1. METABOLISM OF OXYGEN
AND
FREE RADICALS

2. Oxygen acts as a substrate for approx. 200 enzymes.
Based on the enzyme mechanism we discriminate:
OXIDASES
OXYGENASES
OXIDASES
Oxidise substrates without incorporation of oxygen atoms into molecules.
According to the amount of electrons they use we discern:
2-electron oxidases
e.g. oxidase of D-aminoacids:

3. 4-electron oxidases
reduce oxygen to water, e.g. cytochromeoxidase
OXYGENASES
During the reaction incorpotate one or both atoms of oxygen molecule
into substrate and accordingly we discriminate:
DIOXYGENASES
MONOOXYGENASES
DIOXYGENASES
e.g. tryptophan-2,3-dioxygenase

4. MONOOXYGENASES
They are also called mixed function oxidases, or hydroxylases.
The example is steroid hydroxylation.
REACTIONS OF OXYGEN
Gaseous oxygen is in the form of two-atom molecule. For its reduction
to water four electrons are needed. Reduction can proceed stepwise
in one-electron reactions:

5. Some pecularities of oxygen reactivity are due to electron structure of
molecular oxygen and its reduction intermediates:

6. SUPEROXIDE
In vivo originates from autooxidation of: hydroquinones (used by
anti-tumor antibiotics – adriamycin)
flavines
catecholamines
thiols
tetrahydropterines
Also during transition of hemo(myo)globine to methemo(myo)globine
The most important enzymes producing superoxide:
NAD(P)H oxidases (especially of leukocytes)
xanthinoxidase
aldehydoxidase
The sites of production:
cell membrane
endoplasmic reticullum
mitochondria
nucleus

7. HYDROGEN PEROXIDE
Originates from autooxidation of 6-OH-, or 6-aminodopamine (leads to
synapses damage)
Diabetes melitus is experimentally induced in animals bydialuric acid that
autooxidizes to alloxan producing hydrogen peroxide.
Similarly as superoxide, hydrogen peroxide is produced by NAD(P)H
oxidase and xanthinoxidase. Large amounts are produced in peroxisomes
during beta-oxidation of fatty acids.
Other sites of production are similar as for superoxide:
cell membrane
endoplasmic reticullum
mitochondria
Under nanomolar concentration hydrogen peroxide is relatively stabile,
is permeable like a water and it appears that it acts like a signaling
molecule.

8. HYDROXYL RADICAL
Hydroxyl radical belongs among the most reactive compounds. Its life
time is in nanoseconds and it reacts with practically anything in the site
of production before diffusion limit.
Most often originates from Fenton reaction:
Of greatest biological importance are ferrous ions. They may
not appear free in the solution. Autoxidation of ferrous ions gives also
superoxide. Therefore iron is transported and stored in the form of
ferric complex. Fenton reaction is therapeutically used in treatment of
tumors. Bleomycins belong to the group of anti-tumor antibiotics. They
create a complex with ferrous ions and intercalate into DNA helix.
Hydrogen peroxide, difusing from cellular sites of production triggers
destruction of DNA mediated by hydroxyl radicals.

9. REACTIVE NITROGEN SPECIES
Similar effects as the intermediates of oxygen reduction that are
collectively known as reactive oxygen species (ROS), have also the
reactive nitrogen species, derived from nitric oxide. NO, produced by
NO synthases is free radical by itself. Though it produces important
physiological effects, its life time is in microseconds. After that it reacts
with oxygen to form NO2, or with superoside to peroxynitrite ONOO-.
Both compounds are highly reactive and induce cellular damage. Its
marker is nitrotyrosine bound in proteins. As the reactive oxygen and
nitrogen species occure usually together, we speak of RONS.

10. CELLULAR DAMAGE
RONS attack all cellular components. The most important targets are:
membrane lipids, proteins and nucleic acids.
The general mechanism of radical reactions is based on chain reactions,
that have three phases:
initiation
propagation
termination
Initiation can be triggered by any sufficiently reactive radical. Radicals
of the attacked compound then interact with self molecules thus
propagating the initial attack and forming chain reaction. Termination of
the chain is produced during interaction of two radicals or by formation
of low reactive radical that is not able to initiate a new chain. The last
case is the mechanism of antioxidant action.

11. The main target of free radical
attack on membranes are PUFA.Radical of fatty acid is transformed to cojugated diene that reacts with oxygen to give peroxy- derivative. This reacts
with another PUFA and triggers
the chain reaction. Fatty acid
peroxide is unstable and in the
presence of iron ions decompo-
ses. The products are alkans
(pentane in the case of n-6, ethanfrom n-3 PUFA) and the respective aldehyde. Aldehydes
further attack compounds
containing primary amino- group to form the so called lipofuscin-like pigments, LFP.

12. Proteins are attacked on the alpha carbon. Reaction with
oxygen gives peroxy-derivative which decomposes
by several mechanisms. Characteristic products are protein-bound carbonyl
groups that serve as indicator of freeradical damage. Reactions of protein radicals with other molecules of
protein transfer the radical to the side chains.Characteristic products like dityrosine or m-hydroxytyrosine are indicators of free radical
attack.

13. Protein radicals have life time up to 8 days. They can fragment, or
also polymerize. Aldehydes originating from free radical attack on
PUFA can crosslink several protein molecules.
Free radical attack to enzymes can lead to their activation or inhibition.
Important biological consequences are produced after free radical
attack to membrane receptors.

14. Attack to DNA can be initiated either by radicals or by non-radical
compounds. This ensues in
mutation
tumorigenesis
decomposition of DNA (therapeutic use)
RNA is damaged similarly as DNA.
Interaction of ROS with DNA causes with decreasing incidence:
destruction of bases
single chain brakes
two-chain brakes
crosslinks
Hydrogen peroxide specifically produces saturated bond 5-6 in
thymine, and oxidation of ribose to malonaldehyde.
The most important non-radical interaction is addition of aldehydes
on guanine, leading to mutation.

15. OVERVIEW OF CELLULAR INTERACTIONS

16. PROTECTION AGAINST FREE RADICAL DAMAGE
As all the damage depends on ROS formation, their removal protects
the dissemination of the damage. The cell has many specialized enzymes.
Superoxide is destroyed by superoxide dismutase (SOD:
SOD is of several types. They differ by the metal in the active site (Mn,
or Cu+Zn) and by localization either intracellular or extracellular.
Intracellularly is Mn-SOD localized in mitochondria, Cu,Zn-SOD in
cytoplasm. Extracelular Cu,Zn-SOD is glycosylated, which prolongs its
life-time in circulation. As the product of the reaction is hydrogen
peroxide, SOD must cooperate with enzymes decomposing hydrogen
peroxide. The most important are glutathione peroxidase (GPOX) and catalase (KAT). GPOX reduces peroxide to water with the aid of
glutathione, KAT acts in principle like a peroxide dismutase – one
molecule is oxidized, the other reduced.

17. OVERVIEW OF THE ENZYMES ACTING UPON ROS

18. THE ROLE OF RADICALS IN MEDICINE
Besides the protective enzymes, there are low molecular compounds
scavenging free radicals, antioxidants. According to their solubility we
discriminate water-soluble (ascorbate) and liposoluble (vitamine E,
vitamine A) antioxidants.In the food we ingest large amounts of synthetic
antioxidants, which are not necessarily helpful, as they interferre with the
physiological role of ROS as signaling molecules.
THE ROLE OF RADICALS IN MEDICINE
Practically all pathological states are accompanied by free radical
production, that can be the either the cause or epiphenomenon. In both
cases it is complicating factor.
Free radicals are increased during physical activity, by certain drugs,
can be induced by diets or alcohol consumption.
It appears that we are aging due to permanent intracellular production
of free radicals.