Lecture 2 - Reactive oxygen species Flashcards
(27 cards)
ROS: what are they, what are their key features, what do they do, and what are some examples?
Reactive Oxygen Species - Chemically reactive molecules containing oxygen.
- Often highly reactive & short-lived
- Damage cellular components - impair function, can cause mutation or cell death
- Some physiological roles (e.g. immune system, cell signalling)
- Byproducts of some metabolic processes (e.g. respiration)
Examples:
- Superoxide radical (O₂⁻)
- Hydroxyl radical (HO⁻) **
- Hydrogen peroxide (H₂O₂) - less reactive, less damaging, but Fenton Reaction with Fe²⁺ or Cu⁺ produces HO*
- Singlet Oxygen ¹O₂ (O₂ with electrons in a high energy, unstable state (O₂ is the ground state)) **
“*” = free radical
“**” = Highly damaging, no cellular defences
Fenton reaction: what is it?
Reaction of H₂O₂ with Fe²⁺ or Cu⁺ which produces HO*
ROS: how do they cause damage?
- DNA breakage/mutagenesis
- Protein aggregation/(in)activation/fragmentation
- Lipid peroxidation
- Destruction of small molecules
May promote cell death
Detoxification mechanisms: what are the types and what do they do?
Enzyme-free - detoxify ROS using mechanisms that involve molecules that are not enzymes
Enzyme-dependent - detoxify ROS using enzymes
Enzyme-free ROS detoxification: what is it, what is the process, what is an example, and what are some more specific examples?
The detoxification of ROS using mechanisms that involve molecules that are not enzymes
Cellular antioxidants - scavenge radicals (inefficient for HO* and not effective on H₂O₂)
Reduced glutathione - major cellular antioxidant
Vitamin C (ascorbate) - hydrophilic
VItamin E (α-tocopherol) - hydrophobic
GSH: what is it, what is the usual cellular concentration, what does it do, what is its form after doing what it does, can this be reversed, and what is the negative impact of its overusage?
Glutathione
Cellular [GSH] ~10-14mM (<1mM = depleted)
Antioxidant - scavenges radicals
Oxidised glutathione (GSSG): 2 glutathiones linked via S-S bond (GSH:GSSG usually 100:1; ~ 1:1 in oxidative stress)
Converted back to 2 GSH by Glutathione reductase or exported (lost) from cells
- GSH used (destroyed) in Phase II detoxification - most enzyme-dependent oxidant defence mechanisms need it as a cofactor and since cells make GSH slowly (limited by [cysteine]: only ~10µM)
- If GSH removal outstrips replenishment:
- glutathione depletion
- Reduces Phase II detoxification
- Prevents direct ROS removal
- Blocks effects of many enzyme-dependent protection mechanisms (glutathione-dependent protection enzymes)
Enzyme-dependent ROS detoxification mechanisms: what are they, how do they vary between tissues/cells, what are their limitations, what do they require to do what they do, and what are some examples?
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Enzymes that specifically remove particular ROS or reduce oxidized molecules
Expression levels vary between tissues/cell types - some cells are more sensitive to ROS than others.
None are effective for 1O₂ or HO*
- Typically require metal cofactors for the redox chemistry
- Selenium (Se) is essential for some
- Others require GSH (GSH depletion impairs some enzyme-dependent antioxidant defences)
- Some require both Se and GSH
Catalase
Glutathione peroxidase
Peroxiredoxin
Glutaredoxin
Thioredoxin
Superoxide dismutase
Selenium: what may its role be in antioxidant defences, and how may it be affected by selenium deficiency?
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Essential for some antioxidant enzymes - selenoproteins - contain the amino acid Selenocysteine
Se deficiency compromises anti-oxidant defences
Enzyme-dependent ROS detoxification mechanisms - examples
ER
Damaging effects of ROS:
- Lipid peroxidation
- Protein Damage
- DNA damage
- Small molecule damage
*
Lipid peroxidation: what is the process, what do the products do and what are the potential results of this?
- ROS attacks poly-unsaturated fatty acid (PUFA) in the plasma membrane - resulting in the formation of a carbon-centered radical (CCR)
- This radical is unstable and readily reacts with O₂ which forms a peroxyl radical which attacks another PUFA, forming lipid hydroperoxide and another CCR
- Forms a chain reaction which contains until the radical encounters a chain-breaking antioxidant or 2 radicals react together
Lipid hydroperoxide - it decays to reactive aldehydes which cause the adduction of proteins/DNA
Results in:
* Altered membrane fluidity
* Loss of membrane integrity
* Altered membrane protein function
* Adduction of DNA/proteins
Protein damage: what are the types and what do they result in?
Amino acid oxidation (e.g. Cysteine):
* Enzyme inactivation/activation (physiological role but excess ROS can lead to inappropriate alterations)
* Cross-linking/aggregation
Release of Fe⁺ (e.g. aconitase) – promotes Fenton reaction
Adduct formation (eg from a reaction with aldehydes produced from lipid peroxidation) - can provoke an immune response
Creation of carbon-centred radicals - decay to produce aldehydes that form DNA and protein adducts
Cleavage of peptide bonds – fragmentation
DNA damage: what DNA types are damaged more, why, what effects may occur, and what may these damages cause?
Mitochondrial DNA is more easily damaged than nuclear DNA (no histones)
- Strand breaks
- DNA methylation alteration (epigenetic effects)
- Oxidise bases (mainly guanine)
- Abasic sites (bases removed)
DNA damage may result in cell death - if the cell survives the mutations induced, it may contribute to the development of cancer
Small molecule damage: what molecules may be damaged?
Nitric Oxide (NO) - reacts with superoxide to produce peroxynitrite (ONOO-), excessive superoxide can damage the cardiovascular system
Reduced glutathione (GSH) – excessive oxidation causes glutathione depletion:
compromises antioxidant defences
Free dGTP (deoxyGTP) - oxidised; causes mutation if incorporated into DNA
NO: what is it, what does it do, and what does its depletion result in?
Nitric Oxide:
* Antioxidant function - reacts with superoxide to produce peroxynitrite (ONOO⁻)
* Important mediator and neurotransmitter - key role in cardiovascular system health
- Excessive superoxide can damage the cardiovascular system
- ONOO⁻ reacts with proteins
GSH: what is it, what does it do, and what does its depletion result in?
Glutathione (GSH)
It has a protective mechanism, but excessive oxidation causes glutathione depletion:
* Compromises antioxidant defences
* Compromises Phase II detoxification reactions
* Promotes cell death
Toxicants: what processes allow them to generate ROS?
- Redox cycling (intrinsic to toxicant)
- Phototoxicity
- Production by enzymes
- Mitochondrial electron transport chain
- Microsomal electron transport system
Redox cycling: how does it work, why does this produce a radical, and what is an example of a toxicant using this process?
Toxicant doesn’t have a very specific ‘target’, but modifications that occur in the body allow generation of ROS
- Electron donated to toxicant (eg by CYP450 trying to metabolise it) - generates a radical
- Radical donates an electron to O₂, generating superoxide and regenerating the original compound
- Forms a cycle (redox-cycling)
Paraquat – banned defoliant: causes lung fibrosis (enters cells via polyamine uptake system – enriched in the lung); also nephrotoxic, linked to Parkinson’s disease, hearing loss
Phototoxicity: how does it work, how does this produce ROS, and what are some examples of toxicants that do this process?
Toxicant does not interact with a “target molecule” - light converts it into an unstable form that can cause ROS generation, this returns to its ground state which can then be excited by light again and produce more ROS
- Chlorpromazine (anti-psychotic) – hyper-pigmentation of exposed skin
- Hypericin – from St John’s Wort
(herbal remedy for low mood)
Therapeutic exploitation of phototoxicity - Photodynamic therapy: how does this work, what is the process, what uses have been approved, and what type of detoxification is preferred?
Uses phototoxicity to treat disease
* Skin lesions
* Some cancers
* Macular degeneration - intense interest in pathogen destruction
Activation of a photosensitiser drug by light creates ROS: selectively destroys unwanted cells or abnormal blood vessels - photosensitiser must have negligible dark toxicity; applied in the dark; ideally cleared rapidly from healthy tissue this is followed by carefully regulated light (wavelength, intensity, duration) applied to diseased
ALA (5-aminole vulinic acid); is approved for the treatment of cancers - Pro-drug metabolised to protoporphyrin IX
tissue
Unleashes toxic ROS; in tumours, Type 1 decay is preferable (does not need O₂)
Mitochondrial electron transport chain: how does it produce ROS naturally, how frequently does it form ROS, what mechanisms does it have in place to prevent ROS damage, what can mitochondrial damage result in, and why?
In ETC electrons can be donated to O₂ rather than the correct acceptor, producing superoxide
Normally ~2% of O2 is converted to O2-*
Contain antioxidant enzymes - SOD, GPX, PRX3
Mitochondrial damage (especially complexes I and III) increases ROS production
Many iron-rich proteins in mitochondria: oxidative damage releases Fe²⁺ (Fenton reaction - HO* produced)
Mitochondrial damage: what are some compounds that cause it and what do they do?
- Rotenone (plant-derived insecticide) inhibits Complex I - causes Parkinson’s disease in rats, the long-term use in humans correlates with PD
- Barbiturates, antipsychotics (eg haloperidol, chlorpromazine), and some local anaesthetics also inhibit complex I
- Cadmium (heavy metal) inhibits complex III and can displace Fe²⁺ from proteins, promoting Fenton reaction
Microsomal electron transport system: what is it, where is it mostly found, what does it contain, what does it do, and how may this be manipulated by toxicants to produce excess ROS?
Fragments of the ER
Liver (enriched in tissues exposed to xenobiotics)
Consists of a cytochrome P450, flavoprotein reductase + 2 other subunits (function unclear)
Mono-oxygenates compounds – important in Phase I metabolism:
* Uses NADPH as a source of electrons
* Undergoes a reaction of NADPH with O₂, H⁺, and xenobiotics to form an oxidised xenobiotic along with NADPH⁺ and H₂O
- CYP450s are sometimes poorly coupled (‘leaky’) - electrons donated directly to oxygen, releasing O₂*⁻
- “Leakiness” varies: may depend on the substrate
- Xenobiotics may induce CYP450 expression, can further increase O₂*⁻ release
CYP2E1: what is it, what does it do, what may it result in, and what research has been done into this?
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CYP2E1 is a highly inducible CYP450 which generates a lot of superoxide - a very leaky cytochrome
CYP2E1 mainly converts ethanol (~10% normally, up to ~40% in high [blood alcohol]) to acetaldehyde, but due to ‘leakiness’ generates a lot of ROS
- 10-20-fold increase in CYP2E1 activity in ethanol-fed rats; activity markedly increased by moderate ethanol consumption in humans
- CYP450-mediated superoxide production is strongly implicated in alcoholic liver disease (70-80% of UK alcohol-related deaths)
- SOD1 knockout mouse, moderate ethanol consumption causes liver necrosis and inflammation; rodents over-expressing SOD are protected against ethanol-induced liver injury
- CYP2E1 knockout mouse has less oxidative DNA damage than wild-type following ethanol consumption
