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PED3011 Toxicology > Oxidative Stress > Flashcards

Oxidative Stress Flashcards

1
Q

Describe an unstressed cell

A

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

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2
Q

Describe Ca2+ levels in the cell

A

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

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3
Q

How are intracellular Ca2+ levels kept low

A

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

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4
Q

Why are Ca2+ levels kept relatively low in the cell

A

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

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5
Q

What are different examples of cellular stress

A

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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6
Q

How are ROS formed

A

Molecular oxygen has space in orbital shell
Can accept e-

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7
Q

What are some ROS that can be formed

A

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

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8
Q

What is the most and least reactive ROS

A

Most: hydroxyl radical
Least: hydrogen peroxide

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9
Q

Why are ROS important in metabolic processes

A

Vital reactive intermediate

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10
Q

Why are ROS important in physiological control and signalling

A

Nitric oxide - mediator of vasodilation
H2O2 = cell signalling compound

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11
Q

Why are ROS important in defence against g invading pathogens

A

Macrophages use NAPDH oxidase to flood phagosomes with ROS

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12
Q

Where do ROS originate

A

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

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13
Q

What is oxidative stress b

A

Imbalance between cellular production of ROS and ability to detox them

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14
Q

How are ROS generated in the mitochondria

A

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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15
Q

What complexes in the electron chain can leak e-

A

1 and 2 into matrix
3 into both directions

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16
Q

How are the ROS produced from mitochondria detoxified

A

By enzymes that convert radical to H2O2

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17
Q

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

A

Highly oxygen dense organelle

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18
Q

What enzyme deals with H2O2 conversion

A

Glutathione peroxidase

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19
Q

What reaction does glutathione peroxidase mediate

A

H2O2 -> 2x H202

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20
Q

What is the role of SOD

A

Catalyse the reduction of superoxide radicals -> H2O2

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21
Q

Describe the SOD reaction

A

Slow but spontaneous

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22
Q

What is the role of glutathione reductase

A

Maintains the supply of reduced glutathione

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23
Q

Describe the Fentons reactions

A

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

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24
Q

How are Fenton reactions prevented

A

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

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25
Q

What is iNOS

A

The inducible isoform of nitric oxide synthase

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26
Q

What is nNOS

A

Constitutively expressed nitric oxide synthase
Neurons and nerve cells

27
Q

What is eNOS

A

Constitutively expressed nitric oxide synthase
Endothelial cells

28
Q

What switches on iNOS

A

Inflammatory cytokines e.g IL-1, TNFα

29
Q

How is nitric oxide synthesised

A

L-arginine + O2 + Nitric Oxide Synthase = NO

30
Q

What is the function of NO

A

Protect liver from ischaemic damaged
Potent vasodilator

31
Q

What mediates the toxic effects of NO

A

Peroxynitrate (ONOO-)

32
Q

When is NO a key messenger molecule

A

At low concs

33
Q

How is ONOO- formed

A

.NO- + .O2- —> ONOO-

34
Q

Describe the role of peroxynitrate

A

Oxidation of protein thiols, Fe containing proteins, RNA, DNA and lipid peroxidatio

Can induce necrosis and apoptosis

35
Q

What is the effect of the destruction of ceruloplasmin by peroxynitrate

A

Cu2+ release -> fentons -> lipid peroxidation

36
Q

What are the types of DNA damage caused by oxidative stress

A

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

37
Q

Why is 8OHdG seen in the urine after DNA damage

A

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

38
Q

Describe the reaction of hydroxyl radical with DNA

A

Radical binds to guanine
= formation of 8OH double blond

39
Q

What groups are susceptible to oxidative protein

A

SH (thiols)

40
Q

What proteins with thiol groups are affected by oxidative damage

A

Stress proteins, cytokines, antioxidant enzymes and txn molecules

41
Q

What happens to oxidised proteins

A

Scavenged by heat shock proteins

42
Q

What is the role of chaperones in oxidative protein damage

A

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

43
Q

What happens to p53 in healthy cell conditions

A

Bound to mdm2
= Marked for deletion by proteasome

44
Q

What happens to p53 under oxidative conditions

A

SH groups in mdm2 are modified
Releases p53 = no degradation

45
Q

Describe lipid peroxidation

A

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

46
Q

What happens to peroxyl radicals after being formed

A

Rearrange via a cyclisation to endoperoxides
= formation of malondialdehyde

47
Q

What happens if a peroxyl radical reacts with lipids

A

Pyroxylin radical formed

48
Q

What is the result of malondialdehyde (MDA)

A

Damage to other lipids and DNA
Form DNA adducts

49
Q

What protects against oxidative stress

A

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

50
Q

Describe superoxide dismutase (SOD)

A

Metallo enzyme = contain one of the metals

51
Q

What type of SOD is inducible

A

MnSOD in mitochondria
Induced by oxidative stress

52
Q

Describe hydrogen peroxide

A

Not very reactive but can form ROS

53
Q

What can remove hydrogen peroxide

A

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

54
Q

Describe GSH

A

Tripeptide
Reacts w electrophiles directly or under GST

55
Q

What happens GSH conjugates

A

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

56
Q

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

A

central sulfhydryl
Reacts with ROS

57
Q

What are the pivotal roles of GSH

A

Maintain metals in a redox inactive state
Protect against oxidative stress

58
Q

How can you measure oxidative stress

A

Direct methods
Measurements of biological effects of ROS

59
Q

What is a direct way of measuring oxidative stress

A

Redox detecting fluorescent probes
- cells fluoresce when encounter ROS

60
Q

What biological effects of ROS can you measure

A

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

61
Q

Describe the DCFDA assay

A

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

62
Q

Describe the ROS-Glo assay

A

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

63
Q

What is a more sensitive method: luminescence or fluorescence

A

Luminescence

PED3011 Toxicology (11 decks)

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