Lecture 17: ROS & Oxidative Stress

Question 1

What ROS do mitochondria produce, and under what conditions is O2- production increased?

Answer 1

In cpx III, semiquinone at the Q0 site delivers one single e- to O2 to produce superoxide, which is injected into IMS.
Cpx I also generates superoxide, released into matrix. The membs aren’t permeable to this anionic O2-.
O2 prod is inc’d when: High memb pot’l, high NADH/NAD+ ratio, ETC dmg, hypoxia, xenobiotics (ex MPTP, rotenone block e- flow thru cpx I)

Question 2

ROS can also be generated by non-mitochondrial oxidases. Name them.

Answer 2

NADPH oxidases (Nox) expressed in phagocytes to produce superoxide for killing pathogens.  Also produced in cardiomyocytes --possible role in ROS-mediated cellular signaling.
XO and MAO use molecular O2 as an oxidant and produce H2O2.

Question 3

H2O2 can be converted to the highly reactive hydroxyl radical via what rxn? Describe it.

Answer 3

Fenton reaction: Transfers an e- to H2O2 from Fe2+, which is converted to ferric iron. Ferrous iron can be recycled back by ferric reacting with O2-.

Question 4

What is the primary RNOS produced by the cell? How’s it generated? What can it react with to form something that’s much more of a reactive oxidant?

Answer 4

Nitric oxide, generated by nitric oxide synthase in the metabolism of Arg to citrulline. This can react with O2- to form peroxynitrite (ONOO-), and ONOO- can also give rise to the hydroxyl radical.

Question 5

Describe the irreversible dmg caused by the hydroxyl radical in regard to DNA dmg.

Answer 5

DNA is a favored target of the hydroxyl radical gen’d by the Fenton rxn because nucleic acids bind iron well. A common oxidative lesion is the formation of 8-oxodGuo, an indicator for the extent of DNA dmg in the cell. DNA dmg is the first step in mutagenesis and carcinogenesis.

Question 6

Describe the irreversible dmg caused by the hydroxyl radical in regard to lipid peroxidation.

Answer 6

Polyunsat’d acyl chains of phospholipids or PUFAs are highly susceptible to peroxidation. Initiation & propagation occurs. Lipid peroxidation introduces a charged peroxide group in the acyl chain of phospholipids and causes memb dmg. Furthermore, the non-enz bkdn of lipid perox products produces highly reactive aldehydes such as MDA and 4-HNE; biomarkers for lipid perox.

Question 7

Describe the irreversible dmg caused by the hydroxyl radical with regard to protein carbonylation.

Answer 7

Hydroxyl radicals can directly oxidize aa side chains causing protein dmg. More importantly, they mediate proteins carbonylation - the addn of a reactive carbonyl functional group on proteins. 4-HNE (lipid perox product) is one of the most reactive and common carbonyl groups and reacts with certain residues. 4-HNE can diffuse across membs, allowing the reactive aldehyde-containing lipids to covalently modify proteins.

Question 8

H2O2 is damaging mainly due to the hydroxyl radical formed by Fenton chemistry, but what can it oxidize in order to form disulfide crosslinks with other cysteines?

Answer 8

Thiol groups in proteins (cysteinyl residues). This can lead to loss of activity and protein aggregation.

Question 9

What enzyme destroys superoxide?

Answer 9

Superoxide dismutase converts 2 molecules of it into one molecule of O2 and one H2O2. There is a cytosolic (Cu,Zn-Sod1, mutations of which can cause ALS) and mitochondrial (Mn-Sod2, confined to matrix) enzyme.

Question 10

Name the three different enzymes that decompose H2O2 to water and describe how they carry out their function.

Answer 10

Glutathione peroxidase consumes 2 molecules of GSH in converting H2O2 to water. GSSG is reduced back to GSH by glutathione reductase, an NADPH-dependent enzyme.
Peroxiredoxin reduces H2O2 in mito and erythrocytes, itself being reduced by thioredoxin. The oxidized thioredoxin is recycled thru the action of the NADPH-dep enz thioredoxin reductase.
Catalase in peroxisomes uses one molecule H2O2 to reduce another. The AR disorder yielding low levels of catalase is acatalasia.

Question 11

Describe the roles of non-enzymatic antioxidants.

First of all, name them.

Answer 11

Scavenging free radicals and protection of proteins against sulfhydryl oxidation.
Glutathione - Can directly scavenge free radicals like the hydroxyl radical, ultimately generating superoxide.  More imp, GSH keeps sulfhydryls of proteins reduced to maintain their biologic activity (We want a high GSH/GSSG ratio).
Coenzyme Q10 (ubiquinone) - believed to scavenge RO2. radicals and inhibit lipid peroxidation.

Question 12

What physiological roles do ROS/RNOS play?

Answer 12

Moderate levels required for redox signaling that regulates cell growth, diffn, and apoptosis. They also regulate cell processes such as innate immunity, anti-cancer, body wt control and wound healing.

Question 13

Antioxidants can protect healthy people from cancer, but how do they promote growth or pre-initiated tumor cells?

Answer 13

Robust antioxidant protection could desensitize tumor cells to ROS-dependent senescence and apoptosis during cancer therapy.

Question 14

What generates the primary ROS, superoxide?

Answer 14

Metabolism - The mitochondria in the ETC.

Question 15

There are 3 different mechanisms by which cells defend against oxidative stress. What are they?

Answer 15

1. A conversion of superoxide into H2O2
2. H2O2 converted to H2O
3. If #2 fails cells can scavenge (via Vit C, E, GSH, etc) the free radicals (donate a free e- to make radical not reactive)