Oxidative Stress and Anti-Oxidants Flashcards
What causes cellular damage?
ROS and RNS
What is cellular damage by ROS and RNS a significant component in?
A wide range of disease states
Give 10 disease states that cellular damage caused by ROS and RNS is a significant component in?
- Cardiovascular disease
- Alzheimers disease
- Rheumatoid arthritis
- Crohn’s disease
- COPD
- Ischaemia / reperfusion injury
- Cancer
- Pancreatitis
- Parkinson’s disease
- MI
How do the electrons of atoms, molecules and ions usually associate?
In pairs
How does each pair of electrons move?
Within a defined region of space- an orbital
What is a free radical?
An atom or molecule that contains one or more unpaired electrons, and it capable of independent existence
What is used to denote a free radical?
A superscript dot
Are free radicals reactive or inert?
Yes, usually very reactive
What do free radicals tend to do?
Acquire electrons from other atoms, molecules or ions
Why are free radicals damaging?
Because they want to get electrons from another molecule
What does a reaction of a radical with a molecule typically generate?
A second radical
What is the result of a reaction of a radical with a molecule typically generating a second radical?
It propagates damage
What does ROS stand for?
Reactive oxygen species
What are the ROS?
- Molecular oxygen
- Superoxide
- Hydrogen peroxide
- Hydroxyl radical
Regarding radicals, what is molecular oxygen?
Biradical
What is meant by molecular oxygen being biradical?
It has 2 unpaired electrons in different orbitals
What is superoxide?
O 2 º -
When is superoxide produced?
When one electron is added to molecular oxygen
Why is superoxide important?
It’s an important source of other ROS
What is hydrogen peroxide?
H 2 O 2 -
How is hydrogen peroxide formed?
Adding 2H + and e - to superoxide
Is hydrogen peroxide a free radical?
No, but can react to produce free radicals
Is hydrogen peroxide readily diffusible?
Yes
What is the most reactive and damaging free radical?
Hydroxyl radical
What is the hydroxyl radical?
OHº
How is the hydroxyl radical formed?
Adding e - and H + to hydrogen peroxide (which removes H 2 O)
Why is the hydroxyl radical so damaging?
It reactions with anything
How is the hydroxyl radical removed?
By adding e - and H + , which produces water
What are the two reactive nitrogen species?
NAME?
What is nitric oxide?
NOº
Where is nitric oxide important?
Signalling molecule
What happens when nitric oxide is in high concentrations?
It plays a role in the immune system
What role does nitric acid play in the immune system?
It can produce free radicals that damage pathogens
What is peroxynitrate?
ONOO -
When is peroxynitrate formed?
When superoxide reacts with nitric oxide
Is peroxynitrate a free radical?
No, but powerful oxidant that can damage cells
What effect does ROS have on DNA?
It damages them by taking electrons away
What are the two main types of ROS damage to cells?
NAME?
What can the modified base caused by ROS lead to?
Mispairing and mutation
Does ROS react with ribose or deoxyribose sugar?
Either
What can ROS reacting with the sugar in DNA cause?
The strand to break, or mutation on repair
What can be used as a measurement of oxidative damage?
The amount of 8-oxo-dG
What can failure to repair DNA damage lead to?
Mutation, which can lead to cancer
What happens when ROS react with proteins?
Can change backbone or side-chain
What can ROS reaction with side chain lead too?
Modified amino acids
How can ROS modify amino acids?
- Carbonyls
- Hydroxylated adducts
- Ring opened species
- Dimers (e.g. di-tyrosine)
- Disulphide bonds
What can modified amino acids in proteins lead to?
Change in protein structure
What could a change in protein structure lead to?
- Gain of function
- Loss of function
- Protein degradation
What can ROS reacting with backbones of protein lead to?
Fragmentation
What could fragmentation of the protein backbone lead to?
Protein degradation
Where do disulphide bonds play an important role?
In folding and stability of some proteins
What kind of proteins do disulphide bonds usually play an important role?
- Secreted proteins
- Extracellular domain of membrane proteins
Where are disulphide bonds formed?
Between thiol groups of cysteine residues
When can inappropriate disulphide bond formation occur?
If ROS takes electrons from cysteines
What can inappropriate disulphide bond formation lead to?
- Misfolding
- Crosslinking
- Disruption of function
Is inappropriate disulphide bond formation inter-subunit or intra-subunit?
Can be either
How can ROS damage lipids?
Free radical can extract hydrogen atom from polyunsaturated fatty acids in membrane lipids
What can be formed when a free radical reacts with a lipid?
A lipid radical
What can happen to a lipid free radical?
It can react with oxygen to form a lipid peroxyl radical
What is formed when lipid peroxyl radicals are made?
A chain reaction
Why is a chain reaction formed when a lipid peroxyl radical is made?
Because the lipid peroxyl radical can extract hydrogen from a nearby fatty acid
What is the problem with ROS damage to lipid?
NAME?
What are the two types of biological oxidants?
- Endogenous
- Exogenous
What is meant by endogenous?
Within cells
What is meant by exogenous?
Outside cells
Give 7 endogenous sources of biological oxidants
NAME?
Give 4 examples of exogenous sources of biological oxidants
NAME?
Give 3 sources of radiation
NAME?
How does the electron transport chain produce ROS?
- NADH and FADH 2 supply electrons to complexes I, II, III, and IV from metabolic substrates
- e - pass through ETC, reducing oxygen to form water at complex IV
- Occasionally, electrons accidentally escape the chain and react with the dissolved oxygen to form superoxide
How does the electron transport chain deal with the ROS formed
Have protective mechanisms to deal with it
What protective mechanisms does the e.t.c. have to deal with ROS produced?
Enzymes
What are 3 types of nitric oxide synthase (NOS)?
NAME?
What is iNOS?
Inducible nitric oxide synthase
What does iNOS do?
Produces high NO concentration in phagocytes for direct toxic effect
What is eNOS?
Endothelial nitric oxide synthase?
What does eNOS do?
Signalling
What is nNOS?
Neuronal nitric oxide synthase
What does nNOS do?
Signalling
Give the reaction that NOS catalyses
Arginine + NADPH + O 2 → citrulline + NOº + NADP + + H 2 O
Where is NOº used for signalling?
- Vasodilation
- Neurotransmission
- S-Nitrosylation
What is the problem with NOº at high levels?
It has a toxic effect
What happens in a respiratory burst?
Rapid release of superoxide and H 2 O 2 from phagocytic cells
Give 2 examples of cells that perform respiratory bursts
- Neutrophils
- Monocytes
What is the purpose of the ROS and peroxynitrate in the respiratory burst?
It destroys invading bacteria
What is the respiratory burst part of?
The anti-microbal defence system
Describe the process of the respiratory burst
- The membrane bound complex NADPH oxidase converts NADPH to NADP + using oxygen, which is then converted to superoxide
- The superoxide is then converted to hydrogen peroxide
- The hydrogen peroxide is converted to HOClº (bleach), by action of the enzyme myeloperoxidase and the addition of Cl -
- Simultaneously, iNOS produces NOº, which reacts with the superoxide to produce peroxynitrate, which also attacks the bacteria
How is the the enzyme myeloperoxidase, required for respiratory burst, released?
Released from phagolysosome into the phagocytic vesicle by secretory granules
What is chronic granulomatous disease?
A genetic defect in the NADPH oxidase complex
What is the result of the defect in chronic granulomatous disease?
Causes enhanced susceptibility to bacterial infections
Why does chronic granulomatous disease cause an increased susceptibility to bacterial infections?
Because of the reduced capacity for the respiratory burst response
What are the symptoms of chronic granulomatous disease?
- Atypical infections
- Pneumonia
- Abscesses
- Impetigo
- Cellulitis
What are the main cellular defences against ROS?
- Superoxide dismutase (SOD) and catalase
- Glutathione
- Free radical scroungers
What does superoxide dismutase do?
Converts superoxide to H 2 O 2 and oxygen
Where is SOD expressed?
In mitochondria
Why is SOD produced in the mitochondria?
To deal with superoxides produced by accident in the e.t.c.
Why is SOD a primary defence?
Because superoxide is a strong inhibitor of chain reactions
What are the 3 isoenzymes of SOD?
- Cu + - Zn 2+ cytosolic
- Cu + - Zn 2+ extracellular
- Mn 2+ mitochondrial
What does catalase do?
Converts H 2 O 2 to water and oxygen
Where is catalase found?
Widespread
Where is catalase particularly important?
Immune cells
Why is catalase particularly important in immune cells?
To protect against the oxidative burst
Why is SOD alone not sufficient protection?
Because H 2 O 2 still damaging
What tripeptide is synthesised to protect against oxidative damage?
Glycine-Cysteine-Glutamate ; called GSH (reduced form)
What happens when GSH comes into contact with an ROS?
It donates e -
What enzyme is required for the glutathione mechanism of protection from oxidative damage?
Glutathione peroxidase
What does glutathione peroxidase do?
Causes two GSH molecules to react together to form a a disulphide bond, forming the oxidised form- GSSG
What does glutathione peroxidase require?
Selenium
What happens when GSH is converted to GSSG?
H 2 O 2 is converted to H 2 O
How is GSSG reduced back to GSH?
By glutathione reductase
What does glutathione reductase do?
Catalyses the transfer of electrons from NADPH to disulphide bond
Where does the NADPH needed to reduce GSSG come from?
Pentose phosphate pathway
What does the fact that the NADPH required for the reduction of GSSG comes from the pentose phosphate pathway mean?
That the pentose phosphate pathway is essential for protection from free radical damage
What do free radical scroungers do?
Takes hit from free radicals to prevent damage to tissues
Give 6 examples of free radical scroungers?
- Vitamin E
- Vitamin C
- Carotenoids
- Uric acid
- Flavonoids
- Melatonin
What is vitamin E also known as?
α-tocopherol
Is vitamin E water or lipid soluble?
Lipid
Where is vitamin E important?
In protection against lipid peroxidation
What is vitamin C also known as?
Ascorbic acid
Is vitamin C water or lipid soluble?
Water
Where is vitamin C important?
In regenerating reduced form of vitamin E
How do free radical scroungers reduce free radical damage?
By donating a hydrogen atom (and it’s electron) to free radicals in a non-enzymatic reaction
When does oxidative stress occur?
When defences are compromised, or an excessive burden is on antioxidants
What is galactosaemia?
A deficiency is galactokinase, uridyl transferase or UDP-galactose epimerase
What does deficiency in the 3 enzymes in galactosaemia favour?
Conversion of galactose to galactitol
What does increased activity of aldose reductase cause?
An excessive consumption of NADPH
What is the result of an excessive consumption of NADPH?
Compromises defences against ROS damage
How can galactosaemia cause cataracts?
NAME?
What are the symptoms of galactosaemia?
- Heptomegaly and cirrhosis
- Renal failure
- Vomiting
- Seizure and brain damage
- Cataracts
- Hypoglycaemia
What is the G6PDH enzyme essential for?
The pentose phosphate pathway
What does G6PDH deficiency limit?
The amount of NADPH
What is NADPH required for?
Reduction of oxidised glutathione (GSSG) back to reduce glutathione (GSH)
What does lower GSH mean?
Less protection against damage from oxidative stress
What does G6PDH result in a build up of?
H 2 O 2
Why does a G6PDH deficiency cause a build up of H 2 O 2 ?
It’s not converted to water
What does the build up ofH 2 O 2 cause?
- Lipid peroxidation
- Protein damage
What is the result of the lipid peroxidation?
Cell membrane damage
What is the result of cell membrane caused by lipid peroxidation?
Lack of deformity leads to mechanical stress
What is the result of protein damage caused byH 2 O 2 build up?
Aggregates chains of cross-linked haemoglobin, causing Heinz bodies
What can Heinz bodies lead to?
Haemolysis
How do Heinz bodies appear on micrographs?
Dark staining within red blood cells
What are Heinz bodies made up of?
Precipitated haemoglobin
What are the effect of Heinz bodies?
They alter rigidity
How do Heinz bodies alter rigidity?
They bind to the cell membrane
What is the result in the altered rigidity due to Heinz bodies?
Increased mechanical stress when cells squeeze through small capillaries
What removes Heinz bodies?
Spleen
What does the removal of bound Heinz bodies by the spleen result in /
Blister cells
What is the presence of Heinz bodies a clinical sign of?
G6PDH deficiency
Where does metabolism of paracetamol occur?
Hepatocytes
How can paracetamol be metabolised at prescribed dosage?
By conjugation with glucuronide or sulphate
What happens in a paracetamol overdose?
The normal pathway becomes saturated, and a second pathway has to be used
What is the problem with the second pathway of paracetamol metabolism?
The toxic metabolite NAPQI accumulates
Why is NAPQI toxic?
NAME?
What are the direct toxic effects of NAPQI?
Oxidative damage to the liver cell
Why does NAPQI build up deplete glutathione reserves?
It’s used to protect the cell against damage
What is the result of depletion of NAPQI reserves?
Defences are compromised
What is the treatment for paracetamol overdose?
Acetylcysteine
How does acetylcysteine work?
Replenishing glutathione levels
Where is ischaemia reperfusion injury especially relevant?
- Heart
- Brain
- Organ transplantation
What can happen to cells during ischaemia?
They can be reversibly damaged
How can cells damaged by ischaemia recover?
Following restoration of blood flow
What is the problem with reperfusion following ischaemia?
Can result in more damage than caused by initial ischaemia
What is especially sensitive to reperfusion injury?
Mitochondria
What causes ischaemic reperfusion injury?
- Incompletely metabolised products produce ROS on re-introduction of oxygen
- Loss of antioxidants during ischaemia means can’t cope with potential oxidative damage
- Influx of calcium ions upon renewed blood flow
- Recruitment of leukocytes to affected area
