Oxidative Stress Flashcards by Diana Bespalova

Describe an unstressed cell

High levels of reduced GSH
Reduced thiols on proteins
Anti-oxidants and enzymes available
Oxidatively damaged DNA bases are repaired

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Describe Ca2+ levels in the cell

Within cell is low (0.1nM)
Outside relatively high (1.3mM)

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How are intracellular Ca2+ levels kept low

Transported out of the cell via ATPase
Sequestered in intracellular stores in ER and SR bound to calmodulin

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Why are Ca2+ levels kept relatively low in the cell

To prevent formation of insoluble precipitate
-> decreases phosphate levels that switch on and off enzymes

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What are different examples of cellular stress

Elevated temp = proteins become less stable
Hypoxia
Starvation = no substrates for ATP synthesis
Toxins: redox active and contribute to stress
UV radiation

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How are ROS formed

Molecular oxygen has space in orbital shell
Can accept e-

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What are some ROS that can be formed

O2-. (Superoxide anion)
O. (O2 free radical)
OH. (Hydroxyl radical)
H2O2 (Hydrogen peroxide)

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What is the most and least reactive ROS

Most: hydroxyl radical
Least: hydrogen peroxide

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Why are ROS important in metabolic processes

Vital reactive intermediate

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Why are ROS important in physiological control and signalling

Nitric oxide - mediator of vasodilation
H2O2 = cell signalling compound

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Why are ROS important in defence against g invading pathogens

Macrophages use NAPDH oxidase to flood phagosomes with ROS

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Where do ROS originate

Mitochondria respiratory chain
Redox of xenobiotics
Redox active metal ions
UV radiation

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What is oxidative stress b

Imbalance between cellular production of ROS and ability to detox them

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How are ROS generated in the mitochondria

Protons pass through leaky ATP synthase complex V
E- leak into matrix or intermembrane space
Combine with oxygen to form superoxide

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What complexes in the electron chain can leak e-

1 and 2 into matrix
3 into both directions

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How are the ROS produced from mitochondria detoxified

By enzymes that convert radical to H2O2

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Why does ROS production happen at a higher level in the mitochondria

Highly oxygen dense organelle

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What enzyme deals with H2O2 conversion

Glutathione peroxidase

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What reaction does glutathione peroxidase mediate

H2O2 -> 2x H202

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What is the role of SOD

Catalyse the reduction of superoxide radicals -> H2O2

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Describe the SOD reaction

Slow but spontaneous

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What is the role of glutathione reductase

Maintains the supply of reduced glutathione

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Describe the Fentons reactions

.O2- + Fe3+ —> O2 + Fe2+
H2O2 + Fe2+ —> .OH + OH- + Fe3+

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How are Fenton reactions prevented

Free iron levels kept very low
H2O2 + O2- levels are rapidly removed

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What is iNOS

The inducible isoform of nitric oxide synthase

What is nNOS

Constitutively expressed nitric oxide synthase Neurons and nerve cells

What is eNOS

Constitutively expressed nitric oxide synthase Endothelial cells

What switches on iNOS

Inflammatory cytokines e.g IL-1, TNFα

How is nitric oxide synthesised

L-arginine + O2 + Nitric Oxide Synthase = NO

What is the function of NO

Protect liver from ischaemic damaged Potent vasodilator

What mediates the toxic effects of NO

Peroxynitrate (ONOO-)

When is NO a key messenger molecule

At low concs

How is ONOO- formed

.NO- + .O2- —> ONOO-

Describe the role of peroxynitrate

Oxidation of protein thiols, Fe containing proteins, RNA, DNA and lipid peroxidatio Can induce necrosis and apoptosis

What is the effect of the destruction of ceruloplasmin by peroxynitrate

Cu2+ release -> fentons -> lipid peroxidation

What are the types of DNA damage caused by oxidative stress

Oxidative DNA damage Oxidation of bases Marker 8OH deoxyguanosine in urine

Why is 8OHdG seen in the urine after DNA damage

Damaged base is excised by cellular repair mechanism Replaced by unoxidised form = seen in urine

Describe the reaction of hydroxyl radical with DNA

Radical binds to guanine = formation of 8OH double blond

What groups are susceptible to oxidative protein

SH (thiols)

What proteins with thiol groups are affected by oxidative damage

Stress proteins, cytokines, antioxidant enzymes and txn molecules

What happens to oxidised proteins

Scavenged by heat shock proteins

What is the role of chaperones in oxidative protein damage

Assist in non-covalent folding of proteins Prevent and reverse aggregation Assist in ubiquitination

What happens to p53 in healthy cell conditions

Bound to mdm2 = Marked for deletion by proteasome

What happens to p53 under oxidative conditions

SH groups in mdm2 are modified Releases p53 = no degradation

Describe lipid peroxidation

Catalysed by Fe3+ Results in formation of peroxyl radicals (ROO.)

What happens to peroxyl radicals after being formed

Rearrange via a cyclisation to endoperoxides = formation of malondialdehyde

What happens if a peroxyl radical reacts with lipids

Pyroxylin radical formed

What is the result of malondialdehyde (MDA)

Damage to other lipids and DNA Form DNA adducts

What protects against oxidative stress

Antioxidants/GSH Superoxide disumutase Low intracellular [Fe2+] maintained GSH peroxidase GSH reductase Catalase

Describe superoxide dismutase (SOD)

Metallo enzyme = contain one of the metals

What type of SOD is inducible

MnSOD in mitochondria Induced by oxidative stress

Describe hydrogen peroxide

Not very reactive but can form ROS

What can remove hydrogen peroxide

Catalase (Has a high turnover) Glutathione peroxidase (slower and requires reduced GSH)

Describe GSH

Tripeptide Reacts w electrophiles directly or under GST

What happens GSH conjugates

Hydrolysed to S-substituted cysteines Followed by acetylation = mercaputric acid -> excretion

What is the key element of GSH and what is its role

central sulfhydryl Reacts with ROS

What are the pivotal roles of GSH

Maintain metals in a redox inactive state Protect against oxidative stress

How can you measure oxidative stress

Direct methods Measurements of biological effects of ROS

What is a direct way of measuring oxidative stress

Redox detecting fluorescent probes - cells fluoresce when encounter ROS

What biological effects of ROS can you measure

GSH depletion (measure ratio of GSH/GSSG) Measure DNA damage Look for lipid peroxidation

Describe the DCFDA assay

H2DCFDA and methyl ester taken up into cell Esterases cleave -> polar charged compounds Trapped inside cell Oxidation of H2DCF by ROS -> DCF DCF is highly fluorescent = fluoresce

Describe the ROS-Glo assay

Highly specific for hydrogen peroxidase Reacts with H2O2 = precursor for luciferen Add detection solution = luciferan -> luciferase = luminescence

What is a more sensitive method: luminescence or fluorescence

Luminescence