Reviews
The role of free radicals in disease*
T M Florence, DSc
-
A bStraCt
Evidence is accumulating that most of the degen-
erative diseases that afflict humanity have their
origin in deleterious free radical reactions. These
diseases include atherosclerosis, cancer, inflam-
matory joint disease, asthma, diabetes, senile
dementia and degenerative eye disease. The
process of biological ageing might also have a free
radical basis. Most free radical damage to cells
involves oxygen free radicals or, more generally,
activated oxygen species (AOS) which include
non-radical species such as singlet oxygen and
hydrogen peroxide as well as free radicals. The
AOS can damage genetic material, cause lipid
peroxidation in cell membranes, and inactivate
membrane-bound enzymes. Humans are well
endowed with antioxidant defences against AOS;
these antioxidants, or free radical scavengers,
include ascorbic acid (vitamin C), a-tocopherol
(vitamin E), beta-carotene, coenzyme Q I O .
enzymes such as catalase and superoxide dis-
mutase, and trace elements including selenium
and zinc. The eye is an organ with intense AOS
activity, and it requires high levels of antioxidantsto
protect its unsaturated fatty acids. The human
species is not genetically adapted to survive past
middle age, and it appears that antioxidant supple-
mentation of our diet is needed to ensure a more
healthy elderly population.
The Ida Mann Lecture. Presented at the 24th Scientific Congress of The Royal Australian College of Ophthalmologists, Nouember,
1992.
Director, Centre for Enuironniental and Health Science Pty Ltd, Sydne_v, NSW
Reprints: Dr TIM Florence, 112 Georges River Crescent, Oyster Bay, NSW 2225.
The role of free radicals in disease
Key words: Activated oxygen species, ageing,
antioxidants, degenerative disease, eye disease,
free radicals, selenium.
Interest in the role of free radicals in disease
began only about 25 years ago. Originally, most
free radical research was carried out by chemists,
then biochemists became interested and, in the
past five to 10 years, medical researchers have
dominated the scene. The demonstration that
reperfusion injury to organs was entirely the
result of free radical reactions in the reperfused
blood convinced medical researchers that free
radicals are important in human biology.
Free radical pathology is the cornerstone of the
free radical theory of disease, which proposes that
many degenerative diseases are the result of
uncontrolled free radical reactions in the body,
often involving inflammation processes and
autoimmune reactions (Table 1). A corollary of
this theory is that the incidence of degenerative
disease should be decreased by the use of dietary
antioxidants (free radical scavengers). Recent
large-scale epidemiological studies have demon-
strated that antioxidants such as vitamins C and E,
beta-carotene, and selenium and zinc can indeed
significantly influence the rates of some major
diseases. The eye probably has higher free radical
activity than any other organ, and several eye
diseases are undoubtedly the result of excess free
radical production in vulnerable areas of the eye.
3
Table 1. Some free radical diseases agent such as NADPH or GSH to produce the
Cancer Atherosclerosis
hydroxyl radical, OH., the most reactive chem-
Emphysema Arthritis ical species known, which can damage genetic
Asthma Ageing material. A major source of hydrogen peroxide is
Hypertension Cirrhosis the dismutation of superoxide radical: .
Allergy Cataract
Retinopathy Macular degeneration 2 OF.+ 2 H' + H20? + 0 2

This reaction is catalysed by the superoxide

Table 2. Some activated oxygen
species (AOS)
AOS Formula
Molecular oxygen 0 7
Hydroxyl radical OH.
Superoxide radical 0 2 '
Singlet oxygen ' 0 2
Hydrogen peroxide H202
Lipid peroxide LOOH
Alkoxyl radical RO.
Peroxyl radical ROO.
Semiquinone radical Q-.
The nature of free radicals
A simple definition of a free radical is 'any atom
or molecule that has one or more unpaired
electrons'. This definition includes molecular
oxygen, since ground-state 0 2 has two unpaired
electrons. The free radical nature of oxygen
explains its high reactivity. We are saved from the
dangerous oxidative power of oxygen in the air
we breath only by kinetics; the rates of many of
oxygen's reactions are extremely slow, even
though they are thermodynamically favourable.
A free radical will attempt to gain electrons from
other free radicals or molecules (i.e., reducing
agents) so as to 'pair up' its unpaired electrons.
Free radicals include oxygen, superoxide radical,
some transition metal ions such as iron (11) and
copper (11), and carbon and sulphur centred
species (Table 2).?,'
Activated oxygen species (AOS) include both
oxygen free radicals and non-radical species such
as singlet oxygen and hydrogen peroxide (Table
2 ) . These non-radical AOS can be as dangerous
as free radicals if liberated in biological systems.
For example, hydrogen peroxide, HzOz, is only a
relatively weak oxidising agent but, because it
can cross cell membranes almost as easily as
water, it can diffuse unchanged to vulnerable
areas of the cell, such as the nucleus or the
mitochondria. There it can react with a reducing
dismutases. Hydrogen peroxide may then react
with a range of biological reducing agents (e-) to
give the hydroxyl radical.
H202 + e- 4 OH. + OH
This reaction (the Haber-Weiss reaction) is
catalysed by iron complexes such as Fe-ATP and
Fe-ADP.' A particle of iron in the eye is a major
ophthalmic emergency, since serious damage to
the eye will occur unless it is removed quickly.6
The damage undoubtedly results from hydroxyl
radical formed by the iron-catalysed Haber-
Weiss reaction. Another likely source of hydroxyl
radical in biological systems is the peroxynitrite
anion.' Peroxynitrite ( O N 0 0 3 is formed from
superoxide radical and nitric oxide radical:
02-. + ?*TO. O N 0 0
--t
Nitric oxide is the endothelium relaxing factor
(ERF) and is produced by endothelium,
macrophages and neutrophils. It is also a neuro-
transmitter and is formed in brain synapses.
Peroxynitrite anion is a reactive AOS, but it is
also a source of hydroxyl radicals, since it decom-
poses at physiological pH with a half-life of two
seconds:
ON00 +H + OH. + NOz.
Singlet oxygen('02) is produced by the action
of light on photosensitisers such as flavins.
Singlet oxygen may be an important cause of
lipid peroxidation in cell membranes of the eye.
In the retina, the membranes are constantly
bombarded by photons, which react with photo-
sensitisers to yield singlet oxygen and other
AOSeH
Some sources of free radicals in living organ-
isms are shown in Table 3. Free radicals are vital
to life; respiration, phagocytosis and a range of
enzymatic reactions depend on free radical inter-
mediates. They are dangerous only if liberated in
the wrong place or in too high a concentration.

4 Australian and New Zealand Journal of Ophthalmology 1995; 23(1)

Table 3. Sources of free radicals
Electron transport chain
Phagocytosis
Oxidant enzymes (e.g., xanthine oxidase)
Autoxidation (e.g., epinephrine)
Redox drugs (e.g., paraquat)
Tobacco
Sunlight and ionising radiation
Table 4. Some free radical scavengers
Substance Action
- -
Ascorbic acid Destroys nitrosamines and many free
radicals
a-Tocopherol Destroys lipid peroxides
Beta-carotene Destroys singlet oxygen
Catalase Destroys hydrogen peroxide
Superoxide
dismutase Destroys superoxide radical
Glutathione
peroxidase Destroys hydrogen peroxide and lipid
peroxides
Defences against free radicals
Our natural defences against damage by the AOS
are antioxidants, or free radical scavengers
(Table 4). Comparison of the antioxidant ability
of blood serum and tissues of various mammals
shows that maximum lifespan potential (MLP) is
strongly correlated to antioxidant potential.'
Humans have the best antioxidant defences of
any mammal (Table 5), which is one reason we
have the highest MLP. Antioxidants include
enzymes such as catalase and superoxide dismu-
tase, which are synthesised in the body to scav-
enge specific AOS, and molecular antioxidants
such as ascorbic acid (vitamin C) and a-
tocopherol (vitamin E)3 which have a more
general action.
All the components of our antioxidant defence
system combine in a synergistic manner; a
deficiency of one will cause the others to be less
effective. A well-studied example3 is the symbi-
otic relationship between vitamins C and E.
Vitamin C is water soluble, and cannot enter the
cell membrane, whereas vitamin E is lipophilic
and is intercalated in the membrane bilayer, with
its more hydrophilic chroman group on the outer
surface of the membrane. When vitamin E
reduces a lipid peroxide molecule in the
membrane and converts it back to the free lipid,
a vitamin E free radical is formed. Extracellular
vitamin C then reacts with the chroman group to
The role of free radicals in disease
Table 5. Rate of oxidation of brain tissue and
maximum lifespan potential
Lifespan Relative
potential oxidation rate of
(years) brain in normal saline
1.o
~
Man 100
Chimpanzee 50 5
Deer mouse 8 11
Field mouse 3 19
regenerate vitamin E from its free radical. In this
way vitamin E is protected, and allowed to
continue in its vital role of preventing the poten-
tially calamitous cascade of lipid peroxidation in
cell membranes. One vitamin E molecule will
protect 1000 lipid molecules.
The interdependence of antioxidants is often
not understood by researchers. A recent well
publicised study"' found that beta-scarotene and
vitamin E supplements did not protect a group of
Finnish smokers from lung cancer, even though
many previous epidemiological studies had
shown a strong protective effect. However, the
subjects used were moderate to heavy long-term
smokers. Smoking significantly reduces body
stores of vitamin C (one cigarette destroys about
20 mg of vitamin C),3 and many heavy smokers
have marginal scurvy. Vitamin E cannot act as a
biological antioxidant in the absence of vitamin
C, so the study was seriously flawed.
Mitochondria are perhaps the area of a cell
most vulnerable to AOS damage, because they
process more than 90% of the oxygen that our
body uses to produce ATP as a source of energy.
During the four-electron reduction of oxygen,
superoxide radical and hydrogen peroxide are
liberated. Unlike nuclear DNA, which is coiled
and chromatin-packaged, mitochondria1 DNA is
a single, circular, naked molecule, and therefore
highly vulnerable to oxidative damage.' Muta-
gens bind much more strongly (1000 X) to mito-
chondrial than to nuclear DNA. Mitochondria
from aged animals have more fragle membranes
and produce less ATP.
T o prevent oxidative damage, mitochondria
have a complex, multi-layered antioxidant
defence system." The inner membrane (where
oxygen is metabolised) has a high content of
easily oxidised unsaturated fatty acids, but it is
also rich in vitamin E. The centre of the mito-
chondrion (the matrix) contains catalase and the
5
manganese form of superoxide dismutase
(SOD), while the intermembrane space has one
of the two geometric isomers of copper, zinc-
SOD (the other form is in the cytosol).
Hydrogen peroxide, generated in excess in
vulnerable parts of the cell, might be a
particularly dangerous AOS. The enzyme, cata-
lase, is the first line of defence against H202,
dissociating it safely to oxygen and water without
requiring a substrate:
2 Hz02 --t 2 H2O + 0 2 to oxidative damage.
The second line of defence is the enzyme
glutathione peroxidase, which uses glutathione,
GSH, as substrate to dispose of HZOZand lipid
peroxides (LOOH):
HzOz + 2 GSH + 2 GSSG + 2 HzO
LOOH + 2 GSH + 2 GSSG + LOH + H2O
Glutathione peroxidase contains selenium, and
represents one of the biological roles of this
unusual trace element. Vitamin E and selenium
work together to eliminate highly toxic lipid
peroxides. Another role is in thyroid function,
selenium being at the active centre of the
enzyme, type 1 iodothyronine deiodinase, which
catalyses the conversion of T 4 to T3. Selenium is
also believed to delay mitosis, allowing DNA
repair mechanisms time to correct damage to
DNA caused by mutagens. It is also involved in
sperm production, and has anti-viral properties.
Activated oxygen species and
degenerative disease
Many degenerative diseases (Table 1) are now
believed to be caused, or at least exacerbated, by
AOS. Inflammatory reactions and, in some cases,
autoimmune reactions, are involved in arthritis,
asthma, Alzheimers disease, and diabetes. The
familial form of amyotrophic lateral sclerosis
(ALS) results from a defective gene on chromo-
some 2 1.Iz This gene codes for one isomer of Cu,
Zn-SOD, and the brain of patients with familial
ALS have only about half the normal SOD
activity. Lack of SOD would allow the build-up
of toxic superoxide radical in the brain.
Atherosclerosis involves the deposition of fatty
deposits (atheroma) in blood vessels. Atheroma,
however, cannot form unless the endothelium is
first damaged by AOS from white blood cells and
lipid peroxides. These deposits contain choles-
6 Australian and New Zealand Journal of Ophthalmology 1995; 23(1)
terol epoxides, lipid peroxides, and antibodies to
lipid peroxides. In carcinogenesis, the initial
mutagenic event usually involves an AOS or
some oxidative chemical (the ultimate
carcinogen) produced by the cytochrome P-450
system. Metastasised cancer cells cannot attach
to tissue without a concomitant oxidation
process. Likewise, oxidative stress is believed to
be involved in retinopathy and macular degener-
ation. The eye has the most unsaturated lipids
of any organ in the body, and is highly vulnerable
Considering the involvement of AOS in many
degenerative diseases, it is not surprising that
epidemiological and clinical studies have found
that dietary antioxidants have a protective effect
in most cases. Two United States benchmark
epidemiological studies published in 1993 found
that vitamin E can reduce the rate of coronary
heart disease by almost 50%, but only if the daily
intake is at least 100 mg.. This amount of
vitamin E can be obtained only by supplementa-
tion, since even the best diet is unlikely to
provide more than 20 mg daily. An international
epidemiological study on coronary heart disease
and antioxidant intake found that heart disease
was strongly related inversely to the plasma
concentrations of some essential antioxidants,
while a large United States study found that total
mortality was inversely related to vitamin C
intake. With daily vitamin C intakes of more than
700 mg, total mortality was only 40% of the age-
adjusted national average rate.18
Selenium intake has been found to protect
against asthma, cancer and heart disease. New
Zealand, where seleniumintake from the diet is
particularly low, has the worlds highest rate of
asthma. One New Zealand study found a fivefold
increase in asthma incidence if the whole blood
selenium level was below 45 kg/L,19 a level not
unusual in the South Island (mean blood sele-
nium in Australia is 110 kg/L). Cancer and heart
disease rates in several countries were also found
to be inversely related to blood selenium.
The vertebrate retina has a higher specific
oxygen consumption than any other tissue in the
body, by a factor of at least sevenb The high
oxygen and light flux in the rod outer segment
(ROS), combined with its polyunsaturated fatty
acid composition, favours lipid peroxidation as a
result of the formation of AOS. The ROS,
however, is rich in vitamins E and C, carotenoids
and SOD, catalase and glutathione per~xidase.~-~~-?
Selenium also appears to be important to human
&ion; low blood selenium is involved in the
development of rnacular degeneration, poor
colour vision, and impaired blue-cone response.6~2
The rapid progress in identifying specific genes
&at lead to an increased risk of many degenera-
tive diseases has accentuated the need for
preventive medicine. Also, unless the spectrum
of degenerative disease changes, an ageing
population could eventually impose unsustain-
able medical costs on the community. The
obvious answer to this problem is not a con-
tinuous increase in costly medical facilities and
nursing homes, but a reduction in the incidence
of degenerative disease or, at least, a delay in its
onset, SO that the time between onset and death
is shortened. The identification of the involve-
ment of free radicals and other AOS in the
aetiology of many of these diseases provides hope
that the use of antioxidants and other chemo-
preventive techniques might play a major role in
preventive medicine.
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