Ch6 ROS Signalling

Oxidative Stress

Imbalance between ROS formation and antioxidant defence

Major Endogenus ROS Sources

• ETC

• CYP450

• Activated macrophages/PMNs

Major Exogenous Sources

Redox-cycling xenobiotics, UV

Redox Cycling

Repeated reduction/oxidation of a compound with repeated ROS generation

Redox Cycling consequences

↑ ROS, ↓ NAD(P)H, ↓ antioxidants

Superoxide Dismutase mechanism (SOD)

Superoxide → H₂O₂

Catalase mechanism

H₂O₂ → H₂O + O₂

Glutathione Peroxidase (GPX) mechanism

H₂O₂ + GSH → H₂O

GSH

Major intracellular antioxidant; can become depleted

Fenton Reaction mechanism

H₂O₂ + redox-active metal → •OH

Labile Iron Pool (LIP)

Labile iron can promote hydroxyl radical formation

Vitamin E

Lipid-phase antioxidant; prevents lipid radical propagation

Oxidative DNA Damage

Especially guanine; can cause mutations

Protein Oxidation mechanism

Loss of function → cross-linking → fragmentation → aggregates

Lipid Peroxidation mechanism

Chain reaction → membrane damage + toxic aldehydes

Nuclear factor erythroid 2-related factor 2 (Nrf2)

Activates antioxidant/cytoprotective genes via ARE

What does excessive oxidative stress lead to?

Apoptosis/necrosis

Redox Cycling mechanism

Xenobiotic accepts electrons → transfers electrons to O₂ → ROS → xenobiotic regenerated → cycle repeats

Antioxidant Pathway mechanism

O₂•⁻ → SOD → H₂O₂ → catalase/GPX → H₂O

Fenton reaction mechanism

H₂O₂ + transition metal → •OH

Mild Oxidative Stress cell fate

Physiological signalling

Moderate oxidative stress cell fate

Adaptation

Severe oxidative stress cell fate

Apoptosis/necrosis

Nrf2 pathway mechanism

Oxidative stress → Nrf2 → ARE → antioxidant/cytoprotective genes

Physiological ROS functions

• normal cellular signalling

• host defence

• regulation of gene expression

Which molecules are important cellular reductants that provide reducing equivalents in ROS signalling?

NADH and NADPH

Superoxide anion (O₂•⁻)

0.05s; important initial ROS

Hydrogen Peroxide (H₂O₂)

Minutes; relatively less reactive and can travel through cells which is why H₂O₂ is not a radical

Hydroxyl Radical (•OH)

10⁻⁹ s; extremely reactive

When does H₂O₂ become dangerous?

When it participates in the Fenton reaction → hydroxyl radical formation

How is ETC a source of ROS?

• During oxidative phosphorylation, e- are transferred through respiratory complexes

• Some e- can “leak” and prematurely reduce O₂: O₂ → O₂•⁻

• Therefore, mitochondria are both a major source and target of ROS

How are CYP450 enzymes a source of ROS?

• Normally substrate + O₂ + NADPH → CYP-mediated oxidation

• But CYP catalytic cycles can become uncoupled

  • Instead of productive substrate oxidation, e- are transferred to oxygen: O₂ → ROS

  • This is “uncoupling of substrate oxidation” and xenobiotics can increase this uncoupling

Consequence of xenobiotic interacting with CYP450 for ROS

Xenobiotic

→ CYP interaction

→ increased uncoupling

→ ↑ ROS

→ oxidative stress

How can the cell respond to uncoupling of substrate oxidation from CYPS?

• CYP degradation

• down-regulation of CYP expression

Which immune cells are activated to produce ROS as a means of inflammation?

• Macrophages

• Polymorphonuclear cells (PMNs)/neutrophils

Through which enzyme do the activated inflammatory cells produce ROS?

NADPH oxidase

NADPH oxidase inflammation mechanism

• NADPH → NADP⁺ and electron transfer to O₂: O₂ → O₂•⁻

• Inflammatory cells can also generate NO

• Thus, inflammation → ROS/RNS production → oxidative stress

What is the term to describe the compound transferring e- to moleculer oxygen repeatedly?

Electron shuttle

Where and how does benzene undergo bioactivation?

• liver by CYP

• bone marrow by myeloperoxidase (MPO)

What is a relevant metabolite once benzene undergoes bioactivation?

Benzoquinone - can be formed through CYP and MPO activity

How does NADPH quinone oxidoreductase (NQO) detoxify quinones

By reducing them further to hydroquinone

Why can H₂O₂ still be dangerous?

• relatively less reactive than •OH

• longer-lived

• able to cross membranes

• able to travel to other cellular locations

What are the two factors that can increase LIP?

1. Lysosomal damage

2. Strong reducing agents

GSH characteristics

• highly abundant

• contains a reactive sulfhydryl (-SH/thiol) group

• used as a cofactor/substrate by antioxidant enzymes

• important for detoxification of electrophiles and ROS

How are metallothioneins a protective system

• heavy metals bind/oxidise protein thiols

• heavy metals can deplete GSH

• rich in thiol groups

• metallothioneins bind heavy metals

• metal exposure can upregulate metallothionein expression

How is vitamin E an important lipid-phase antioxidant?

It’s hydrophobic:

• its tail sits in the membrane

• its head can react with lipid radicals

How can vitamin E be regenerated

With help from vitamin C and GSH

What happens when guanine is oxidised?

Incorrectly pair with adenine instead of cytosine

What can repair oxidised guanine?

Base excision repair (BER)

Why is mtDNA especially vulnerable?

• lacks histones

• contains few/no non-coding introns

• is close to ROS production

• has relatively inefficient repair mechanisms

What does mitochondria have that provides some redundancy?

• multiple copies of mtDNA

• multiple mitochondria per cell

What can ROS oxidise in terms of oxidative protein damage?

• cysteine residues

• methionine

• aromatic amino acids

Oxidative protein damage consequences

• protein-protein cross-links

• fragmentation

• denaturation

• oxidation of active centres

• loss of protein function

• toxic protein aggregates

Heat Shock Proteins (HSPs)

Help refold damaged/misfolded proteins

Proteasome

Helps degrade damaged proteins

Lipid peroxidation chain reaction mechanism

Lipid radical

↓

reacts with O₂

↓

peroxyl radical

↓

attacks another lipid

↓

new lipid radical

↓

chain reaction

What can lipid peroxidation generate?

Toxic aldehydes

Lipid peroxidation consequences

membrane disruption

altered membrane fluidity

altered membrane proteins

loss of membrane integrity

formation of secondary toxic aldehydes

Which eg of a molecule has a high affinity for lipids and can cause primary peroxidative stress?

Carbon tetrachloride (CCl₄)

Which signalling pathways can ROS activate?

• MAP kinases

• protein kinase C

• AP-1

• Nrf2

• NF-κB

What is NF-κB associated with?

• inflammatory signalling

• cytokine production

• cellular stress responses

What is AP-1 associated with?

• stress responses

• gene regulation

• cell survival/death signalling