6. Oxidative Stress

what is oxidative stress?

the imbalance between pro- and antioxidants in the cell, leading to accumulation of oxidative damage

what is a free radical?

any molecular species capable of independent existence that contains one or more unpaired valance electrons not contributing to intramolecular bonding

what do free radicals have?

an unpaired electron

what do free radicals do?

abstract one electron from target (oxidation). this produces a new free radical (chain reaction). free radicals are highly reactive

What are ROS?

oxygen-centered free radicals plus molecules that contribute to their formation (and are stong oxidants themselves)

what is SOD?

enzyme that alternately catalyses the dismutation of the superoxide radical into ordinary molecular oxygen and hydrogen peroxide

What is GPX?

Cytosolic enzyme that catalyses reduction of hydrogen peroxide to water and oxygen as well as catalysing the reduction of peroxide redicals to alcohols and oxygen

What is PRX?

ubiquitous family of antioxidant enzymes that also control cytokine-induced peroxide levels and thereby mediate signal transduction in mammalian cells

What is TRX?

small ubiquitous redox protein that plays key role in redox signalling and oxidative stress responses

What is superoxide like?

Highly reactive; half life is concentration dependent and it is soluble in water so cannot cross membrane

Where is superoxide generated?

Mitochondria

What can superoxide combine with to form peroxynitrate?

Nitric oxide free radial (RNS)

What is hydrogen peroxide like?

Low reactivity, half life seconds to minutes and is soluble in water and lipids so acts as messenger between reactive oxygen species that can transfer the action from one place to another

what is hydroxyl radical like?

Very highly reactive, half life is u seconds and is soluble in water. Reacts with first molecule it hits - can’t really travel anywhere due to short half life

How are superoxides detoxified?

Carried out by SOD enzyme. Transfers superoxide radical into hydrogen peroxide

Why is the formation of peroxynitrate toxic?

Damages DNA and causes necrosis (oxidative stress)

1 Detoxifying hydrogen peroxide

Direct catalytic reaction with water catalysed by catalase enzyme

2 Detoxifying hydrogen peroxide

Oxidation of glutathione into glutathione disulphide catalysed by glutathione peroxidases

How does the detoxification of hydrogen peroxide occur?

2 possible pathways. Reactivity measured by half life. Lost glutathione has to be regenerated. Done by oxidation and reduction cycles (NADPH)

What are antioxidants?

Cell’s primary defence against ROS

What are the types of antioxidants?

Enzymatic; non-enzymatic ‘scavengers’ and metal chelators

What are the enzymatic antioxidants?

SODs, catalase, GPXs, TRXs

What are the lipid soluble non-enzymatic scavengers?

VitE, Carotenoids, Q10

What are the water soluble non-enzymatic scavengers?

VitC, GSH, uric acid

What do non-enzymatic antioxidants do?

Terminate chain reactions by reacting with oxidised products.

What are scavengers?

Non-enzymatic antioxidants

What do metal chelators do?

Bind divalent cations reducing risk of fenton reaction - create highly reactive ROS

What SOD is found in the mitochondrial matrix?

Manganese SOD (SOD-2)

What SOD is found in the cytoplasm?

Copper zinc SOD

1 Removal of hydroxy radicals

Chelating bivalent cations (removal of toxic metal from body)

2 Removal of hydroxy radicals

Blocking the chain reaction from the peroxidation products using scavengers (non-enzymatic antioxidants)

What are the types of DNA damage in cells

SS breaks (2000 per hr) or ds breaks (10 per day)

Depurination and depyramidation

Deamination

Glycation by lipid peroxidation products

What is the cells secondary defence to ROS?

DNA repair

What are the methods of DNA repair?

Base excision repair (BER); nucleotide excision repair (NER), mismatch repair, double strand break repair

What is base excision repair?

Cuts oxidised base out producing a single stranded break in DNA and repairs that - repairs primary base oxidations and single stranded breaks

What are the types of nucleotide excision repair?

Transcription coupled and genome wide

What are the types of double strand break repair?

Non-homologous end joining and homologous recombination

What is mismatch repair?

Important in cancer. Fixes damage not usually caused by oxidative stress

what is XPD?

helicase subunit of TF2H, functions in NER and transcription, mutated in trichodiodystrophy

What happens if you delete XPD?

Get short lived mice and shows signs of ageing at young ages e.g. baldness. Still goes through ageing process in the 3 weeks its alive

what is XPA?

Necessary for NER but not for transcription

What happens if you knockout XPA?

Mice continues to live, doesn’t show premature ageing.

What are the cellular secondary defences?

Protein turnover by the chaperone-ubiquitin-protease system and protein turnover in the lysosme

what is macroautophagy?

autophagosomes deliver cytoplasmic constituents to endosomes or lysosomes

what is microautophagy?

lytic organelles take up cytoplasm directly

what is chaperone mediated autophagy (CAM)?

Lysosomal pathway of proteolysis

How is oxidative stress linked to ageing?

It accumulates with ageing and shortens telomeres

How can mitochondrial dysfunction shorten lifespan without ROS increase?

polG mutation. Polymerase gamma - responsible for DNA replication and proof reading in mitochondria only. Can have mitochondrial induced premature ageing but without oxidative stress

what is hormesis?

describes any process in a cell or organism that exhibits a biphasic response to exposure to increasing amounts of a substance or condition

What is lipofuscin?

Non-degradable material that accumulates in lysosome over time.

What happens if lysosome membrane becomes leakyy?

Contents can spill into cytoplasm. Affects pH of cytoplasm. Hydrolases can damage cytoplasm

What is the free radical theory of ageing?

Damage from FRs increases with age. Manipulations that increase lifespan decrease damage. Decreasing oxidative stress slows ageing

What is the effect of oxidative stress on telomeres?

Shortens them. Healthy individuals have slower telomere shortening. Vascular dementia = telomeres shorten quicker

What happens to cells kept under hyperoxia?

Increased rate of telomere shortening

What happens to cells kept under antioxidant pBN?

Decreased rate of telomere shortening

What is the current theory on mitochondria and ROS related ageing?

Decreased Mt function and increased oxidative damage is associated with ageing. There is reduced oxidative damage with age in long-lived mutants

Oxidative stress and cells possibility 1

ROS oxidises sulfhydryl (SH) groups in protein

Causes cross linking and disulphide bond formation

Protein becomes damaged and non-functional

Oxidative stress and cells possibility 2

ROS can function as an important signal transducer

SH group in cysteine of peroxiredoxin protein becomes oxidised into sulfinic acid

Changes structure of protein & gives it a new function

Instead of a peroxidase it becomes a chaperone (this can be reversed by sulfiredoxin)

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What do chaperones do?

Protect proteins from oxidative damage by helping misfolded proteins refold correctly

Oxidative stress and cells possibility 3

ROS can signal in response to damage

If proteins are damaged chaperones will be activated & influence the proteasome = death

However it can also trigger hormetic responses (adaptive reactions). Can lead to senescence or rejuvenation

What is biphasic?

Low does gives beneficial effect whilst high dose gives toxic effect (2 phases)