Session 5: Oxidative Stress and Antioxidant Defence Mechanisms

Oxidation is the ___ of electrons and reduction is the ___ of electrons

Oxidation is the loss of electrons and reduction is the gain of electrons (OILRIG)

An oxidising agent (oxidant) ___ something else. A reducing agent (reductant) ___ something else

An oxidising agent (oxidant) oxidises something else. A reducing agent (reductant) reduces something else

What is oxidative stress

Condition in which the rate of generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) exceeds body’s ability to protect itself against them

What does oxidative stress result in?

Increase in oxidative damage to molecules (proteins, lipids and DNA)

Leading to cell/tissue damage

Oxygen can gain an electron to form ___

Oxygen can gain an electron to form superoxide

Superoxide can react with nitric oxide to form ___

Superoxide can react with nitric oxide to form peroxynitrite

___ radicals are considered the most reactive and damaging free radical as it reacts with anything

Hydroxyl radicals are considered the most reactive and damaging free radical as it reacts with anything

Hydrogen peroxide can react with ___ ions to produce free radicals

Hydrogen peroxide can react with Fe2+ ions to produce free radicals

A free radical is an atom, molecule or ion that contains one or more ___ electrons, capable of independent existence

A free radical is an atom, molecule or ion that contains one or more unpaired electrons, capable of independent existence

What are some examples of endogenous sources of biological oxidants?

Electron transport chain, nitric oxide synthases, NADPH oxidases

What are some examples of exogenous sources of biological oxidants?

Radiation (cosmic rays, x-rays, UV light), pollutants, drugs (primaquine, paracetamol), toxins (paraquat herbicide, polonium-210)

What are the two main types of ROS damage to DNA?

1) ROS reacts with a base = modified base can lead to mis-pairing & mutation

2) ROS reacts with a sugar = ribose/deoxyribose causing strand break & mutation

When ROS reacts with DNA and causes DNA damage - failure of repair of this damage can lead to mutations which cause ___

When ROS reacts with DNA and causes DNA damage - failure of repair of this damage can lead to mutations which cause cancer

Reaction of unsaturated lipids with ROS is termed ___ ___.

Reaction of unsaturated lipids with ROS is termed lipid peroxidation.

What does lipid peroxides do to cell membranes?

Damage

ROS damage to lipids is thought to occur in the early stages of ___ disease

ROS damage to lipids is thought to occur in the early stages of cardiovascular disease

___ is produced by the body to protect against oxidative damage.

Glutathione is produced to synthesised by the body to protect against oxidative damage.

What are Heinz bodies a clinical sign of?

Glucose-6-Phosphate Dehydrogenase (G6PDH) deficiency

Describe the production of superoxide radicals in the mitochondria

The superoxide radical is produced by adding an electron to molecular oxygen

Is hydrogen peroxide a free radical? How is it produced? Why is it bad to accumulate hydrogen peroxide?

Hydrogen peroxide is NOT a free radical but it can react with iron ions (Fe2+) to produce free radicals...

H2O2 + Fe2+ --> Hydroxyl free radical

What is the most damaging free radical?

Hydroxyl free radical (OH*)

How is nitrogen oxygen species (NOS) produced?

Superoxide radicals can react with other free radicals such as nitric oxide (NO*) to produce peroxynitrite (ONOO-)

Peroxynitrite is not a free radical but it is a powerful oxidant that can damage cells

Oxidative stress

a condition in which the production of oxidants (ROS and RNS) and free radicals exceeds the body's ability to handle them and prevent damage

Oxidative stress can damage molecules such as...

Proteins, lipids, DNA

Cellular damage by ROS and RNS is indicated in multiple diseases including...

- CVD

- Type 1 Diabetes (T1D)

- Rheumatoid Arthritis (RA)

- Crohn's disease

- COPD

- Ischaemia

- Cancer

- Pancreatitis

- Parkinson's disease (PD)

- Multiple sclerosis (MS)

What are some sources of endogenous biological oxidants?

Electron transport chain, nitric oxide synthases, NADPH oxidases

What are some sources of exogenous biological oxidants?

- Radiation (UV light, x-rays)

- Pollutants

- Drugs e.g., Primaquine (anti-malarial) and Paracetamol

- Toxins e.g., Paraquat (herbicide) and Polonium-210

What are the two forms of DNA damage by ROS and RNS?

1) Modified base (ROS + base) = leading to mispairing and mutation

2) Modified pentose sugar (deoxyribose or ribose) = leading to strand breakage and mutation as result of repair

How does protein damage occur via ROS and RNS?

- Side chains of amino acids or protein backbone can react with ROS

- Leads to modification of protein structure

- Loss of protein function

What is one of the most significant types of protein damage by ROS/RNS?

When ROS takes electron from a cysteine residue leading to formation of inappropriate DISULPHIDE bond

This leads to misfolding, cross-linking & disruption of protein function

An example of ROS damage to protein leading to inappropriate cross-linking and a clinical sign of G6PDH

Heinz bodies - precipitated haemoglobin within RBC altering their rigidity

How does lipid damage occur by ROS and RNS?

- ROS reacts with lipids in lipid peroxidation

- Lipid peroxides (lipid peroxyl radicals) formed

- Lipid peroxyl radicals react with nearby fatty acids & hydrophobic environment of bilayer disrupting its integrity

- Damage to cell membrane integrity

What is the name of the process which describes oxidative damage by ROS in lipids specifically?

Lipid peroxidation

What process of oxidative damage is significant in the aetiology of atherosclerosis (early CVD)?

Lipid peroxidation is significant in the aetiology of atherosclerosis

Outline the two antioxidant defense systems

1) Enzymatic defense systems = superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (cGPx)

2) Non-enzymatic defense system = glutathione (GSH)

Enzymatic defense system

Superoxide radicals are converted to hydrogen peroxide by superoxide dismutase enzyme

O* -> H2O2 [superoxide dismutase, SOD]

Hydrogen peroxide is converted to water and molecular oxygen by catalase enzyme

H2O2 -> H2O + O2 [catalase, CAT]

Glutathione (GSH)

Helps to prevent damage by reactive oxygen species generated in the course of metabolism

Which vitamins are the 'free radical scavengers'?

Vitamins A, C and E (ACE)

What trace elements are considered to be 'free radical scavengers'?

Melatonin, Selenium, Carotenoids, Polyphenols, Flavonoids

Free radical scavengers have a high ___ ___ absorbance capacity (ORAC)

Free radical scavengers have a high oxygen radical absorbance capacity (ORAC)

Free radical scavengers like vitamins A, C and E are thought to reduce the risk of...

Chronic degenerative disorders

How do free radical scavengers protect the cell?

By donating a H+ atom and its electron to free radicals in a non-enzymatic reaction

Respiratory burst

Some phagocytic immune cells (neutrophils & monocytes) release ROS & RNS when stimulated. This is part of the antimicrobial defense system.

Which phagocytic immune cells carry out respiratory burst?

Neutrophils and monocytes

What membrane-bound enzyme is involved in respiratory burst process?

NADPH oxidase

Where is NADPH oxidase found

In the membrane of phagosomes of phagocytic immune cells (neutrophils & monocytes)

What disorder is caused by a genetic defect in the NADPH oxidase enzyme?

Chronic granulomatous disease

What occurs in chronic granulomatous disease?

Enhanced susceptibility to bacterial infections...

Atypical infections, pneumonia, abscesses, impetigo, cellulitis

Give an example of a disorder which arises from oxidative damage to proteins in the cell?

Aggregation of cross-linked haemoglobin producing Heinz bodies - this can lead to haemolysis and anaemia

Vitamin E

Lipid-soluble antioxidant

Protection against lipid peroxidation

Vitamin C (ascorbic acid)

Water-soluble antioxidant

Important role in regenerating reduced form of Vitamin E

How is the Electron Transport Chain considered to be an endogenous source of ROS?

NADH and FADH2 donate electrons which pass through transport chain onto oxygen to form H2O.

Occasionally these electrons can escape the chain and react with dissolved oxygen to form superoxide radicals.

Superoxide radicals give rise to other ROS.

Free radical

Atom, molecule or ion that contains one or more unpaired electrons, capable of independent "free" existence

A ___ radical is any atom, molecule or ion that contains one or more unpaired ___. They are very reactive and they able to acquire ___ from other molecules causing cellular ___.

A free radical is any atom, molecule or ion that contains one or more unpaired electrons. They are very reactive and they able to acquire electrons from other molecules causing cellular damage.

Oxidative stress refers to a cellular state at which cells do NOT have a sufficient antioxidant power to cope with ROS

True or false?

True

What is ROS

Reactive oxygen species

Name the inclusions of the red blood cells which form as a result of oxidative damage to haemoglobin

Heinz bodies

What are the three key antioxidant vitamins?

A,C,E

Which one of the following is ROS?

A) All of the above except hydrogen

B) Hydrogen

C) Hydroxyl radical

D) Hydrogen peroxide

E) All of the above

F) Superoxide radical

A) All of the above except hydrogen

Which one of these cellular components is MOST affected by ROS?

A) Nucleus

B) Golgi

C) Endoplasmic reticulum

D) Mitochondria

D) Mitochondria

Which key enzyme catalyses the conversion of superoxide to hydrogen peroxide and oxygen

Superoxide dismutase

Oxidative stress is thought to be involved in many disease. Name one of them which is associated with deficiency of an enzyme. Involved in the metabolism of carbohydrates

Galactosaemia

People with G6PDH deficiency (reduced G6PDH activity) in the pentose phosphate pathway produce ___ GSH

Lower GSH

- This means less protection against oxidative damage from oxidative stress