Oxidative stress Flashcards
the un stress cell contains (4)
- High levels of reduced glutathioone
- Reduced thiols on protein
- Anti-oxidants and enzyme available to protect cells
- Oxidatively damaged DNA bases are repaired
What are the levels of Mg2+ and Ca2+ in a normal cell
- Calcium with cell is low
- Mg2+ conc are almost equivalent
What does magnesium do in the cell
forms a complex of ATP and avoid over hydrolysis of ATP
what is the problems with Ca2+ in the body
- Ca2+ precipitates phosphate, which is a key modulator of enzyme activity
(Bone and teeth made of this calcium phosphate mineral) (Phosphate is needed for many different pathways) - Ca2+ is itself a key modulator of molecular shape and charge (Co-ordinated with side chains)
how is calcium stored in the body
transported out of the cell/sequestered in intracellular stores such as ER and SR
Ca2+ binds to calmodulim (calcium modulated protein)
True/False - calcium is locally acting
True
what can cause cellular stress
elevated temp, hypoxia, starvation and toxins (ROS, heavy metals, Alcohols), UV radiation
how doe ROS normally generate and what are the most common
molecular oxygen can accept electrons to give ROS
O2-,O-, OH.
what is H2O2 hydrogen peroxide
much less reactive than other ROS but still a problem because of its participation in Fenton’s reactions - still problems if high conc
how to macrophages use ROS to fight pathogens
macrophages use NAPDH oxidase to flood phagosomes with ROS to destroy engulfed pathogenic microorganisms
how does ROS metabolise exogenous compounds
act as intermediates
how do ROS originate
- Mitocondrial respiratory chain (1-3% of oxygen molecule in mitocondiria are in the form H2O2)
- Redox of xenobiotics (Xenobiotics have a range of abilty in redox generators)
- Redox active metal ions (Metals able to exist in mutiple redox states)
- UV radiation
how does mitocondrial genome damage occur
- Mitochondrial oxidative phosphorylation complexes generate the proton motive force (move protons from the matrix to the intermembrane space).
- protons then pass through complex V (ATP synthase) to generate ATP
- however these complexes are ‘electron leaky’
- high energy electrons that are used in oxidative phosphorylation to generate the PMF, rather than remaining in the membrane, can leak into the matrix (or in case of complex III) also into the intermembrane space.
- Here they can combine with oxygen to generate superoxide. Because the mitochondrion is a highly oxygen dense organelle, this reaction occurs at a higher level.
- Superoxide dismutase converts the free radical to hydrogen peroxide which creates a short term solution
how does the body deal with small with small amounts of ROS
scavenged and metabolsied/inactivated by eith antioxidants (from diet), Vit C/E, selenium and carotenoids
how are ROS linked to diseases
neurodegenerative = Alzheimers, cancer
how does NRF2 interact with ROS
activated by oxidative stress to leave cellular partner and go to nucleus and encourage read through (check)
what occurs when there is iron present with oxidative stress
Fentons reaction = H202 production
Relies on good supply of glutathione
H2O2 and O2- (superoxide radical) are toxic because
they lead to the production of hydroxy; radical which is extremely reactive
how is free iron level kept low
ferritin
what occurs in arthritis sufferers
elevated levels of superoxide appaers to release iron from ferritin in the synovial fluid
what is nitric oxide ‘tied up?’ with
Calcium
what are the positive uses of nitric oxide
protects liver for ischemic damage
potent vasodilator
what are the 3 chronic inflammatory conditions linked to nitric oxide
nNOS = Constitutively expressed in neurons, nerve cells & type 2 skeletal muscle.
iNOS = inducible isoform, expressed in immune & cardiovascular systems
eNOS = constitutively expressed in endothelial
what is switched on by inflammatory cytokines IL-1 & TNFa
iNOS
How do Nitric oxide turn toxic
Reacts with O2- to produce peroxynitrite (ONOO-) which is a potent cytotoxic oxidising agent
what does peroxynitrite do in the body
Oxidation of protein thiols; iron-containing proteins, RNA, DNA & lipid peroxidation
Can induce necrosis and apoptosis
Shown to destroy ceruloplasmin
(Transport protein; destruction leads to copper ion release>fentons>Lipid peroxidation)
How does thiol oxidation effects Ca2+ homeostasis
Oxidation of thiols by ROS in calcium ion pump proteins & Ca++ ATPases in plasma membrane = increased intracellular Ca++
How does an increase in intracellular calcium effect the cell
Stimulation of lipases; proteases; caspases
Increased intracellular oxidative stress - Oxidation of reduced thiols
Altered ATP production leads to changes in cytoskeleton and cell blebbing = leading to cell death by apoptosis
Generation of ROS causes (5) and how to these overall effect the cell
Oxidative damage to DNA
Oxidative damage to protein
Oxidative damage lipids
Depletion of reduced glutathione
Loss of cellular homeostasis
= decrease in membrane integrity
How does oxidative damage effect DNA
Oxidation of base especially guanosine
(8OH deoxyguanosine (8OHdG) in urine is an important marker)
C8-OH radical binds to guanosine
how does oxidative damage effects protein
- Misfolding of protien (Protein no longer works and is identified by scavenger protein and tagged for destruction)
= Or protein can gain different function and do someting unknown
(SH (thiols) groups particularly susceptible =Damage leads to aberrant folding)
= Proteins affected include stress proteins, cytokines, antioxidant enzymes, transcription molecules
= Oxidised proteins are scavenged by heat shock proteins (HSP)
= Chaperones: assist in non-covalent folding of proteins, prevent and reverse aggregation, assist in ubiquitination (“tagging” of proteins for degradation by…)
= Proteasomes (multi-catalytic proteases) or autophagy (DNA binding transcription factors eg AP-1, p53 are controlled by the redox state of cysteinyl thiols)
how does oxidative damage to proteins effect p53 functioon
P53 under “healthy cell” conditions is bound to mdm2, a ubiquitin ligase.
This “marks” p53 for deletion by the proteasome.
Under oxidative conditions, the SH groups in mdm2 are modified releasing p53, which means it is no longer degraded
how does lipid peroxidation from oxidative damage to lipids effect the cell
lipid peroxidation is catalysed by iron
Results in the formation of peroxyl radicals (ROO.) These rearrange via a cyclisation reaction to endoperoxides, leading to formation of malondialdehyde (MDA). MDA can react with DNA bases to form DNA adducts (role in cancer aetiology)
How does Superoxide dismutase protect against oxidative stress
Metallo enzymes (Cu, Zn, Mn, Fe) reacts with SODs creating MnSOD in mitochondria inducible by oxidative stress. 2 molecules superoxide give hydrogen peroxide plus molecular oxygen controls level of superoxide (but H2O2 can give rise to ROS)
How else, bar antioxidants and MnSOD and cytosolic SOD2, does the body protect against oxidative stress
Low intracellular Fe conc is maintained, Glutathione peroxidase and glutathione reductase are presence. Also catalase which rapidly removes hydrogen peroxide peroxisomes
Tripeptide glutamyl-cysteinyl-glycine (Glutathione) reacts with _ directly or under _ catalysis
electrophiles, GST
how are glutathione (GSH) conjugates sorted by -
hydroxylsed to S-substituted cysteines followed by acetylation to give mercapturic acids that are excreted
how does the body use glutathione balance to protect against oxidative stress
- glutathione (GSH)/ glutathione disulphide (oxidised form)(GSSG) ratio kept high
-GSH is depleted by conjugation
-depleted by transport of GSSG from cell - GSH compartmentalisation in nucleus and cytosol
what occurs when the body is exposed to heavy metals or excess paracetamol
glutathione is drained and the ratio is thrown off
how is oxidative stress measured
direct = redox detecting fluorescent probes
Biological effects of ROS = GSH depletion, oxidative DNA damage, lipid peroxidation
how does the DCFDA cellular ROS detection assay work
Supply of methyl diester making the compound lipophilic.
- The diacete form (H2DCFDA) and its acetomethyl ester (H2DCFDA-AM) are taken up by cells where nonspecifc cellular esterases act upon it to cleave off the lipophilic groups, resulting in a charged compound believed to be trapped inside the cell
- Oxidation of H2DCF by ROS converts the molecule to 2’, 7’ dichlorodihydrofluorescein (DCF), which is highly fluorescent
- The reported wavelengths for the measurement of DCF fluorescence are 498 nm for excitation and 522 nm for emission
how does the ROS-Glo assay
- Claimed to be highly specific for hydrogen peroxide (DCFDA isn’t)
- Compound reacts with H2O2 with produced luciferin precursor
- Then detection solution is added to create luciferin with emits light
