Role Of Free Radicals In Tissue Injury

Role Of Free Radicals In Tissue Injury

Ischaemia-reperfusion injury occurs due to excessive accumulation of free radicals or reactive oxygen species.

The mechanism of reperfusion injury by free radicals is complex but following three aspects are involved:

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1. Calcium overload.
2. Excessive generation of free radicals (superoxide, H₂O₂, hydroxyl radical, pernitrate).
3. Subsequent inflammatory reaction. These are discussed below.

1. Calcium Overload:

• Upon restoration of blood supply, the ischaemic cell is further bathed by the blood fluid that has more calcium ions at a time when the ATP stores of the cell are low.
• This results in further calcium overload on the already injured cells, triggering lipid peroxidation of the membrane and causing further membrane damage

Contrasting features of reversible and irreversible cell injury:

2. Excessive Generation Of Free Radicals:

Although oxygen is the lifeline of all cells and tissues, its molecular forms as reactive oxygen radicals or reactive oxygen species can be most devastating for the cells. Free radical-mediated cell injury has been extensively studied and a brief account is given below.

Oxygen-free radical generation: 

Normally, a reduction-oxidation (redox) reaction in the metabolism of the cell involves the generation of ATP by an oxidative process in which biradical oxygen (O₂) combines with hydrogen atom (H), and in the process, water (H₂O) is formed.

• This normal reaction of O₂ to H₂O involves ‘four electron donation’ in four steps involving the transfer of one electron at each step. Free radicals are intermediate chemical species having a single unpaired electron in their outer orbit.
• These are generated within the mitochondrial inner membrane where cytochrome oxidase catalyzes the O₂ to H₂O reaction.

Three intermediate molecules of partially reduced species of oxygen are generated depending on the number of electrons transferred :

• Superoxide oxygen (o^–₂): one electron
• Hydrogen peroxide (H₂O₂): two electrons
• Hydroxyl radical (OH^–): three electrons

These are generated from the enzymatic and non-enzymatic reactions as under:

• Superoxide (o^–₂):  Superoxide anion (O^–₂) May be generated by direct auto-oxidation of O₂ during mitochondrial electron transport reaction. Alternatively, O^–₂ is produced enzymatically by xanthine oxidase and cytochrome P450 in the mitochondria or cytosol.
• Hydrogen peroxide (H₂O₂) (O^–₂):  So formed as above is catabolized to produce H₂O₂ by superoxide dismutase (SOD). H₂O₂ is reduced to water enzymatically by catalase (in the peroxisomes) and glutathione peroxidase, GSH (both in the cytosol and mitochondria).
• Hydroxyl radical (OH^–): OH^– radical is formed by 2 ways in biological processes by radiolysis of water and by reaction of H₂O₂ with ferrous (Fe++) ions; the latter process is termed as Fenton reaction. Fenton reaction involves the reduction of normal intracellular ferric (Fe⁺⁺⁺) to ferrous (Fe⁺⁺) form, a reaction facilitated by (O^–₂).

Other free radicals In addition to superoxide, H₂O₂ and hydroxyl radicals generated during the conversion of O₂ to H₂O reaction, a few.

Other free radicals active in the body are as follows:

• Nitric oxide (NO)  and peroxynitrite (ONOO) NO is a chemical mediator formed by various body cells (endothelial cells, neurons, macrophages, etc), and is also a free radical.
• NO can combine with superoxide and forms ONOO which is a highly reactive free radical.
• Halide reagent (chlorine or chloride) released in the leucocytes reacts with superoxide and forms hypochlorous acid (HClO) which is a cytotoxic free radical.
• Exogenous sources of free radicals include some environmental agents such as tobacco and industrial pollutants.

Cytotoxicity of free radicals:

Free radicals are formed in physiologic as well as pathologic processes. Oxygen radicals are unstable and are destroyed spontaneously.

• The rate of spontaneous destruction is determined by the catalytic action of certain enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH).
• The net effect of free radical injury in physiologic and disease states, therefore, depends upon the rate of their formation and the rate of their elimination.
• However, if not degraded, then free radicals are highly destructive to the cell since they have electron-free residue and thus bind to all molecules of the cell; this is termed oxidative stress.
• Out of various free radicals, hydroxyl radical is the most reactive species. ‘

Free radicals may produce membrane damage by the following mechanisms:

• Lipid peroxidation Polyunsaturated:
  • Fatty acids (PUFA) in the membrane are attacked repeatedly and severely by oxygen-derived free radicals to yield highly destructive PUFA radicals—lipid hydroperoxy radicals and lipid hydroperoxides.
  • This reaction is termed lipid peroxidation.
  • The lipid peroxides are decomposed by transition metals such as iron.
  • Lipid peroxidation is propagated to other sites causing widespread membrane damage and destruction of organelles.
• Oxidation of proteins:
  • Oxygen-derived free radicals cause cell injury by oxidation of protein macromolecules of the cells, cross-linkages of labile amino acids as well as by fragmentation of polypeptides directly.
  • The result is a degradation of cytosolic neutral proteases and cell destruction.
  • DNA damage Free radicals cause breaks in the single strands of the nuclear and mitochondrial DNA.
  • This results in cell injury; it may also cause malignant transformation of cells.
  • Cytoskeletal damage Reactive oxygen species are also known to interact with cytoskeletal elements and interfere in mitochondrial aerobic phosphorylation and thus cause ATP depletion.

Conditions with free radical injury:

• Currently, oxygen-derived free radicals have been known to play an important role in many forms of cell injury:
• Ischaemic reperfusion injury
• Ionizing radiation by causing radiolysis of water
• Chemical toxicity
• Chemical carcinogenesis
• Hyperoxia (toxicity due to oxygen therapy)
• Cellular ageing
• Killing of microbial agents
• Inflammatory damage
• Destruction of tumor cells
• Atherosclerosis.

 

Antioxidants :

Antioxidants are endogenous or exogenous substances that inactivate the free radicals. These substances include the following:

• Vitamins E, A and C (ascorbic acid)
• Sulfhydryl-containing compounds for example, Cysteine and glutathione.
• Serum proteins e.g. ceruloplasmin and transferrin.

3. Inflammatory Reaction:

• Ischemia-reperfusion event is followed by an inflammatory reaction. Incoming activated neutrophils utilize oxygen quickly (oxygen burst) and further release large excess of oxygen free radicals.
• Ischaemia is also associated with the accumulation of precursors of ATP, namely ADP, and pyruvate, which further build up the generation of free radicals.

Stress Proteins In Cell Injury:

• When cells are exposed to stress of any type, a protective response by the cell is by release of proteins that move molecules within the cell cytoplasm; these are called stress proteins.
• There are 2 types of stress-related proteins: heat shock proteins (HSP) and ubiquitin (so named due to its universal presence in the cells of the body).

HSPs:  These are a variety of intracellular carrier proteins present in most cells of the body, especially in renal tubular epithelial cells. They have been classified into various groups depending on their molecular weight and their migration pattern on electrophoresis.

They normally perform the role of molecular chaperones (house-keeping) i.e. they direct and guide metabolic molecules to the sites of metabolic activity for example.

• Protein folding
• Disaggregation of protein-protein complexes
• Transport of proteins into various intracellular organelles (protein kinesis).

However, in response to stresses of various types (e.g. toxins, drugs, poisons, ischemia, cancer), their level goes up, both inside the cell as well as into the plasma where they leak out, and hence the name stress proteins.

For example:

• In myocardial infarction, HSPs have been shown to limit tissue necrosis in ischaemic reperfusion injury, termed ischaemic preconditioning.
• They have also been shown to have a central role in protein aggregation in amyloidosis.

Ubiquitin:  This is another related stress protein that has a ubiquitous presence in human body cells.

• Like HSPs, ubiquitin too directs intracellular molecules for either degradation or synthesis.
• Ubiquitin is involved in a variety of human degenerative diseases by activation of genes for protein synthesis, especially in the nervous system in aging for example, In Alzheimer’s disease, Creutzfeldt-Jakob disease, Parkinson’s disease.

Pathogenesis Of Chemical Injury:

Chemicals induce cell injury by one of the two mechanisms: by direct cytotoxicity, or by conversion of chemical into reactive metabolites.

Direct Cytotoxic Effects:

Some chemicals combine with components of the cell and produce direct cytotoxicity without requiring metabolic activation.

• The cytotoxic damage is usually greatest to cells that are involved in the metabolism of such chemicals for example, In mercuric chloride poisoning, the greatest damage occurs to cells of the alimentary tract where it is absorbed and the kidney where it is excreted.
• Cyanide kills the cell by poisoning mitochondrial cytochrome oxidase thus blocking oxidative phosphorylation.
• Other examples of directly cytotoxic chemicals include chemotherapeutic agents used in the treatment of cancer, and toxic heavy metals such as mercury, lead, and iron.

Conversion To Reactive Toxic Metabolites:

This mechanism involves metabolic activation to yield the ultimate toxin that interacts with the target cells. The target cells in this group of chemicals may not be the same cells that metabolized the toxin.

• An example of cell injury by conversion of reactive metabolites is toxic liver necrosis caused by carbon tetrachloride (CCl₄), acetaminophen (commonly used analgesic and antipyretic), and bromobenzene.
• Cell injury by CCl₄ is a classic example of an industrial toxin (earlier used in the dry-cleaning industry) that produces cell injury by conversion to a highly toxic free radical, CCl₃, in the body’s drug-metabolizing cytochrome P₄₅₀ enzyme system in the liver cells.
• Thus, it produces profound liver cell injury by free radical generation. Other mechanism of cell injury includes direct toxic effect on the cell membrane and nucleus.

Pathogenesis Of Physical Injury:

Injuries caused by mechanical force are of medicolegal significance. But they may lead to a state of shock. Injuries by changes in atmospheric pressure (for example, Decompression sickness).

• Radiation injury to humans by accidental or therapeutic exposure is of importance in the treatment of persons with malignant tumors as well as may have carcinogenic influences
• The killing of cells by ionizing radiation is the result of the direct formation of hydroxyl radicals from the radiolysis of water.
• These hydroxyl radicals damage the cell membrane and may also attack the DNA of the target cell.
• In proliferating cells, there is inhibition of DNA replication and eventual cell death by apoptosis (for example, Epithelial cells).
• In non-proliferating cells, there is no effect of inhibition of DNA synthesis and in these cells, there is cell membrane damage followed by cell death by necrosis (for example Neurons).

Pathogenesis of Cell Injury:

• Irrespective of the type of cell injury, a common underlying mechanism involves mitochondrial damage by ATP depletion and cell membrane damage.
• Hypoxic-ischaemic cell injury is the prototype; it may be reversible or irreversible.
• Reversible cell injury occurs due to decreased cellular ATP causing initially impaired aerobic respiration, followed by anaerobic glycolytic oxidation.
• Other changes are intracellular lactic acidosis, damage to membrane pumps (Na⁺-K⁺ and Ca⁺), and dispersal of ribosomes.
• Irreversible cell injury is due to the continuation of earlier changes of reversible cell injury; it includes further cytosolic calcium influx and its excess in the mitochondria, and further damage to membranes, cytoskeleton, and nucleus.
• Lysosomal damage causes the release of hydrolytic enzymes which can be estimated in the blood as indicators of cell death, for example, SGOT, SGPT, LDH, CK-MB, cardiac troponins, etc.
• Ischemia-reperfusion injury is due to the release of reactive oxygen species or free radicals. These include OH^– as the most potent radical; others are O^–₂ and H₂O₂.
• Free radical injury occurs when their generation exceeds their elimination and is implicated in the mechanism of cell injury from various etiologic factors.
• Stress proteins (heat shock proteins and ubiquitin) are released as a form of protective response to environmental stresses.
• In general, cell injury from chemicals and drugs may occur from either direct toxicity or their conversion into toxic metabolites, most often in the liver.
• Cell injury from ironing radiation may cause cell death (apoptosis or necrosis); it may induce mutations in proliferating cells.