Oxidative Stress Flashcards
What is oxidative stress?
A chronic imbalance between levels of reactive oxygen species/reactive nitrogen species and endogenous antioxidant defences
What respiratory diseases are linked to oxidative stress?
ARDS Asthma COPD Pulmonary Hypertension Pulmonary Fibrosis
Is all oxidative stress bad?
No, acute reactive oxygen species can be protective and promote adaptive responses
Is all free radical production is bad?
No, free radical production is required for host defences and cell function and signalling
When does oxidative stress lead to disease?
Chronic oxidative stress will lead to pathology
What is a free radical?
A species with one or more unpaired electrons that is free to partake in chemical bonds
What is a reactive oxygen species?
Species that are usually derived from O2 radicals (superoxide) which may or may not be radicals
What is a reactive nitrogen species?
Species that are usually derived from NO radicals which may or may not be radicals
What are the sources reactive oxygen species?
Smoking Industrial airborn pollution Car exhaust fumes Enclosed cooking fired Ionising radiation UV radiation Coal mining
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)
Where are xanthine oxidases found?
Lung epithelial lining fluid
Sites of inflammation
Implicated in pathology of several cardiovascular diseases
Where is myloperoxidase (MPO) found?
Released by neutrophils for pathogen killing
makes phlegm and snot green
What do ROS do?
Lipid damage (peroxidation) Protein damage (modification) DNA damage
How do ROS damage lipids?
Unsaturated fatty acids undergo addition of O2 in a chain-reaction.
Essentially this is akin to butter going rancid
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
How do ROS damage DNA?
Most common is 8-oxo-guanine.
Mutation occurs when DNA polymerase makes a mistake when replicating the damaged section
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)
