3. Oxidative Stress

Question 1

Name examples of diseases involving cellular damage caused by oxidative stress.

Answer 1

CVD, COPD, Alzheimer’s, rheumatoid arthritis, Crohn’s disease, multiple sclerosis, Parkinson’s, pancreatitis, cancer, ischaemia/reperfusion injury

Question 2

What is a free radical?

Answer 2

Any atom. molecule or ion that contains 1 or more unpaired electrons (usually move in pairs within an orbital) and is capable of independent (“free”) existence.

Question 3

How do free radicals cause cellular damage?

Answer 3

• Are very reactive within cells and tend to “steal” electrons from other molecules. causing damage (e.g. to protein, lipid or DNA).
• Reaction can also generate a second radical - propagates damage.

Question 4

Explain how Reactive Oxygen Species are produced by the ETC.

Answer 4

• Molecular oxygen = biradical - has 2 unpaired electrons in different orbitals (so stable).
• In the mitochondrial ETC, the final destination for an e- is an O2 molecule (which is then combined with protons to produce water).
  > But 0.1-2% of e- don’t reach end of chain and prematurely reduce O2 to form SUPEROXIDE (O2•-) - important source of other ROS.
  > Superoxide + 2H+ and e- = HYDROGEN PEROXIDE (H2O2) - not a free radical but can react with Fe2+ to produce free radicals.
  > Hydrogen peroxide can catalyse formation of HYDROXYL RADICAL (OH•), eg. from glutathione - most reactive and damaging free radical.

Question 5

Describe the formation of Reactive Nitrogen Species.

Answer 5

Superoxide can react with signalling molecule NITRIC OXIDE (NO•) to produce PEROXYNITRITE (ONOO-) - not a free radical but powerful oxidant that can damage cells.

Question 6

Name different sources of biological oxidants.

Answer 6

Endogenous:

1. ETC
2. Nitric oxide synthases
3. NADPH oxidases

Exogenous:

1. Ionising radiation (UV light, X-rays, cosmic rays)
2. Pollutants
3. Drugs (eg. Primaquine anti-malarial)
4. Toxins (eg. Paraquat herbicide)

Question 7

Describe the enzymatic production of nitric oxide.

Answer 7

Oxidation of L-arginine by nitric oxide synthase (requries NADPH) to produce citrulline + NO•

3 types of NOS:

• iNOS - inducible NOS; produces high NO concentrations in phagocytes for direct toxic effect
• eNOS - endothelial NOS; signalling
• nNOS - neuronal NOS; signalling

Question 8

Describe the roles of NO• as a signalling molecule.

Answer 8

1. vasodilation
2. neurotransmission
3. s-nitrosylation

Question 9

Explain the 2 ways ROS causes damage to DNA.

Answer 9

1. ROS reacts with nitrogenous base… modified base can lead to mispairing and mutation.
2. ROS reacts with ribose/deoxyribose sugar… can cause strand break and mutation on repair.

Question 10

How can one measure the amount of oxidative damage in a cell?

Answer 10

Amount of 8-oxo-dG present in cells (form of deoxyguanosine oxidised by ROS).

Question 11

Why is mtDNA particularly sensitive to ROS damage?

Answer 11

1. mtDNA is situated near inner MT membrane where ROS are formed.
2. Unlike nuclear DNA, mtDNA is not protected by histones.

Question 12

Explain how ROS damage proteins?

Answer 12

1. React with protein backbone… fragmentation… protein degradation.
2. React with protein side chain… modification of amino acid… chain in protein structure… protein degradation, loss of function or gain of function.

Question 13

Describe an example of amino acid modification caused by ROS.

Answer 13

ROS sequesters e- from cysteine residue… formation of disulphide bond between thiol groups of cysteine residues… protein misfolding, cross-linking and/or function disruption.

Question 14

Explain how ROS damage lipids.

Answer 14

Via lipid peroxidation:

1. Free radical (eg. OH•) sequesters hydrogen atom from a polyunsaturated fatty acid in lipid membrane… forms a lipid radical.
2. Lipid radical can react with O2… forms a lipid peroxide radical.
3. Chain reaction is formed as lipid peroxyl radicals extract hydrogen from nearby fatty acids.
4. Hydrophobic environment of bilayer is disrupted and membrane integrity fails.

Question 15

In which condition does lipid peroxidation by ROS play an important part?

Answer 15

atherosclerosis

Question 16

Under which conditions is ROS production beneficial for health?

Answer 16

During respiratory burst = stimulated phagocytes (eg neutrophils and monocytes) rapidly release ROS to destroy invading bacterial/fungal cells (phagocyte is usually also destroyed).

Question 17

How are ROS produced during the respiratory burst?

Answer 17

NADPH oxidase in phagosome membrane transfers e- from NADPH to O2… produces superoxide…

i) reacts with NO• (from iNOS) to produce peroxynitrite (ONOO-)
ii) reacts with Cl- via myeloperoxidase (from secretory granules) to form hyperchlorite (HOCl•)

Question 18

What does a genetic defect in NADPH oxidase complex cause?

Answer 18

Chronic granulomatous disease: enhanced susceptibility to bacterial infections

• atypical infections
• pneumonia
• abscesses
• impetigo
• cellulitis

Question 19

Name 3 types of cellular defences against oxidative stress.

Answer 19

1. superoxide dismutase and catalase
2. glutathione
3. free radical scavengers

Question 20

What is the function of superoxide dismutase?

Answer 20

Converts superoxide to hydrogen peroxide and oxygen.
Primary defence because superoxide is strong initiator of chain reactions.

3 SOD isoenzymes (cytosolic, extracellular and mitochondrial)

Question 21

What is the function of catalase?

Answer 21

Converts hydrogen peroxide to water and oxygen.

Widespread enzyme important in immune cells to protect against oxidative burst.

Question 22

Suggest a theory for the greying of hair with age.

Answer 22

Declining levels of catalase in hair follicles.

Question 23

What is the function of glutathione?

Answer 23

Is a tripeptide containing cysteine.
1- Cysteine thiol group of reduced form (GSH) donates e- to ROS and reacts with another GSH to form a disulphide bond - oxidised form (GSSG). Reaction catalysed by glutathione peroxidase.

2- GSSG reduced back to GSH by glutathione reductase which catalyses transfer of e- from NADH to disulphide bond.

Question 24

Why is the pentose phosphate pathway essential for protection against free radical damage?

Answer 24

Provides the NADPH required to reduce GSSG back to GSH - ie recycling it.

Question 25

Why is selenium an essential dietary trace element?

Answer 25

Acts as co-factor for glutathione peroxidase.

Question 26

Explain the role of free radical scavengers. Give examples.

Answer 26

Reduce ROS damage by donating a hydrogen atom (and its electron) to free radicals in a non-enzymatic reaction. - Vitamin E (alpha-tocopherol) = lipid-soluble antioxidant important for protection of lipid bilayer against lipid peroxidation. - Vitamin C (ascorbic acid) = water-soluble antioxidant important in regenerating reduced form of vitamin E. - Also inc. carotenoids, flavonoids, uric acid and melatonin.