Ch17 Mitochondria

What happens if IM permeability is increased by xenobiotics?

Membrane potential will collapse due to rapid backflow of protons across membrane

What happens if ETC is directly inhibited?

Energy supply for driving proton pumps is impaired

List the possible sites of xenobiotic attack in mitochondria

• blocking citrate cycle (no NADH production)

• inhibition of substrate uptake (no available fuel)

• inhbition of fatty acyl β-oxidation (no acetyl-CoA produced and fatty acids accumulate)

• blocking ETC

• specific inhibition of ATP synthetase activity

What is PCP?

Pentachlorophenol (PCP) is a weak acid and therefore dissociated at physiological pH

• also a protonophore (transports photons)

Describe PCP toxicity (uncoupling of oxidative phosphorylation)

1. Penetrates permeable OM and reach intermembrane space, where it is protonated due to high proton density

2. This uncharged lipophilic molecule can cross the IM, where it is readily dissociated (loses proton) due to inside negative potential

3. Negatively charged PCP anion is then expelled back to intermembrane space, and cycle repeates: proton gradient between matrix and intermembrane space is lost

4. Results in uncoupling of oxidative phosphorylation which leads to increased O2 consumption and increased energy loss as heat

What is NADH

Major reducing equivalent and is used to provide electrons to maintain proton pump function to reduce O2 and water, and to synthesise ATP from ADP and Pi

What is β-oxidation?

Fatty acids are progressively shortened into acetyl-CoA products

As enzymes in β-oxidation are in the matrix, where do fatty acids must cross?

To the IM

How do short-, medium- and long-fatty acids cross membrane?

• Short and medium = easily cross membrane

• Long = transport system

What is carnitine?

Shuttling molecule that transports long-chain fatty acids across membrane and reactivates them by binding again to CoA

What does β-oxidation produce?

Acetyl-CoA and NADH

What are the two mechanisms in VPA toxicity?

1. VPA and VPA metabolites decrease free CoA cellular levels, CoA depletion = major cause for β-oxidation inhibition

2. Direct inhibition of enzymes involved in β-oxidation

• Metabolite of VPA (4-ene VPA) is activated by CoA and which itself undergoes first step of β-oxidation

Consequences of impairment of NADH production (β-oxidation impairment)

• Lack of NADH

• Lack of electron transport to oxygen

• Because β-oxidation of fatty acids = primary source of energy in some organs in fasting state (lack of glycogen), sustained β-oxidation inhibition → severe energy crisis

• Fatty acid accumulation → microvesicular steatosis

Which xenobiotic acts as a pseudosubstrate for citrate cycle?

Fluoroacetate

What is fluoroacetate?

Analog of acetate

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Why does fluoroacetate acting as a pseudosubstrate considered a lethal synthesis?

Organism synthesis substrate analogs that eventually block the metabolism and irreversibly damage the organism

Brief normal ETC process and ROS generaton

1. NADH and FADH₂ donate electrons

2. Protons transported to create proton gradient

3. O₂ reduction

4. ATP and ROS production

What are the two types of ETC inhibitors?

1. Block electron transport by binding to one of the components of ETC eg rotenone

• Complex I inhibition; ubiquinone-like compounds

2. Stimulate electron flow through initial parts of ETC, but which at one site or another divert electron flow from its normal path by accepting elctrons themselves

What are the two ways complex I inhibition can be brought about?

1. Non-specific inhibitors (eg compounds that inhibit dehydrogenases)

2. Specific inhibitors that bind to a subunit of a complex eg rotenone

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Which molecules are responsible for cell death?

• Cytochrome c

• Smac/DIABLO

• AIF

• EndoG

Describe MOMP induction mechanism

1. Induced by members of Bcl-2 protein family → recruits Bak and Bax

2. Bak and Bax translocate into OM, forming protein aggregates and eventually megapores

3. Death proteins located in IM can escape through pores

4. IM integrity remains fully intact, so mitochondria continues to maintain proton gradient across IM and to couple respiration with ATP production

5. Apoptosis

Describe mPT pore opening mechanism

1. Composed of proteins including VDAC, ANT, cyclophilin-D

2. Complex associated with Bax and Bcl-2 also

3. Complex forms pore that is normally closed

4. Pore regulates Ca2+ by periodically and transiently ridding mitochondria of excess metabolites and ions, including Ca2+

5. Accessory protein Bax binds to pore proteins and further increases permeability of opened pore

Which molecules inhibits mPT pore opening?

• Cyclosporin A

• Ro-68-4300

• Bongkrekic acid

• L-deprenyl

Consequences of rapid pore opening

• IM is depolarised and mitochondrial membrane potenital (proton gradient) collapses. So oxidative phosphorylation is uncoupled → no ATP production

• Intramitochondrial solutes release (Ca2+)

• Mitochondria swell due to rapid water influx

• Initiated energy crisis

• Necrosis

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What can protect against cholestasis-induced liver damage?

Oral administration of another bile acid, ursodeoxycholic acid (UDCA)

• Prevents Bax recruitment from cytosol and inhibits Bax from being associated with megapore

List reasons why mtDNA is highly susceptible to oxidation by ROS?

• Close to large amounts of ROS

• Almost no nonsense sequences

• No protective histones

• Less effective DNA repair

Describe the mechanism of Doxorubicin toxicity

• Directly oxidises and damages mtDNA

• Accumulation of oxidised DNA bases could be causally linked with irreversible cardiomyopathy, as expression of mitochondrial genome is crucial to integrity of respiratory chain

Why may oxidative stress continue long after discontinuation of doxorubicin?

Newly synthesised dysfunctional mitochondria from oxidised DNA bases may continue to produce high levels of ROS → continuous source of oxidative damage to mtDNA

List the possible consequences of mtDNA damage

1. Gradually induce symptoms very similar to those associated with diseases that are based on genetic defects in mtDNA

• Eg certain antiviral nucleoside analogs that damage mtDNA decrease expression of critical genes required to sustain oxidative phosphorylation → injury in organs with high energy demands

2. mtDNA replication is independent of nuclear DNA synthesis, mitochondrial proliferation can occur at any tiem of cell cycle

• Thus xenobiotics that interfere with mtDNA function can compromise the function of rapidly and slowly dividing cells

Why can toxicity exhibit a delayed onset after chronic exposure to xenobiotic?

• Effects clinically manifest after a lag time as long as severela months

• Cells harbour hundreds/thousands of mitochondria (threshold effect for toxicity)

  • Large number of mtDNA needs to be damaged before there is a reduction in overall energy supply

Describe the mechanism of tacrine-induced mitochondrial toxicity and liver dysfunction (acute; uncoupled oxidative phosphorylation)

1. Positively charged tacrine is transported across IM to matrix, where the cationic drug accumulates in negatively charged environment

2. Due to protonophoretic activity of tacrine, mitochondrial electron transport becomes uncoupled from oxidative phosphorylation

• Tacrine = uncoupler at high concentrations

3. Acute rather than delayed toxicity

Describe the mechanism of tacrine-induced mitochondrial toxicity and liver dysfunction (delayed)

1. Tacrine can inhibit DNA topoisomerases

2. mtDNA replication inhibitions and ultimately cell injury

3. Prevents resealing of DNA nick ends

4. Unwinds supercoiled mtDNA → relaxed circular mtDNA and nhbits mitochondrial replication

5. mtDNA slowly but gradually depleted

6. Double-strand breaks cause certain “genotoxic stress” → activation of p53, Bax, and other pro-apoptotic factors

7. Depending on remaining amounts of ATP in hepatocyte, cell ultimately undergoes apoptosis/necrosis

Why does doxorubicin selectively damages myocardium?

1. Heart needs high and continuous ATP supply for its uninterrupted function and is thus extremely vulnerable

2. Heart has low antioxidant capacity → vulnerable to oxidative stress

• Eg GS-peroxidase and catalase activity in heart muscle = very low

3. Doxorubicin can alter cardiac-specific transcription

What can doxorubicin alter in terms of intracellular Ca2+ in heart?

Indirectly alters protein kinase C-mediated regulation of intracellular Ca2+ and downregulate expression of several nuclear genes encoding enzymes in energy production

• Includes heart- and muscle-specific isoform of adeneine nucleotide transporter (ANT) as well as components of ETC