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OXYGEN TOXICITY – FROM MICROBES TO MAN
Oxygen is toxic to aerobic (and anaerobic) organisms, yet paradoxically oxygen is essential
for their survival. Today terrestrial aerobes (both animals and plants) have successfully
adapted to live in an atmosphere composed of approximately 21% oxygen and can survive
minor fluctuations in the level of respired oxygen without disastrous consequences. True
anaerobes, on the other hand, tolerate oxygen poorly, and some cannot survive even a brief
exposure to atmospheric oxygen (Table 1.1).
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Anaerobes were the first living organisms on the planet. These evolutionary simple organisms show
a wide range of oxygen tolerance.
Strict or obligate anaerobes will only grow if oxygen is absent.While some obligate anaerobes are
killed almost immediately following exposure to oxygen (aerophobic) (e.g., Clostridia species)
others can survive for many days but cannot reproduce (e.g., Bacteroides fragilis).
Another group of organisms, microaerophiles actually require some oxygen for growth but cannot
survive when exposed to atmospheric oxygen concentrations.
Most bacteria that reduce nitrate (producing nitrite, nitrous oxide or nitrogen) are called facultative
anaerobes as they are not affected by exposure to oxygen and in fact will preferentially use oxygen,
rather than nitrate, during respiration.
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Anaerobes can be found in any environment where oxygen levels are decreased to less toxic
levels including muds and other sediments; bogs and marshes; polluted waters; certain
sewage-treatment systems; rotting material; deep underground areas such as oil pockets; the
sources of springs; decaying teeth and gangrenous wounds; the colon; and inappropriately
canned foods.
Rather than using oxygen during respiration (they usually lack terminal cytochromes that
transfer electrons to oxygen) they use other electron acceptors such as ferric ions, sulfate or
carbon dioxide which become reduced to ferrous ions, hydrogen sulfide and methane,
respectively, during the oxidation of NADH (reduced nicotinamide adenine dinucleotide is a
major electron carrier in the oxidation of fuel molecules) (Figure 1.1).
Oxygen is toxic to anaerobes as it can affect the organism’s internal homeostasis by altering its
reductive capacity, consuming compounds such as NAD(P)H, thiols and other chemicals
essential for biosynthetic reactions and inactivating key enzymes.
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Although anaerobes had free range during the early stages of the evolution of living organisms, this
was eventually curtailed by the success of oxygen- producing photosynthetic plants. With the levels
of oxygen rising in the atmosphere, anaerobes had three choices, adapt, find niches where oxygen
would not penetrate, or die. Organisms eventually evolved that not only survived in an oxygenenriched atmosphere but prospered.
Evidence suggests that the atmospheric oxygen levels have fluctuated markedly over time,
increasing from 15-18% in the late Devonian to as high as 35% in the late Carboniferous and early
Permian periods. This hyperoxia has been suggested to be one of the possible causes of the mass
extinction of terrestrial vertebrates (Graham et al. 1995). Atmospheric oxygen finally stabilized at
today’s level (at least to date).
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Obligate aerobes (e.g., higher plants and animals) use oxygen in respiration and for the
biosynthesis of a variety of biomolecules. All higher organisms are obligate aerobes but they
can make use of both anaerobic and aerobic processes.
For example, many tissues such as the red blood cell, the cornea of the eye, the skin, the
kidney medulla and type IIb (fast twitch-glycolytic) skeletal muscle fibers make use of
anaerobic glycolysis. Here the two molecules of ATP produced by the anaerobic conversion of
glucose to lactate is sufficient to supply most of these tissues’ normal energy needs.
However, as the average human requires more than 40kg/day of ATP, and as much as 0.5kg/
minute when undergoing strenuous exercise, anaerobic respiration simply cannot keep pace
with this demand. Rather, higher organisms must obtain the vast majority of their energy from
aerobic respiration, and that is why oxygen is essential for their survival.
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Obligate aerobes are very oxygen sensitive. A total lack of oxygen is referred to as anoxia and
rapidly results in cell death. For example, brain damage can result from perhaps as little as
three minutes of anoxia.
An acute decrease in respired oxygen leads to hypoxia, a situation where oxygen is still
delivered to the tissue, but at a rate insufficient to maintain normal cellular processes. The
effects of hypoxia depend upon the tissue and the degree and duration of the hypoxic event.
For example, the brain is a very aerobic tissue and is exquisitely sensitive to oxygen tension.
In higher animals an acute reduction in arterial oxygen tension leads to altered mental
function, analgesia and loss of muscle coordination (Blass and Gibson (1979); Gibson and
Blass (1976); Gibson et al. (1978; 1981)). A more marked drop can result in unconsciousness,
progressive depression of the central nervous system, circulatory failure and death.
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Ischemia is a consequence of mechanical disruption of blood flow to a tissue resulting in
decreased oxygen, glucose and ATP levels. For example, the occlusion of essential blood
vessels to the heart (a consequence of atherosclerosis and/or blood clots) results in ischemia.
This leads to myocardial damage and heart attack. It has been estimated that irreversible
myocardial damage can occur after about 20 minutes of ischemia (Sobel (1974)). The affected
tissue eventually dies.
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Exposure to elevated levels of oxygen results in hyperoxia and is deleterious to aerobic
microorganisms, plants and animals. The growth of aerobic bacteria is inhibited following
exposure to pure oxygen. Plants show decreased chloroplast development and leaf damage
when exposed to oxygen levels above normal. Animals exposed to 100% oxygen show a
variety of symptoms depending upon the duration of exposure (Crapo et al. (1980); Francica
et al. (1991)). Humans suffer chest soreness, coughing and sore throats following several
hours of exposure to pure oxygen. Longer periods cause alveolar damage, edema and
permanent irreversible lung damage. Hyperoxia also leads to damage to most of the major
organs.
Unfortunately, earlier this century unintentional retinal damage and blindness (retrolental
fibroplasia) was caused to premature babies when they were maintained on high oxygen
levels in their incubators. Fortunately, the level of oxygen to which premature babies are
exposed is now more carefully monitored. It should be noted, however, that hyperoxia can
also be beneficial. For example, hyperbaric oxygen is used to treat gangrene because of its
toxicity to the obligate anaerobes that cause it.
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WHY IS OXYGEN TOXIC?
Over the years, several theories have been put forward to explain oxygen’s toxicity. This
subject was reviewed recently by Gilbert (1999) so only an overview will be presented here.
•! One early hypothesis as to oxygen’s toxicity was that oxygen exerted its action through
enzyme inhibition. For example, oxygen can inhibit nitrogenase and the first enzyme in the
dark reactions of photosynthesis, ribulose 1,5-bisphosphate decarboxylase, and at high
concentrations some thiol-containing enzymes (Haugaard (1946); Stadie et al. (1944)).
However, enzyme inhibition is far too slow and limited to explain oxygen’s toxic effect, and not
all enzymes are affected by oxygen.
• Abundant evidence showed that irradiation caused DNA damage and cancer through a free
radical mechanism and that oxygen had a sensitizing effect (von Sonntag (1991) and
references therein).
•! In the mid 1950s Gerschman and Gilbert proposed that oxygen, itself a diradical, may exert
its toxic action through the formation of free oxygen radicals. These could then damage
biologically important macromolecules such as DNA, proteins and lipids (see Gerschman
(1981); Gerschman et al. (1954); and reviews by Gilbert (1999); Halliwell and Gutteridge
(1993)). This breakthrough proposal, however, was initially strongly criticized by researchers
who proposed that free radicals were far too reactive to exist in any great quantity in biological
materials. These objections were finally laid to rest by the detection of free radicals both in dry
biological tissues and in living organisms by electron spin resonance (Commoner et al. (1954,
1957)).
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• Free radicals were further implicated by the discovery of the enzyme superoxide dismutase
(SOD). Fridovich theorized that the superoxide radical anion was the major toxic form of
oxygen and that SOD protected against it (Fridovich (1983, 1986a,b); McCord and Fridovich
(1969)).
The superoxide theory of oxygen toxicity, though not completely correct, was responsible for a
great deal of experimental work and a better understanding of the field as a whole (reviewed in
Halliwell and Gutteridge (1993)).
We now know that oxygen mediates its toxic effects through a variety of compounds, not just
free radicals, many of which contain other atoms in addition to oxygen.
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FREE RADICAL PRO-OXIDANTS.
The term radical originally used by chemists referred to an ionic group that had either positive
or negative charges associated with it (e.g., carbonate, sulfate etc.). A free radical is now
defined as an atom or molecule that has one or more unpaired electrons (i.e., electrons that
occupy atomic or molecular orbitals by themselves) and is capable of independent existence.
In the strictest sense the free of free radical, is redundant. It may come as some surprise that
oxygen is a free radical (in fact a diradical) as are metals that have incomplete 3d shells (e.g.,
transition metals and their various oxidation (valency) states) (Table 1.2).
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Free radicals can be formed when a non-radical either gains or loses a single electron (Table
1.3). Free radicals can be formed during homolytic fission of covalent bonds. The energy
required to cause bond dissociation can be brought about by several different processes,
including exposure to heat or electromagnetic radiation, or by chemical reaction.
Remember that covalent bonds are formed when two atoms share electrons (usually one from
each atom). During homolytic fission one electron of the bonding pair is retained by atom A,
while the other is retained by atom B forming the free radicals A• and B•, respectively. During
homolysis of water, for example, the hydroxyl free radical (HO•) and the hydrogen atom (H•)
are produced.
Radical reactions are much more common in the gas phase and at high temperatures, e.g.,
combustion. Readers should be aware that many radical reactions found in the literature
(especially chemistry texts) may be for gas phase reactions and are not always applicable to
biological systems. Having said this, gas phase free radical chemistry is extremely important to
those investigating the effects of atmospheric pollution and cigarette smoke on biological
systems.
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In biological systems the most infamous free radical cascade is the lipid peroxidation chain
reaction (Table 1.5). Here a single initiation process can lead to the destruction of many polyunsaturated fatty acid molecules. Unfortunately, not only does this affect membrane fluidity and
thus many biochemical processes, but it can also lead to the production of cytotoxic carbonyl
breakdown products (Chapter 3).
Lipid peroxidation is also the major process responsible for food spoilage. Like any other chain
reaction, lipid peroxidation consists of three phases termed a) initiation, b) propagation and c)
termination. Biological systems are equipped with several mechanisms designed to prevent
lipid peroxidation. Such processes include prevention of radical formation (inhibiting initiation)
or interception of fatty acid radicals once formed (inhibiting propagation). Biological systems
are also capable of repairing damage that occurs.
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Table 1.6 The Different Pro-Oxidants And Other Species Of Importance To
Biological Systems. (L – alkyl; 1∆g and 1Σg+ represent the two forms of singlet oxygen; X• – a radical species).
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Table 1.7 Pro-oxidants Are Beneficial Too. (Halliwell and Gutteridge (1999) and references therein; and other references at the end of this chapter).
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HOW DO AEROBIC ORGANISMS SURVIVE EVEN WHEN PRO- OXIDANTS ARE BEING
CONTINUOUSLY PRODUCED?
The cells of aerobes are constantly being exposed to pro-oxidants. Consequently, their DNA,
proteins, and lipids are continuously being damaged. During evolution one option would have
been to prevent the formation of pro- oxidant species. This, however, would be virtually
impossible to achieve in an oxygen-enriched environment as pro-oxidants are unavoidable side
reactions of other important biochemical processes. Instead nature accepted that pro- oxidants
would be produced so protective mechanisms evolved to repair and replace damaged
molecules. In addition we are equipped with a suite of antioxidant defenses designed to
prevent the formation of pro-oxidants, or to intercept and destroy them if formed. Interestingly,
aerobes also make good use of pro-oxidants as messengers, signals and defense molecules
(Table 1.7).
Under normal conditions the production of pro-oxidants is presumed to be in balance with
antioxidant defenses. However, the overproduction of pro-oxidants and/or decreased
antioxidant protection can lead to tissue damage and disease. Thus, in individuals with a
genetic predisposition or for those exposed to environmental stressors such as cigarette
smoke, sunlight and pollution, the pro- oxidant/antioxidant balance can be upset (Figure 1.6).
The overproduction of pro- oxidant species or the failure of antioxidant defenses results in a
condition called oxidative stress, a causal, or at least ancillary, factor in the pathology of many
diseases (Sies (1985, 1997)).
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