].
41
Fig. 5. Role of active oxygen species in inflammation.
Superoxide as an reducing agent
One route to generate hydroxyl radicals in biological systems
is mediated by O
2
through a metal-catalyzed Haber–Weiss
reaction [36–39];
O
•−
2
+ H
2
O
2
Fe
2+
−→ O
2
+ OH
+ OH
(3)
which is an overall reaction and consists of two steps:
Fe
3+
+ O
•−
2
+→Fe
2+
+ O
2
(4)
Fe
2+
+ H
2
O
2
Fe
3+
+
OH + OH
(5)
It is proposed that the above mechanism in vivo proceeds
through catalytic activity of metal ions bound to biomolecules
(Biol) in the close proximity of the target molecule (DNA)
Biol-M
n+1
+ O
•−
2
Biol-M
n+
+ O
2
(6)
Biol-M
n+
+ H
2
O
2
Biol-M
n+1
+
OH + OH
(7)
DNA +
OH damaged-DNA (8)
Superoxide as an oxidant
In 1976, Klug-Roth and Rabani [40] published the kinetics of
copper(II)-catalyzed decomposition of superoxide to dioxy-
gen, with the following proposed mechanism:
Cu
2+
+ O
•−
2
Cu
+
+ O
2
(9)
Cu
+
+ O
•−
2
+ 2H
+
Cu
2+
+ H
2
O
2
(10)
Cu
2+
+ O
•−
2
+ 2H
+
Cu
3+
+ H
2
O
2
(11)
Cu
3+
+ O
•−
2
Cu
2+
+ O
2
(12)
A similar mechanism applies to iron(III) involving
Fe
3+
/Fe
2+
and Fe
3+
/Fe
4+
couples [41]. The above reactions
imply that the superoxide anion is a precursor of Cu
3+
or
Fe
4+
formation. These cations are strong oxidants and there-
fore may be involved in oxidative damage to biologically
important molecules, as follows:
Biol-M
n+
+ O
•−
2
+ 2H
+
Biol-M
n+1
+ H
2
O
2
(13)
Biol-M
n+1
(damaged-Biol)-M
n+
(14)
Hydroxyl radical
Hydroxyl radical is highly reactive with a half-life in aqueous
solution less than 1 ns. Thus when produced in vivo it reacts
close to its site of formation. It can be generated through a
variety of mechanisms. Ionizing radiation causes decompo-
sition of H
2
O, resulting in formation of
OH and hydrogen
atoms.
OH is also generated by photolytic decomposition
of alkylhydroperoxides. Production of OH close to DNA
could lead to this radical reacting with DNA bases or de-
oxyribosyl backbone of DNA to produce damaged bases or
strand breaks. It has been proposed that the extent of DNA
strand breaking by
OH is governed by the accessible sur-
face areas of the hydrogen atoms of the DNA backbone
[42].
The majority of the hydroxyl radicals generated in vivo
comes from the metal catalyzed breakdown of hydrogen per-
oxide, according to the reaction [43–46]
M
n+
( = Cu
+
, Fe
2+
, Ti
3+
, Co
2+
) + H
2
O
2
M
(n+1)+
× ( = Cu
2+
, Fe
3+
, Ti
4+
, Co
3+
) +
OH + OH
(15)
where M
n+
is a transition metal ion. The most realistic in
vivo production of hydroxyl radical according to reaction
(15) occurs when M
n+
is iron or copper [47–49]. The Fe
2+
-
dependent decomposition of hydrogen peroxide is called the
42
Fenton reaction [46, 50–52]. In addition to reaction (15), the
following reactions may occur:
OH + H
2
O
2
H
2
O + H
+
+
O
2
(16)
O
2
+ Fe
3+
Fe
2+
+ O
2
(17)
OH + Fe
2+
Fe
3+
+ OH
(18)
Generally, the hydroxyl radical may react by (i) hydrogen ab-
straction, (ii) electron transfer and (iii) addition reactions. The
reaction of hydroxyl radical with a biomolecule will produce
another radical, usually of lower reactivity. As a result of the
high reactivity of
OH, it often abstracts carbon-bound hy-
drogen atoms more or less non-selectively, e.g. from glucose.
Production of
OH close to an enzyme molecule present in
excess in the cell, such as lactate dehydrogenase, might have
no biological consequences. However, attack by
OH on a
membrane lipid can cause a series of radical reactions that can
severely damage the membranes [52, 53]. Hydroxyl radical
also causes addition to DNA bases leading to generation of a
variety of oxidative products. The interaction of
OH with
guanine leads to the generation of 8-oxo-7,8-dihydro-20-
deoxyguanosine (8-oxo-dG) and 2,6-diamino-5-formamido-
4-hydroxypyrimidine (FAPy-G) (Fig. 6) [54, 55]. Adenine
reacts with
OH in a similar manner to guanine, although
oxidative adenine lesions are less prevalent in DNA damage
[56]. It has been demonstrated that in the presence of Fe(III)
or Fe(III)–EDTA complex, endogenous reductants such as
ascorbate, GSH, and the reduced form of NADH, caused
DNA damage at every type of nucleotide with a slight dom-
inance by guanine [57]. Specifically, NADH in the presence
of Fe(III)–EDTA and H
2
O
2
generated
OH, lead to formation
of 8-oxo-dG [58]. The DNA damage was inhibited by typi-
cal
OH scavengers and by catalase [59–61], suggesting that
these reductants cause DNA damage via the Fenton reaction.
Fig. 6. Structures of various products formed from the C8-OH–adduct radical of guanine, which itself is formed by attack of
OH on the C8-position of guanine.
Peroxyl radicals
The reactions of peroxyl radicals (ROO
) are prevalent in all
aspects of life, ranging from interactions with DNA [62] to
“knocking” in the internal combustion engines of automo-
biles [63]. They are high-energy species, with a reduction
potential ranging from +0.77 to +1.44 V, depending on the
R group. Several methods, ranging from chemical and physi-
cal to enzymatic techniques, may be used to generate peroxyl
radicals [64].
The simplest peroxyl radical is dioxyl radical HOO
, the
conjugate acid of superoxide,O
2
. There are many, more
complex peroxyl radicals, including cholesterol derivatives,
fatty acids, etc. [65]. The chemistry of this type of molecule
is variable due to the identity of the R group, the local
environment, and the concentration of oxygen and other
reactants.
Perhaps the most interesting feature of peroxyl radicals is
the diversity of biological reactions in which they participate.
The detection and measurement of lipid peroxidation is the
evidence most frequently cited to support the involvement
of free radical reactions in human disease and toxicology.
Peroxyl radicals are involved in DNA cleavage [66] and pro-
tein backbone modification [67]. Peroxyl radicals synergis-
tically enhance the induction of DNA damage by superoxide
[68].
The pathway of reactions of peroxyl radicals is as follows
[69]. Chain initiation refers to the attack of any species that
has sufficient reactivity to abstract a hydrogen atom from a
methylene group. Hydroxyl radical is sufficiently reactive to
do this, although superoxide is not.
CH
2
−+
OH →−
CH−+H
2
O
(R
i
= Rate of initiation) (19)
43
Under aerobic conditions the radical
CH
produced in
(19) reacts with dioxygen to yield a peroxyl radical:
CH
+ O
2
→−CHOO
(Rp1 = rate of propagation)
(20)
It should be noted that a very low oxygen pressure might favor
the self-reaction of carbon-centered radicals, terminating the
process.
2CHOO
→{Non-radical products, i.e. termination}
(21)
or 2CH
→{Non-radical products, i.e. termination}
Peroxyl radicals produced in (20) are capable of abstracting
hydrogen from another adjacent lipid molecule to propagate
the process.
CHOO
+−CH
2
→−CHOOH +−CH
× (Rp2 = rate of propagation) (22)
Electron paramagnetic resonance spectroscopy
Electron Paramagnetic Resonance (EPR; also known as Elec-
tron Spin Resonance, ESR) is a spectroscopic technique
which allows us to detect free radicals (molecular fragments
or atoms possessing a single unpaired electron) and iden-
tify and quantify these species even in complex biological
systems where multiple radicals and other non-radical com-
ponents are present [70]. The technique is routinely used
to measure free radicals such as superoxide, hydroxyl and
nitric oxide. EPR also enables measurement and imaging of
physiologically pertinent tissue parameters (functional imag-
ing) such as tissue perfusion, oxygenation, metabolism, re-
dox state, viability, pH, etc., using appropriate spin probes.
Short-lived free radicals of reaction oxygen species can be
determined using stabilizer molecules called spin traps. The
detection of spin-trapped radical adducts by EPR is a partic-
ularly powerful technique for the sensitive and specific de-
tection, identification, and relative quantitation of short-lived
free radicals [70].
Mechanisms of free radical-induced
mutagenesis and DNA base modification
It has been estimated that one human cell is exposed to ap-
proximately 10
5
oxidative hits a day from hydroxyl radical
and other such species [71–76]. Permanent modification of
genetic material resulting from these “oxidative damage” in-
cidents represents the first step of carcinogenesis involved in
mutagenesis and aging [1]. DNA alterations caused by rad-
icals are removed by specific and non-specific repair mech-
anisms. Repair of DNA base damage is thought to occur
mainly by base-excision [77]. However, misrepair of DNA
damage could result in mutations such as base substitution
and deletion, also leading to carcinogenesis [75, 76]. Mu-
tagenic potential is directly proportional to the number of
oxidative DNA lesions that escape repair. It is known that
repair mechanisms decay with age and thus DNA lesions ac-
cumulate with age. The sequence specificity of DNA damage
sites affects the mutation frequency [73]. Therefore, investi-
gation of the sequence specificity of DNA damage would be
beneficial for cancer prevention.
The specific mechanism by which oxidative stress con-
tributes to the development of carcinogenesis is largely un-
known. However, at least two different mechanisms are
thought to play a role in oxidative damage and in the de-
velopment of carcinogenesis.
The first mechanism by which oxidative damage can af-
fect carcinogenesis is through the modulation of gene expres-
sion. Epigenetic effects on gene expression can lead to the
stimulation of growth signals and proliferation [78]. Chro-
mosomal rearrangements are thought to result from strand
breakage misrepair, contributing to genetic amplifications, al-
terations in gene expression and loss of heterozygosity, which
in turn may promote neoplastic progression [79]. Active oxy-
gen species have been demonstrated to stimulate protein ki-
nase and poly(ADP ribosylation) pathways, thus affecting
signal transduction pathways. This further can lead to mod-
ulation of the expression of essential genes for proliferation
and tumour promotion [80]. Recently, Lander et al. [81] sug-
gested that free radical signall may be mediated through ras
signal transduction pathways.
In the second mechanism, radicals induce genetic alter-
ations, such as mutations and chromosomal rearrangements,
which can play a role in the initiation of carcinogenesis
[82, 83]. Oxidative DNA damage results in a wide range
of chromosomal abnormalities, causing a blockage of DNA
replication and wide cytotoxicity [84]. Mutations can occur
through misrepair or due to incorrect replication, while chro-
mosomal rearrangements can result from strand breakage
misrepair [71]. The initiation potential of oxidants may be
contributing to carcinogenesis due to their ability to induce
DNA base changes in certain oncogenes and tumour suppres-
sor genes [85]. It has been demonstrated that the hydroxyl
radical is able to activate certain oncogenes, such as K-ras
and C-Raf-1. The activation proceeds through the induction
of DNA point mutations in GC base pairs and N-terminal
deletions in these genes [85]. Base point mutations in CpG
dinucleotides are also frequently found in certain tumour sup-
pressor genes, such as p53 and retinoblastoma, leading to
their inactivation [86, 87]. Furthermore, hydroxyl radical at-
tacks or cells containing mutant or absent p53, resulted in
44
afailure to arrest in G
1
, reducing their capacity to repair
damaged DNA [85]. This increase in replication errors can
initiate additional oncogene activation and tumour suppres-
sor gene inactivation, ultimately contributing to malignancy.
Free radical-induced cytotoxicity may also contribute to the
initiation of carcinogenesis by depleting the normal cell pop-
ulation, promoting the clonal expansion of more resistant-
initiated cells, thus increasing the probability of mutation
[78].
Oxygen radicals and genotoxicity of DNA damage
ROS-induced DNA damage can be described both chemi-
cally and structurally and shows a characteristic pattern of
modifications. It is well established that in various cancer
tissues free radical-mediated DNA damage was found [88].
The majority of these changes can be reproduced in vitro. The
forms of DNA damage produced by ROS experimentally in-
clude the following: modification of all bases, production of
base-free sites, deletions, frame shifts, strand breaks, DNA–
protein cross-links, and chromosomal rearrangements. An
important reaction involved in DNA damage involves gener-
ation of hydroxyl radical, e.g. through Fenton chemistry [89].
Hydroxyl radical is known to react with all components of
the DNA molecule: the purine and pyrimidine bases as well
as the deoxyribose backbone [90, 91]. In terms of oxidative
DNA damage, major interest has focused on modifications to
DNA bases, with over 20 products identified, however only
afew have been investigated in detail.
Hydroxyl radical is able to add to double bonds of DNA
bases at second-order rate constants of 3–10 × 10
9
M
1
s
1
and abstracts an H-atom from the methyl group of thymine
and each of the five carbon atoms of 2
deoxyribose at rate
constants of 2 × 10
9
M
1
s
1
[92, 93]. While OH–adduct
radicals of DNA bases are generated via addition reactions,
the allyl radical of thymine and carbon-centered sugar radi-
cals are formed from abstraction reactions. Peroxyl radicals
are generated in oxygenated environments via oxygen addi-
tion to OH–adduct radicals and also to carbon-centered radi-
cals at diffusion-controlled rates [92, 93]. Further reactions of
base and sugar radicals generate a variety of modified bases
and sugars, base-free sites, strand breaks, and DNA–protein
cross-links.
Hydroxyl radical adds to pyrimidines: to the C5 and C6 po-
sitions of thymine and cytosine, generating C5-OH– and C6-
OH–adduct radicals, respectively. Oxidation reactions of the
C5-OH–adduct radicals of thymine and cytosine followed by
addition of water or OH
and deprotonation lead to the forma-
tion of glycols of cytosine and thymine, respectively [94, 95].
Oxygen adds to C5-OH–adduct radicals to give 5-hydroxy-6-
peroxyl radicals that may eliminate superoxide followed by
reaction with water, giving rise to cytosine glycol and thymine
glycol. Oxidation of the allyl radical of thymine generates 5-
(hydroxymethyl)uracil (5-OHMeUra) and 5-formyluracil. In
the absence of oxygen, 5-hydroxy-6-hydro- and 6-hydroxy-
5-hydropyrimidines are formed by reduction of 5-OH– and
6-OH–adduct radicals of pyrimidines, respectively, followed
by protonation.
Hydroxyl radical is also able to add to purines giving
rise to C4-OH–, C5-OH–, and C8-OH–adducts [96, 97].
One-electron oxidation and one-electron reduction of C8-
OH–adduct radicals yields 8-hydroxypurines (7,8-dihydro-
8-oxopurines) and formamidopyrimidines, respectively [94,
96, 98, 99]. The most extensively studied of these oxidised
DNA products is 8-oxo-deoxyguanosine (8-oxo-dG), mainly
because it is the most easily detectable. The presence of 8-
oxo-dG in human urine was first reported by Shigenaga et al.
[100]. This base modification occurs in approximately one in
10
5
guanidine residues in a normal human cell [101].
An example illustrating the mechanisms of the forma-
tion of 8-hydroxyguanine (7,8-dihydro-8-oxoguanine, 8-OH-
G) is given in Fig. 6. 8-Hydroxyguanine and 8-hydroxy-
29-deoxyguanosine undergo keto–enol tautomerism, which
favours the 6,8-diketo form. Hence, 8-OH-G is often called 8-
oxy-7-hydroguanine or 8-oxoG. The nucleoside is then called
8-oxo-7-hydro-29-deoxyguanosine or 8-oxo-dG (therefore,
8-oxo-dG and 8-OH-dG are the same compounds). Through-
out this paper 8-oxoguanine (8-oxoG) or deoxynucleosides
(8-oxo-dG) will be used. We note that the analogous reactions
of adenine yield 8-hydroxyadenine (8-OH-Ade).
Multiple methods for measuring oxidative DNA dam-
age exist; a popular method employs enzymatic digestion
of DNA, which liberates 8-hydroxypurines for analysis by
HPLC usually with electrochemical detection (HPLC-EC)
[102–106]. Another method employs acidic hydrolysis of
DNA, which liberates the free base, because the glycosidic
bond is cleaved by acid. Detection is via HPLC or, after con-
version to volatile derivatives, by gas chromatography mass
spectrometry (GC-MS) [107].
The 8-oxoG lesion is important because it is relatively
easily formed and is mutagenic, therefore is a potential
biomarker of carcinogenesis [108]. The experimental pro-
posed mutagenic potential of 8-oxo-dG is supported by a
loss of base pairing specificity, misreading of adjacent pyrim-
idines, or insertion of adenine opposite the lesions [109]. Mu-
tations that may arise due to formation of 8-oxo-dG involve
GC TA transversions [110]. Previous work has shown
that the mispairing of 8-oxo-dG with adenine appears to be
possible due to the energetically favoured syn glycosidic con-
formation, whereas pairing with dG assumes the anti form.
Measurements by Kasai [109] demonstrated that factors
such as hard physical labour, day–night shift work, smoking,
low meat intake, and low BMI (<21.8) significantly increased
the 8-oxo-dG level, while moderate physical exercise, such
as sports, reduced its level. These results were comparable
with previous data obtained from studies on rats [111]. These
45
findings suggest that the lifestyle may significantly affect the
level of oxidative damage.
Another important mechanism of mutations is formation
of 2-oxy-dA in the nucleotide pool and its incorporation into
DNA. It has been presented that the incorporation of 2-oxy-
dA opposite G induced GC TA transversions in the chro-
mosomal lac I gene [112]. It is known that the human saniti-
zation enzyme hMTH1 hydrolyzes 8-oxy-dGTP and prevents
mutations. In a series of experiments testing sanitization en-
zymes for 2-oxy-dA it has been unexpectedly evidenced that
2-oxo-dATP is also hydrolyzed by hMTH1 [113].
Lipid peroxidation and DNA damage
While major attention has focused on direct DNA damage
by free radicals because of the genetic consequences of such
damage, reactive radical species may also cause damage to
other cellular components [114]. Cell membrane phospho-
lipids are very sensitive to oxidation and have been found
to be frequent targets of radical-induced damage that enables
them to participate in free radical chain reactions. Many of the
fatty acids are polyunsaturated, containing a methylene group
between two double bonds that makes the fatty acid more
sensitive to oxidation. The high concentration of polyunsat-
urated fatty acids in phospholipids enables them to partici-
pate in free radical chain reactions [115]. The most common
fatty acid in cells is linoleic acid. The best biomarker of lipid
peroxidation is a set of arachidonic acid oxidation products
termed isoprostanes [116, 117]. They can readily be detected
by GC-MS.
The initial products of unsaturated fatty acid oxidation are
short-lived lipid hydroperoxides. When they react with met-
als they produce a number of products (e.g. aldehydes and
epoxides) which are themselves reactive.
Malondialdehyde (MDA) is one of the major aldehyde
products of lipid peroxidation [118]. It is mutagenic in mam-
malian cells and carcinogenic in rats [119, 120]. MDA can
react with DNA bases dG, dA, and dC to form adducts M
1
G,
M
1
A and M
1
C (Fig. 7). M
1
G has been detected in human
liver, white blood cells, pancreas, and breast tissue [121].
The M
1
Glevel corresponds approximately to 6500 adducts
per cell. Several studies concluded that M
1
Gisareactive
electrophile in the genome [122]. N
2
-Oxo-propenyl-dG, the
product of rapid and quantitative ring-opening of M
1
G, is
also electrophilic, but targets regions of DNA different from
M
1
G. Thus the interconversion of N
2
-oxo-propenyl-dG and
M
1
G may reveal varying reactive groups of DNA that could
participate in the formation of DNA–DNA interstrand cross-
links or DNA–protein cross-links.
It has been demonstrated that hydroxypropanodeox-
oguanosines (OH-PdGs) are present in human and rodent
liver DNA [123]. It has been suggested that these propano
adducts are mediated by the reaction of DNA with acrolein
and crotonaldehyde, which in turn are products of lipid perox-
idation. Acrolein and crotonaldehyde are mutagenic in bac-
teria and mammalian cells [124]. There is little known about
the repair of OH-PdGs. PdG is a substrate for the nucleotide
excision repair complex of E. coli and mammalian cells and
is recognized and repaired by the mismatch repair system
[125].
Several exocyclic etheno DNA adducts arising from lipid
peroxidation have been detected by
32
P-post-labeling and
GCMS analysis in DNA from healthy human volunteers
[126]. The most important involves etheno-dA, etheno-dC
and etheno-dG. Their biological activity has been widely
studied by site-specific mutagenesis experiments. Etheno-
dA and etheno-dC were found to be strongly genotoxic but
weakly mutagenic in single-stranded E. coli [127, 128]. How-
ever, the same adducts were found to be highly mutagenic
when introduced into monkey kidney cells, implying that
marked differentials exists in mutagenic potency between
bacterial and mammalian cells [129]. The kind of cell system
used to evaluate the mutagenic activity of a given lesion is
therefore of key importance.
Inflammation, alcohol, smoking and DNA damage
Recent data have expanded the concept that inflammation is
a critical component of tumour progression [130]. It is now
becoming clearer that the neoplastic process, proliferation,
survival, and migration is linked with tumour microenvirone-
ment synchronized with inflammatory cells.
Several studies have demonstrated a direct link between
chronic inflammation and DNA damage [131]. Liver tissue
from patients suffering chronic inflammatory diseases such
as hepatitis, hepatitis B, and cirrhosis exhibit increased levels
of 8-oxo-dG compared to control liver tissue [132]. Similar
findings were reported for patients infected with Helicobacter
pylori [133]. Chronic atrophic gastritis shows also increased
level oxidised bases, however, chronic non-atrophic gastritis
does not.
An inverse correlation between alcohol consumption and
lymphocyte levels of 8-oxo-dG in humans has been recently
presented in an international experimental study [134]. The
study was conducted in four different regions of Europe, in-
cluding Potsdam (Germany), Turin (Italy), Malm¨o (Sweden)
and Granada (Spain). Mean 8-oxo-dG levels differed signifi-
cantly across study centres, with the highest levels in Granada
and lowest levels in Turin [134]. Mean levels of total alcohol
intake and of types of alcoholic beverages consumed (wine,
fortified wines, beer and cider) also differed across the study
centres, with the highest total alcohol consumption in Turin,
and the lowest intake in Granada [134]. When combining all
the data, but adjusting for study centre, individual 8-oxo-dG
level correlated inversely with alcohol intake. The finding of
a relationship between alcohol consumption and 8-oxo-dG
46
Fig. 7. Synthesis of malondialdehyde (MDA) and its reaction with DNA bases.
in lymphocytes was unexpected and not based on a prior
hypothesis. This finding consequently requires confirmation
from a randomised intervention study. Additional progress
in understanding alcohol’s enhancing effect on DNA dam-
age will depend on a better understanding of the interactions
between alcohol and other risk factors and on additional in-
sights into the multiple biological mechanisms involved. The
enhancing effect of alcohol may also be affected by other di-
etary factors (such as low folate intake), lifestyle habits (such
as use of hormone replacement therapy), etc.
Recently published trials have concluded that cigarette
smoking has a low impact upon certain pathways involved
in DNA damage and the antioxidative defence system [135].
Various markers of oxidative DNA damage and repair, and an-
tioxidative defence mechanisms have been studied in smok-
ers. Lymphocytic 8-oxo-dG levels were significantly lower
in smokers as compared with non-smokers. The levels of
oxidised pyrimidine bases in lymphocytes of smokers quan-
tified by the endonuclease III-modified comet assay were
non-significantly lower than those of non-smokers. Urinary
excretion levels of 8-OH-dG assessed by enzyme-linked im-
munosorbent assay did not differ significantly between smok-
ers and non-smokers. Plasma antioxidative capacity mea-
sured by the Trolox equivalent antioxidant capacity assay was
slightly higher in smokers as compared with non-smokers,
and it was significantly related to lymphocytic 8-oxo-dG
levels [135].
Repair of DNA lesions
Oxygen radicals may induce a number of DNA base alter-
ations that can lead to mutagenesis. However, there are spe-
cific and general repair mechanisms that can repair DNA base
modifications [83, 136].
The first evidence of a repair mechanism for the 8-oxo-dG
lesion was observed in irradiated mouse liver, where levels
of this lesion were found to decrease with time [137]. A re-
pair enzyme was partially purified from E. coli [138] and was
later found to be identical to the cloned DNA repair enzyme,
formamidopyrimidine–DNA glycosylase FPG protein, previ-
ously isolated from E. coli. This enzyme has both glycosylase
and apurinic endonuclease activity. The repair pathway for
this oxidative lesion in E. coli includes at least three path-
ways characterized by mutant strains: MutM, which lacks
the Fpg protein and exhibits increased G T transversions
[139, 140]; MutY, that recognizes dA mismatches with 8-
oxo-dG and removes the adenosine inserted opposite to the
8-oxo-dG [141]; and MutT, which degrades (hydrolyses) the
nucleotide pool of 8-oxoGTP [142]. These three proteins co-
operate in the prevention of spontaneous oxidative mutations
in E. coli and represent the multilevel security against 8-oxo-
dG-related damage [143].
Two separate glycosylase and endonuclease enzymes re-
sponsible for repair of the 8-oxo-dG lesion were isolated from
mammalian cells [144]. These two enzymes are the mam-
malian counterparts to the MutM repair enzyme in E. coli.
Some endonucleases without glycosylase activity could
recognise sites damaged by free radicals. The endonuclease
with glycosylate activity, DNA glycosylase endonuclease III,
recognizes thymine glycol and a selection of other oxidative
and non-oxidative base modifications. An intriguing obser-
vation linked to DNA damage by free radicals is that nitric
oxide inhibits some DNA repair enzymes including FAPY
glycosylase that removes 8-oxo-dG [145].
Antioxidant defense system
Antioxidant is a classification of several organic substances,
including vitamins C, E and vitamin A (which is converted
from β-carotene), selenium (a mineral), and a group known as
the carotenoids (CAR) [146–149]. CAR, of which β-carotene
is the most popular, are pigments that add colour to many
fruits and vegetables for example, without them carrots
would not be orange. These substances at the molecular and
cellular level are thought to be effective in helping to de-
activate free radicals and prevent cancer, heart disease and
47
stroke. Antioxidants play the housekeeper’s role, “mopping
up” free radicals before they get a chance to cause damage.
Despite numerous studies carried out on the role of antioxi-
dants in cancer and heart disease prevention, the jury is still
out as to which groups of people, if any, benefit from taking
antioxidant supplements [76, 150, 151]. Some studies have
shown that smokers with diets high in CAR have a lower rate
of lung cancer development than their smoking counterparts
whose CAR intake is relatively low [152]. However, a recent
trial indicated that some β-carotene takers (see below), pri-
marily smokers, actually had higher death rates [153]. Other
research efforts have suggested that diets high in CAR may
also be associated with a decreased risk of breast cancer [154,
155]. Also, vitamin C has been found to prevent the forma-
tion of N-nitroso compounds, the cancer-causing substances
from nitrates and nitrites found in preserved meats and in
some drinking water [156].
Vitamin C
Vitamin C’s major role is to make collagen, the main protein
substance of the human body that holds connective tissues
together in skin, bone, teeth, and other parts of the body.
Vitamin C is also critical for the proper function of our im-
mune system, for manufacturing certain nerve transmitting
substances and hormones, and for the absorption and utiliza-
tion of other nutrients, such as vitamin E and iron [157, 158].
Vitamin C is also a very important and powerful antioxidant
that works in aqueous environments of the body, such as the
lungs and lens of the eye [159]. Its primary antioxidant part-
ners are vitamin E and the carotenes (β-carotene), as well as
works along with the antioxidant enzymes [160, 161]. The
recommended daily allowances (RDAs) for adults are 40 mg
per day in the Europe and 60 mg per day in the US.
Ascorbic acid has two ionizable hydroxyl groups and there-
fore is a di-acid (AscH
2
). Because the pK
a
of the first is
4.25 and the pK
a
of the second is 11.8, formation of the
mono-anion is favoured at physiological (cellular) pH (see
Fig. 8) [162–164]. At physiological pH, 99.9% of vitamin
Cispresent as AscH
, and only very small proportions as
AscH
2
(0.05%) and Asc
2
(0.004%). The antioxidant chem-
istry of vitamin C is thus the chemistry of AscH
and we will
use the term ascorbate throughout the paper to refer to this
species. The initial product of ascorbate oxidation by many of
these species is the semi-dehydroascorbate radical (Asc
),
a poorly reactive radical that can either be converted back to
ascorbate by NADH-dependent enzymes or undergo dispro-
portionation to form dehydroascorbate (DHA) [162–164].
AscH
is a donor antioxidant and donates a hydrogen atom
to an oxidising radical to produce the resonance stabilized
tricarbonyl ascorbate-free radical (AFR). AscH
has a pK =
–0.86 thus it is not protonated and is present in the form of
Asc
(see Fig. 8).
In practically all metabolic activities, ascorbate reduces
transition metal ions (Cu and Fe). Since Fe(III) has very low
solubility, the ability of ascorbate to reduce Fe(III) to Fe(II)
has significance in iron sorption in gut [165]. Ascorbate also
reduces Cu(II) to Cu(I).
Ascorbate converts ROS into poorly reactive ascorbate-
derived products that is, ascorbate acts as one of the many
antioxidants that can protect biomolecules against damage
by such species in vivo. Asc
is considered to be a terminal,
small-molecule antioxidant and the level of this radical is a
good measure of the degree of oxidative stress in biological
systems [163, 166].
It its known that vitamin C protects membranes against
oxidation. Low-density lipoprotein (LDL) is well known to
play an important role in atherosclerosis, the underlying cause
of coronary heart disease and strokes. Oxidatively modified
LDL is taken up by macrophages via scavenger receptors at
a much greater rate than native LDL [167, 168]. This leads
to the formation of cholesterol-laden foam cells, which are
characteristic of many atherosclerotic lesions. Two mecha-
nisms for a protective role of vitamin C against LDL oxi-
dation have been suggested by Retsky et al. [169]. In one
mechanism, ascorbic acid may scavenge free radicals in the
aqueous phase. In the other, dehydroascorbic acid (the oxi-
dation product of ascorbic acid), or its decomposition prod-
ucts, might modify LDL such that copper can bind less
strongly to the LDL particle, thus increasing the resistance of
LDL to oxidation by copper. However, very recently, Ret-
sky et al. [170] have shown that when LDL has already
been partially oxidised by copper, then ascorbate and dehy-
droascorbate no longer protect against further oxidation by
copper.
The intake of high doses of vitamin C, initially suggested
by Linus Pauling, has been a subject of the intense debate over
years [171, 172]. The benefit of a high intake of vitamin C has
never been established. However, a positive effect of vitamin
C intake was reported from clinical trials of stomach cancer
incidence and cardiovascular disease [173, 174]. Vitamin C
cooperates with vitamin E to regenerate α-tocopherol from
α-tocopheryl radicals in membranes and lipoproteins [175]
(see also below).
Besides the positive role of ascorbate it should be noted
that there are studies exploring pro-oxidant properties of
ascorbate [176, 177]. Concerns have been raised over poten-
tially deleterious transition metal ion-mediated pro-oxidant
effects. It has been determined that vitamin C induces
the decomposition of lipid hydroperoxides into the DNA-
reactive bifunctional electrophiles 4-oxo-2-non-enal, 4,5-
epoxy-2(E)-decenal, and 4-hydroxy-2-non-enal [176]. The
compound 4,5-epoxy-2(E)-decenal is a precursor of etheno-
2
-deoxyadenosine, a highly mutagenic lesion found in hu-
man DNA [176]. Vitamin C-mediated formation of genotox-
ins from lipid hydroperoxides in the absence of transition
48
Fig. 8.Forms of ascorbate at various pH levels and its reaction with radicals.
Fig. 9.Vitamin E and its derivatives: compound I, where R
1
= R
2
= R
3
=
Me, is α-tocopherol; compound II, where R
1
= R
3
= Me; R
2
= H, is β-
tocopherol.
metal ions could help explain its lack of efficacy as a cancer
chemoprevention agent.
Vitamin E
Vitamin E is a fat-soluble vitamin that exists in eight different
forms. Each form has its own biological activity, the mea-
sure of potency or functional use in the body. α-Tocopherol
(Fig. 9) is the most active form of vitamin E in humans,
and is a powerful biological antioxidant [178]. Antioxidants
such as vitamin E protect cells against the effects of reactive
radicals, which are potentially damaging byproducts of the
body’s metabolism [179].
The term vitamin E should be used as the generic descrip-
tor for all tocol and tocotrienol derivatives exhibiting qual-
itatively the biological activity of α-tocopherol. This term
should be used in derived terms such as vitamin E deficiency,
vitamin E activity, vitamin E antagonist.
The term tocol is the trivial designation for 2-methyl-2-
(4,8,12-trimethyltridecyl)chroman-6-ol (R
1
= R
2
= R
3
=
H). Compound I (R
1
= R
2
= R
3
= Me) is designated α-
tocopherol (Fig. 9) or 5,7,8-trimethyltocol. Compound II (R
1
= R
3
= Me; R
2
= H), is designated β-tocopherol or 5,8-
dimethyltocol [180].
α-Tocopherol is considered to be the major membrane-
bound antioxidant employed by the cell [181] and its main
antioxidant activity is protection against lipid peroxidation
[182, 183]. As mentioned above, ascorbic acid is regarded
as the major aqueous-phase antioxidant [184]. Recent evi-
dence suggests that α-tocopherol and ascorbic acid function
together in a cyclic-type reaction [178]. During this process,
α-tocopherol is converted to a radical by donating a labile hy-
drogen to a lipid or lipid peroxyl radical [181, 182]. The oxi-
dised α-tocopherol radical is energetically stable and has low
reactivity with other molecules within the membrane (Fig.
10). Oxidised α-tocopherol can then be re-reduced to its origi-
nal form by ascorbic acid (see also above). This regeneration
of reduced α-tocopherol presumably occurs at the surface
49
Fig. 10. Reaction of vitamin E with peroxyl free radicals and regeneration of vitamin E radical (tocopheroxyl radical) through one-electron oxidation of
vitamin C.
of the membrane where ascorbic acid and α-tocopherol can
meet [181]. Along with acting as a reducing agent for α-
tocopherol, ascorbic acid is also considered a preventative
antioxidant because of its ability to scavenge for reactive
radicals.
Some studies advocate vitamin E to inhibit the incidence
of stomach cancer, however a recent trial on North American
populations found that there is no association between stom-
ach cancer mortality and regular use of vitamin E [185]. It
should be noted that in North American populations, stom-
ach cancer rates are relatively low, so the results do not rule
out a beneficial effect of vitamin supplementation in areas
in which stomach cancer rates are high and stomach cancer
etiology may differ.
A long-term epidemiological study of cancer incidence
was carried out by the US National Institute of Health in Fin-
land (Alpha-Tocopherol/Beta-Carotene, ATBC Trial) [186].
The results show no reduction in lung cancer and 40% de-
creased incidence of clinically significant prostate cancer in
volunteers taking vitamin E. Prostate cancer mortality also
decreased 41%.
In conclusion, vitamin E, in particular together with sele-
nium have been shown to decrease cancer incidence and mor-
tality in large-scale, well-controlled prospective randomised
clinical trials.
Carotenoids
CAR are pigments that are found in plants and microorgan-
isms but are not synthesized by animals [187]. They are re-
sponsible for the red, yellow, and orange colour of fruits and
vegetables. There are over 600 CAR occurring in nature,
which can be grouped into carotenes, xanthophylls (CAR
Fig. 11. Structures of carotenoids.
containing oxygen), and lycopene. In the last decade sev-
eral epidemiological studies have indicated that CAR may
prevent or inhibit certain types of cancer, artherosclerosis,
age-related muscular degeneration, and other diseases [188].
The antioxidant activity of CAR arises as a result of the
ability of the conjugated double bond structure (Fig. 11) to de-
localise any unpaired electrons. This is primarily responsible
for the excellent ability of β-carotene to physically quench
singlet oxygen without degradation and for the chemical
50
reactivity of β-carotene with free radicals such as peroxyl
radical (ROO
), hydroxyl radical (
OH), and superoxide rad-
ical (O
2
) [189, 190]. In general, the longer the polyene chain,
the greater the peroxyl radical stabilizing ability. It has been
shown that the peroxyl radical (ROO
)was about 100–1000-
fold more reactive with CAR than with the allylic hydro-
gen sites on polyunsaturated fatty acids, hence at sufficient
concentrations, CAR could protect lipids from peroxidative
damage [191].
In the ATBC Trial [186] supplemental β-carotene was ad-
ministered to 29,133, 50–69-year-old male smokers in Fin-
land for 5–8 years. The dosage of 20 mg per day was substan-
tially higher than what is typically contained in the Finnish
diet. In the men taking β-carotene, there was a significant,
18%, increase in the incidence of lung cancer, which con-
tributed to an 8% excess in total mortality! The ATBC trial
results imply that β-carotene or, more specifically, the all-
trans isomer of β-carotene in a water-soluble beadlet is not
correlated with the reduced cancer risk associated with veg-
etable and fruit intake [192]. However, these results do not
totally rule out a protective role for β-carotene; for example,
if it is given in higher dosages or earlier in the process of lung
carcinogenesis.
Another multi-centre lung cancer prevention trial Beta-
Carotene and Retinol Efficiacy (CARET) [193] was started.
The trial was randomised, double blind, and placebo con-
trolled. In this trial 18,314 smokers, former smokers, and
workers exposed to asbestos were examined. The treatment
group has a relative risk of lung cancer of 1.28 compared
with the placebo group. However, the trial was stopped
early.
Antioxidant mechanisms of carotenoids
Generally three mechanisms are discussed for the reaction
of free radicals (ROO
,R
) with CAR: (i) radical addition,
(ii) hydrogen abstraction from the CAR, and (iii) electron
transfer reaction.
It has been proposed that peroxyl radicals add to CAR
polyene chain to form a resonance-stabilized, carbon-
centered CAR radical adduct [194]:
ROO
+ Car ROO-Car
(23)
which then interacts with another ROO
:
ROO-Car
+ ROO
ROO-Car-ROO
(non-radical products) (24)
yielding a non-radical product terminating the chain reaction.
A second type of reaction is hydrogen abstraction from the
CAR forming the neutral CAR radical, Car
:
R
+ Car(H) RH + Car
(25)
and finally an electron transfer reaction [195]:
R
+ Car R
+ (Car)
+•
(26)
Recently, there has accumulated a growing body of evi-
dence which suggests that scavenging of lipid ROO
, where
Risanaliphatic group, by β-carotene may not proceed via
an electron transfer mechanism, reaction (26), but rather by
adduct formation (reactions (23) and (24)) and/or hydrogen
abstraction (reaction (25)).
Pro-oxidant mechanisms of carotenoids
Burton and Ingold [190] were among the first to propose that
β-carotene might participate in lipid peroxidation as a pro-
oxidant. The term ‘pro-oxidant activity’ involves the ‘ability’
of β-carotene to increase the total radical yield in the system
[196–200].
The key factors in converting carotenoids from antiox-
idants to pro-oxidants are the partial pressure of dioxy-
gen ( pO
2
) and carotenoid concentration. At higher pO
2
,a
carotenoid radical, Car
(generated through the hydrogen ab-
straction reaction (25)) could react with dioxygen to generate
a carotenoid-peroxyl radical, Car-OO
[201]:
Car
+ O
2
Car-OO
(27)
This is an autoxidation process and Car-OO
could act as
a pro-oxidant by promoting oxidation of unsaturated lipid
(RH):
Car-OO
+ RH Car-OOH + R
(28)
The pro-oxidant mechanism of β-carotene has also been
reported for the radical adduct reactions (reactions (27) and
(28)). Liebler and his group reported [202, 203] that at higher
pO
2
the radical adduct ROO-Car
can react directly with O
2
forming ROO-Car-OO
radical (reaction (27)). Alternatively,
it has been reported that decomposition of non-radical prod-
uct ROO-Car–ROO yields carotenoid-epoxide (5,6-epoxy-
β,β-carotene) and reactive RO
, reaction (29) [202]:
ROO-Car
+ O
2
ROO-Car-OO
(29)
ROO-Car-ROO Car-epoxide + RO
(30)
The experimental conditions, such as pO
2
, may determine
which of these simultaneous reaction pathways would be the
primary one.
51
Although there were some discernible trends in carotenoid
reactivity for individual radicals, rate constants varied by no
greater than a factor of 2.5. The mechanism and rate of scav-
enging is strongly dependent on the nature of the oxidising
radical species but much less dependent on the carotenoid
structure [204].
Conclusions and perspectives
The generation of OFR is a consequence of aerobic life. OFR
represent a constant source of assaults to our genetic mate-
rial that can be either enhanced or reduced by nutritional,
hormonal, and environmental influences. At present, we do
not know the exact role that damage by oxygen radical plays
in carcinogenesis and its synergetic role with other forms
of genetic events accelerating cell transformation and malig-
nant progression. However, it is known that oxidative stress
can participate in the initiation of proliferation of tumour
cells. The effect of oxidative stress at a certain stage of car-
cinogenesis is directly related to the type and the reactivity
of the radicals involved. Antioxidant enzymes together with
non-enzymatic antioxidants are involved in OFR conversion.
However, antioxidant protection against free radicals should
be taken with caution since the antioxidant action might actu-
ally stimulate cancer progression through the enhanced sur-
vival of tumour cells.
In preventing OFR-related cancer, the key role seems to
be reduction of endogenous and exogenous sources of oxida-
tive stress and the elimination of environmental carcinogens.
There is also the possibility that cancer treatment could make
use of the results of OFR studies. Two classes of genes, re-
ferred to as oncogenes and tumour suppressor genes, have
been demonstrated to participate in the sequence of events
that results in pathological cell growth. Therefore, the in-
volvement of oncogenes in all stages of radical-induced car-
cinogenesis seems to open the possibility of gene therapy for
OFR-related cancer.
Acknowledgements
We thank VEGA (Grant #1/9256/02) and APVT (20-005702)
for financial support and NATO for a collaborative linkage
grant (LST.EAP.CLG980614). We also thank Dr. Miroslav
Tatarko for his help with the manuscript preparation.
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