Fundamental and Molecular

Mechanisms of Mutagenesis
ELSEVIER Mutation Research 350 (1996) 103-108

Antioxidant defences against reactive oxygen species causing

genetic and other damage
Diana Anderson

Accepted 3 June 1995

K~~rwrds: Free radical damage: Antioxidant; DNA peroxidation; Cancer

1. Introduction other molecules, chain reactions are often initiated

Oxygen. present as 20% of the atmosphere, is a
terminal oxidant essential for respiration and other
oxidative reactions in all aerobic organisms. During
the reduction of molecular oxygen, reactive oxygen
species are formed. These free radicals are impli-
cated in many diseases including atherosclerosis
(Witzum. 19941, respiratory tract disorders (Cross et
al., 1994), neurodegenerative disease (Jenner, 1994),
inflammatory bowel disease (Grisham, 1994), cancer
(Ames. 1989: Cerutti. 1994) and also in ageing
(Ames, 1989. 1988a, b).
A free radical is any atom or molecule that con-
tains one or more unpaired electrons (Halliwell, 1994;
Halliwell and Gutteridge. 1989). The unpaired elec-
trons alter the chemical reactivity of an atom or
molecule, usually making it more reactive than the
corresponding non-radical. The chemical reactivity
of radicals varies enormously. The hydrogen radical
(H ‘) containing one proton and one electron is the
simplest free radical. By the removal of (H ‘) from
Corresponding author. Fax: + 44 I8 1 66 17039.
e.g. during lipid peroxidation. In the human body.
solar radiation or low wavelength electromagnetic
radiation. such as gamma-rays. from the environment
can split water to generate the hydroxyl radical,
OH ‘, which is very reactive at the site of formation.
The body also makes another oxygen radical where
the unpaired electron is located on oxygen. superox-
ide (02 ). but this is poorly reactive. Active phago-
cytes including neutrophils, monocytes. macrophages
and eosinophils. generate large amounts of superox-
ide in the killing of foreign organisms. However,
with chronic inflammation this normal protective
mechanism may itself be damaging (Grisham, 1994).
Another physiological free radical is nitric oxide
(NO ‘) which is made by the vascular endothelium as
a relaxing factor, but excessive nitric oxide can be
toxic (Moncada and Higgs. 1993). Superoxide can
react with iron and copper ions to make hydroxyl
radicals (Fig. I) or can combine with nitric oxide
Oi- + NO + ONOO- (peroxynitrite). This decom-
poses into toxic products including nitrogen dioxide
gas (NO,), hydroxyl radical and nitronium ion
(NO; ).
Since most molecules in the body are not radicals.
any reactive free radical generated is likely to react

0027-5 107/96/$I5.00 0 1996 Elsevier Science B.V. All rights reserved

SSIII 0017-5 107(95)00096-8
 hydroperoxides. The glutathione peroxidase that re-

:
6-l
Woo*
moves hydrogen peroxide contains selenium which
is essential for catalytic function at its active site.
+ M’+ Mn The non-selenium dependent GSH peroxidase does
,’
SPONTANEOUS SOD not detoxify H,O, but has a high activity towards
I / DNA hydroperoxide (Ketterer and Meyer, 1989).
GSH also has important roles in leukotriene synthe-
sis and xenobiotic metabolism and is found at minute
concentrations in all human cells (Chance et al..
1979). Reactive radicals, such as NO,, OH’ or
CAT
CCI,O;. abstract an atom of hydrogen from poly-
unsaturated fatty acid side chains in membranes or
lipoproteins. This leaves an unpaired electron on
HP0 + %O> carbon. The carbon radical reacts with oxygen and
the resulting peroxyl radical attacks adjacent fatty
Fig. I. The interaction of active oxygen species. M, metal ion:
SOD. huperoxidr dismutase: CAT. catalase; GP. zlutathionr per-
acid side chains to generate new carbon radicals and
oxidase. so the chain reaction continues. The attack of one
reactive free radical can oxidise multiple fatty acid
side chains to lipid peroxides, damaging membrane
proteins. This makes the membrane leaky and even-
with a non-radical. Attack of reactive radicals on tually causes complete membrane breakdown (Hal-
membranes or lipoproteins starts lipid peroxidation liwell, 1994).
which is implicated in atherosclerosis development Since iron and copper ions are powerful promot-
(Witzum, 1994). ers of free-radical damage causing formation of hy-
droxyl radicals and accelerating lipid peroxidation,
there is a complex system of storage and transport
2. Antioxidant defences against reactive oxygen proteins to ensnare these essential metals. Iron within
species ferritin which is the usual storage form of iron will
not stimulate free-radical reactions (Halliwell and
Defence mechanisms have evolved within the Gutteridge. 1989).
body to limit the levels of reactive 0, species and Repair enzymes also exist that destroy free-radical
the damage they induce. Superoxide dismutase (SOD) damaged proteins (Stadtman and Oliver. 1991). re-
can convert superoxide to hydrogen peroxide (H 202) pair free-radical damage to DNA (Breimer, 1991)
(30,-+ 2HL+ H,O, + 0,). These enzymes are with excretion of oxidised bases in urine (Ames.
found in mitochondria and cytosol. Catalases remove 1989: Stillwell et al.. 1989) and remove oxidised
hydrogen peroxide. are found in peroxisomes in most fatty acids from membranes (Maiorino et al., 1991).
tissues and probably remove peroxide generated by Such reactions are intracellular.
peroxisomal oxidase enzymes (Chance et al.. 1979). Some antioxidant defences are extracellular in-
Glutathione transferases and glutathione peroxidases cluding the plasma iron-transport-protein transferrin
are usually associated with detoxication by conjuga- and the iron-binding protein lactoferrin. When iron is
tion of xenobiotic electrophiles (Ketterer and Meyer. bound in this way it cannot catalyse free-radical
1989). They remove hydrogen peroxide generated by damage (Halliwell and Gutteridge. 1989). These pro-
SOD in the mitochondrial and cytosol (Chance et al.. teins are found in body secretions, such as tears.
1979). They transform the tripeptide glutathione Albumin can scavenge several radicals and binds
(GSH) into its oxidised form (GSSG) 2GSH + H ?O, copper ions (Halliwell and Cutteridge. 1989) and
+ GSSG + 2H,O. Two types of GSH peroxidase caeruloplasmin is a safe transport form of copper and
occur in the cell, both of which detoxify fatty acid helps load iron onto transferrin (Gutteridge and
hydroperoxides. thymine hydroperoxide and DNA Stocks. 1981). Carnosine which is present in high
 D. Atldersm/ Mutntion Research 350 flYY61 103-108 105

concentrations in human muscle and brain chelates three levels of defence (Ames. 1989). First, nuclear
copper and iron ions so as to inhibit oxidative reac- DNA is compartmentalised away from mitochondria
tions (Kohen et al., 1988). and peroxisomes where most oxidants are probably
Other antioxidant defences are found both intra- generated. Secondly, most non-replicating nuclear
and extracellularly. a-Tocopherol occurs in mem- DNA is surrounded by histones and polyamines
branes and lipoproteins. It prevents the chain reac- which may protect against oxidants. Thirdly, most of
tion of lipid peroxidation by scavenging intermediate the types of DNA damage produced can be repaired
peroxyl radicals. Vitamin C can convert the toco- by efficient enzyme systems (Breimer, 1991; Still-
pherol radical. which is much less reactive in attack- well et al.. 19891. The overall result of this multi-level
ing adjacent fatty acid side-chains, back to (Y- defence is that nuclear DNA is very well, but not
tocopherol. a-Tocopherol is important in protection completely. protected from oxidants.
against cardiovascular disease and neurodegeneration When hydroxyl radicals are generated close to
(Muller and Goss-Sampson. 1990). Reduced glu- DNA, purine and pyrimidine bases are attacked so
tathione is found in trace amounts in the body except causing mutations where, for example, guanine is
in the respiratory tract (Slade et al.. 1993) where it converted to 8-hydroxyguanine and other products
scavenges oxidative toxins such as NO;. ozone and (Dizdaroglu. 1993). One view of the somatic damage
free radicals in cigarette smoke. Vitamin C also theory of cancer and ageing is that the amount of
helps in this instance as does p-carotene. Urate is maintenance and repair of somatic tissues is always
the end product of purine metabolism and it scav- less than that required for indefinite survival. Thus
enges free radicals (Kaur and Halliwell. 1990). some DNA damage in somatic cells induced by
Ascorbate, a-tocopherol, GSH and water remove endogenous mutagens will accumulate with time
free radicals by reacting directly with them non-cata- (Ames. 1989).
lylically. There are 4 important endogenous processes lead-
Plants contain many phenolic compounds, such as ing to significant DNA damage. These are oxidation
flavonoids. that inhibit lipid peroxidation and (Totter. 1980; Ames. 1983: Cutler, 1984), methyla-
lipooxygenases (Laughton et al.. 1991). A diet rich tion. deamination and depurination (Saul and Ames.
in vegetables. fruits. nuts and grains with a reduction 1986). Supporting this are the existence of specific
in fat intake seems to protect against several human DNA-repair glycosylases for oxidative, methylated
diseases including cardiovascular disease and cancer and deaminated adducts. and a repair system for
(Ames. 1983; Coghlan. 1991; Caragay. 1992). An apurinic sites produced by spontaneous depurination
increased dietary intake of vitamin E seems to de- (Lindhal, 1982). Ames (1989) has shown that oxida-
crease death from myocardial infarction (Byers. tion is a major type of DNA damage and has calcu-
1993). Epidemiological studies on serum anti- lated that the total number of oxidative hits of DNA
oxidants and diet suggest that an increased level of per cell per day in man may be more than 10’. He
vitamin E and p-carotene reduces mortality from has promoted the use of a simple urinary assay for
cancer in the lung and colon (Menkes et al.. 1986; measuring the oxidised base 8-hydroxydeoxyguano-
Blot et al., 1993). sine (oh8dG) (Cundy et al., 1988; Shigenaga et al.,
1989). This base is present at a level of I / 130 000
bases in nuclear DNA and l/8000 bases in mito-
chondrial DNA. This very high level in mitochon-
3. Radical interactions resulting in genetic drial DNA may be caused by the extensive oxygen
damage metabolism, relatively inefficient DNA repair and
the absence of histones in mitochondria. Energy
Because of the time between generation of oxi- supplies to the cell could be compromised since
dants and their destruction by a defence mechanism, mitochondria might be accumulating mutations with
low levels can exist for sufficient time to produce age (Ames, 1989).
damage to cellular macromolecules (Chance et al.. During DNA peroxidation. DNA peroxides so
1979). For nuclear DNA, the mammalian cell has formed reside in the pyrimidine fraction and involve
thymine. These are either products of the reaction of
O2 with radicals resulting from the addition of OH.
across the thymine 5, 6-double bond to give ring-
saturated thymine hydroxy-hydroperoxides or with
the radical produced by hydrogen abstraction from
the thymine 5-methyl group to give %hydroper-
oxymethyl acid (Schulte-Frohlinde and von Sonntag.
1985). These hydroperoxides are relatively unstable
and in the presence of metal ions, notably copper
ions, are mutagenic (Thomas et al.. 1976).
If mild oxidative stress occurs. tissues can re-
spond by increasing antioxidant defences, but severe
oxidative stress can cause cell injury and death. Such
cell death can progress as necrosis or apotosis. Cer-
tain cells appear to encode free radical scavengers in
anti-apoptosis genes (Sarafian and Bredesen. 1994).
Several possibilities relate oxidative endogenous
DNA damage to cancer (Ames. 1989; Cerutti, 1994)
and also ageing (Ames. 1989). One is that mutagens
react with nuclear DNA to produce somatic muta-
tions, including point mutations and somatic events
such as deletions. Mutagenic oxidants could include
long-lived ones generated outside the nucleus in the
mitochondria and cytoplasm which are capable of
crossing the nuclear membrane, lipid soluble oxi-
dants generated in the nuclear membrane itself and
oxidants generated within the nucleus. Spontaneous
methylation of DNA could cause both point muta-
tions and apurinic sites which lead to breaks and
clastogenic events. Somatic mutation could disrupt
cells by altering their regulation or gene production.
Another possibility is that the high rate of oxidative
damage to mitochondria causes an accumulation of
mutations with age in mitochondrial DNA that re-
sults in energy deficiencies in old cells. In addition
to mutation, DNA damage can prevent DNA replica-
tion leading to cell death. This can cause neighbour-
ing cells to proliferate which can be a promotional
stimulus for carcinogenesis in cells that do not un-
dergo DNA replication.
Mutation can occur in cancer-related genes. The
most important cancer-related genes are ras-family
proto-oncogenes and the pS3 tumour suppressor gene.
Skin cancer is related to solar radiation and near-ul-
traviolet light (290-380 nm) damages DNA partly
by oxidative mechanisms. G + T transversions are
frequently observed in the middle position of hot
spot codon 12 of Ki-ras and H-t-as in non-melanoma
skin tumours (Daya-Grosjean et al., 1993). G + T
transversions have also been observed in several ~53
codons in smoking related lung carcinoma (Taka-
hashi et al.. 1989) and could be produced by the
release of oxy-radicals into the tissue by inflamma-
tory leucocytes (Cerutti. 1994).
Oxidation or methylation could also cause a loss
of S-methylcytosine, an epigenetic change. S-Meth-
ylcytosine is involved in turning off genes in differ-
entiation so that its loss by DNA damage could
cause de-differentiation and contribute to cancer and
ageing (Wilson et al.. 1987; Holliday. 1987). Simi-
larly, %hydroxyguanosine and 7-methylcytosine are
likely to interfere with maintenance methylation at
neighbouring or base-paired cytosines and cause loss
of 5-methylcytosine.
4. Importance of oxidative damage to cancer and
other disease states
Halliwell and Gutteridge (1984) emphasised that
oxidative damage could be just as much a conse-
quence of tissue injury as a cause of it. Tissue injury
almost certainly leads to oxidative stress. This could
then contribute significantly to worsening the tissue
injury or it might be irrelevant. However, the disease
states listed earlier have accumulating evidence that
free-radical damage is important. It is also important
to understand how antioxidant defences are useful in
protecting against such diseases, e.g.. carcinogenesis
is a multiple stage event and antioxidants could act
at any stage but they may not be anticarcinogenic in
every instance. High antioxidant capacity shields
DNA from oxidative damage and mutagenesis. but at
the same time it may protect initiated cells from
excessive oxidant toxicity and apoptosis and favour
their clonal expression in tumour promotion (Cerutti
and Trump, 1991). No clear answer can be given
about the role of individual antioxidants on human
cancer. The outcome may depend on the multiple,
interacting oxidant components of the target tissue
and the particular carcinogen to which it is exposed
(Cerutti, 1994). However, antioxidant defences per-
haps can be enhanced by appropriate diets (Ames.
1983; Coghlan. 1991; Caragay. 1992) and vitamin
supplementation; see earlier.
 D. Anderson/Mutation
5. Conclusion
Thus, it would seem that reactive oxygen species
play an important role in cancer and various other
disease states in man, and means are now available
for beginning to understand their contribution to
such diseases. In addition, the role of antioxidant
defences in preventing such diseases is becoming
better understood. Although in vitro methods have
been used to examine oxygen-mediated genetic dam-
age for many years (e.g., Phillips et al., 19841, new
and simple methods such as the COMET assay are
available for measuring genetic damage caused by
oxidative events in human cells and the modifying
responses produced by antioxidants (Anderson et al.,
1994).
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