METABOLISM OF OXYGEN AND FREE RADICALS

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:

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

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:

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

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

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.

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.

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.

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.

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.

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.

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.

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.

OVERVIEW OF CELLULAR INTERACTIONS

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.

OVERVIEW OF THE ENZYMES ACTING UPON ROS

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.