Oxidative Stress Flashcards by Nicola Reed

What is oxidative stress?

A chronic imbalance between levels of reactive oxygen species/reactive nitrogen species and endogenous antioxidant defences

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What respiratory diseases are linked to oxidative stress?

ARDS
Asthma
COPD
Pulmonary Hypertension
Pulmonary Fibrosis

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Is all oxidative stress bad?

No, acute reactive oxygen species can be protective and promote adaptive responses

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Is all free radical production is bad?

No, free radical production is required for host defences and cell function and signalling

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When does oxidative stress lead to disease?

Chronic oxidative stress will lead to pathology

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What is a free radical?

A species with one or more unpaired electrons that is free to partake in chemical bonds

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What is a reactive oxygen species?

Species that are usually derived from O2 radicals (superoxide) which may or may not be radicals

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What is a reactive nitrogen species?

Species that are usually derived from NO radicals which may or may not be radicals

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What are the sources reactive oxygen species?

Smoking
Industrial airborn pollution
Car exhaust fumes
Enclosed cooking fired
Ionising radiation
UV radiation
Coal mining

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What are the most common cellular sources of reactive oxygen species?

NADPH oxidases
Mitochondria (upto 25% of cellular ROS - electron leakage)
Peroxidases (MPO, EPO)
Oxidases (Xanthine oxidase, DUOX)

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Where are xanthine oxidases found?

Lung epithelial lining fluid
Sites of inflammation
Implicated in pathology of several cardiovascular diseases

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Where is myloperoxidase (MPO) found?

Released by neutrophils for pathogen killing

makes phlegm and snot green

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What do ROS do?

Lipid damage (peroxidation)
Protein damage (modification)
DNA damage

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How do ROS damage lipids?

Unsaturated fatty acids undergo addition of O2 in a chain-reaction.

Essentially this is akin to butter going rancid

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How do ROS damage proteins?

Several amino acids can be modified. The most common are:
Cysteine or methionine oxidation
Tyrosine to dityrosine, nitrotyrosine, or chlorotyrosine
Protein carbonyl formation
Lysine adduction by lipid-oxidation products

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How do ROS damage DNA?

Most common is 8-oxo-guanine.

Mutation occurs when DNA polymerase makes a mistake when replicating the damaged section

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Define carbonyl stress

The increased burden in reactive carbonyl species resulting in protein modification

What are the consequences of oxidative stress?

Autoimmune induction
Pro-inflammatory signalling
Mucus hypersecretion
Bronchial constriction
Mitochondrial damage
Altered cell fate (Cancer/death)
Extracellular remodelling (fibrosis)
Muscle dysfunction
Impaired cell function (phagocytosis)
Impaired drug function (corticosteroid)

How are ROS removed?

Target the oxidative stress:
    Induce endogenous antioxidant defences
    Supplement antioxidants (pharmacological/dietary)

What does myeloperoxidase do?

It makes hypochlorous acid (HOCl) from Cl- ions for pathogen killing

(A variant, eosinophil peroxidase, (EPO) prefers Br- ions, to make HOBr)

What is the sequence of events in lipid peroxidation

Initiation (initial radical attack)
Propagation (addition of oxygen)
Cycling (fatty acid radical attacks adjacent fatty acid)
Lipid peroxide
Fragmentation (formation of Reactive Carbonyl Species)

What is lipid peroxidation?

ROS mediated damage of lipids

What fatty acids are affected by lipid peroxidation?

Affects polyunsaturated fatty acids in the lipid membranes

The most susceptible common fatty acids are:

Linoleic acid
Linolenic acid
Arachidonic acid

What are the different sources of endogenous antioxidant defences?

Enzyme/protein antioxidants
Metal binding proteins
Chain breaking antioxidants
Antioxidant Response Element (ARE)

List the different enzyme/protein antioxidants

``` Superoxide dismutase (3 isoforms: cytosolic, mitochondrial, and extracellular) Catalase Glutathione peroxidase (many isoforms) Caeruloplasmin (prevents Fenton chemistry) Thioredoxin (acts like GSH) ```

List the different Metal binding protiens

Transferin Ferritin Lactoferin

List the different chain breaking antioxidants

Lipid phase: - Tocopherols - Ubiquinol - Carotinoids - Flavinoids Aqueous phase: - Ascorbate - Urate (uric acid) - Glutathione and other thiols (Can also target carbonyls)

Describe the properties of Glutathione (GSH)

It is an important extra and intracellular antioxidant throughout the body It is a simple tripeptide It conjugates and detoxifies electrophiles directly, or enzymatically (GST) It maintains essential REDOX and thiol status of the cells and is involved in cysteine storage

What determines protein susceptibility to Carbonyl stress

Concentration and half life of target protein Is the target protein expressed at the site of oxidative/carbonyl stress Can the protein be modified and where is the modification Does modification impact on function (inactivate enzyme, increase or prevent degradation)

What determines the outcome of Carbonyl stress?

Low level carbonylation tags proteins for proteosomal degradation High level carbonylation inhibits the proteosome and cause accumulation of the dysfunctional protein

Give examples of how oxidative stress can be measured

Measurement of glutathione (reduced and oxidised) Measurement of total protein carbonyls Immunoassay based detection of protein carbonyls

Describe the features of the measurement of oxidative stress by measurement of glutathione

Colourimetric assay - quantitative Measures both reduced and oxidised glutathione GSSG levels indicate oxidative burden GSSG/GSH ratio is a better measure of oxidative stress Higher ration=more oxidative stress

Describe the features of the measurement of oxidative stress by measurement of total protein carbonyls

Spectrophotometric DNPH assay - quantitative Requires only 50ng of protein Sensitivity ~ 1pmol

Describe the features of the measurement of oxidative stress by immunoassay based detection of protein carbonyls

ELISA Western blot 2D gel electrophoresis Immunochemistry

What are the 2 key enzymes involved in glutathione synthesis?

Glutamate-cysteine ligase | Glutathione synthetase

What is the key enzyme involved in glutathione regeneration?

Glutathione reductase

When is NF-E2 related factor 2 (Nrf2) active?

Nrf2 is only active when released for complex with KEAP1

Describe the features of Nrf2

Nrf2 activity declines with age and COPD severity Carbonyl stress reduces Nrf2 activity and expression Nrf2 loss increases inflammation and pathology - Cigarette smoke model: increases inflammation and pathology

How does the body prevent oxidative stress?

Antioxidant and Carbonyl scavengers

What is the clinical evidence for oxidative stress in airway disease?

Clinical evidence: - DIRECT: Smokers/Individuals exposed to high pollution/ROS most suscetable to developing COPD - DIRECT: increased ROS, RNS, TBARs, Carbonyls – correlate to disease severity - INDIRECT: increased MΦ & Neutrophil activity in COPD  release more ROS - INDIRECT: Reduced dietary anti-oxidant intake = decreased lung function

What is the mechanistic evidence for oxidative stress in airway disease?

Mechanistic evidence - Chronic Smoke exposed mice/rats get COPD-like pathology (Inflammation & lung destruction) - Chronic O3 exposure in mice get COPD-like pathology (Inflammation & lung destruction) - Antioxidants protect from Cigarette smoke induced Inflammation & emphysema in mice

What is the therapeutic based evidence for oxidative stress in airway disease?

Therapeutic based evidence......(Limited evidence) - NAC 600mg bid oral: Reduced exhaled H2O2 & exacerbation frequency (Various studies) - Erdosteine 300mg bid: Less exacerbations & hospitalisation, improved QoL - Anti-oxidant rich diet: Reduced COPD mortality risk & improved lung function (Various studies)