MCP Lecture 8

Question 1

What are ROS? Give 3 examples

Answer 1

chemically reactive species formed upon incomplete reduction of oxygen - unpaired electron = free radicals

Superoxide (.O2-) = moderate activity
Hydrogen peroxide (H2O2) = low activity (oxidant - not free radical)
Hydroxyl Radical (.OH) = Highest reactivity

Question 2

Where are superoxides generated?

Answer 2

mitochondria (byproduct of ATP synthesis)
complex III: superoxide - injected into intermembrane space
complex I: exclusively released into the matrix

Question 3

Under what conditions is O2- production increased?

Answer 3

1. high membrane potential = slower transfer of electrons leading to a higher reduction level of electron carriers at the Q site and increases electron leak to O2
2. High NADH/NAD+ ratio: over reduction of the electron transport chain
3. Electron transport chain damage - alters electron flow accuracy
4. Xenobiotics: compounds can block electron transport which increases reduction level of electron carriers located upstream
5. electron backflow in complex I - over-accumulated succinate during ischemia rapidly oxidized by complex II - drives electrons back through complex I and increases superoxide production

Question 4

What is MPTP and what is an adverse effect?

Answer 4

an inducer of Parkinson’s disease - xenobiotic

blocks electron flow to the Q site and increases electron leak in complex I

Question 5

What is the Fenton reaction?

Answer 5

.O2- + H2O2 —> O2 + H2O + .OH
presence of iron is catalytic - converts hydrogen peroxide into hydroxyl radical and water (so overaccumulation of free iron causes oxidative stress)

Question 6

What does ionizing radiation cause?

Answer 6

homolytic fission of the O-O bond in H2O2

H2O2 —> 2 .OH

Question 7

How is nitric oxide generated in the cell?

Answer 7

nitric oxide synthase which metabolizes arginine to citrullin

Question 8

How is peroxynitrite formed?

Answer 8

reaction of NO. with superoxide to form peroxynitrate - very reactive (this can give rise to hydroxyl)

Question 9

What are the forms of oxidative damage?

Answer 9

1. DNA damage - nucleic acids bind iron well - target of the .OH from the fenton reaction
2. lipid peroxidation - produce lipid peroxide which causes membrane damage
3. hydroxyl radicals directly oxidize amino-acid side chains which cause protein damage

Question 10

What is used as an indicator for the extend of DNA damage in the cell? (used for diagnosis of Friedreich’s ataxia)

Answer 10

8-hydroxy-2’-deoxyguanosine - due to oxidative leasion

Question 11

Which FA are most susceptible to peroxidation?

Answer 11

arachidonic acid and linoleic acid due to hydrogens close to the double bonds being highly reactive and prone to lose electron to .OH

Question 12

What are the most reactive/common carbonyl groups generated from lipid peroxidation? Example? What does it react with?

Answer 12

aldehydes = 4-hydroxy-(2E)-nonenal which reacts with the side chain of cysteine, histidine, and lysine residues

Question 13

How can cells convert primary ROS (superoxide) into hydrogen peroxide? From hydrogen peroxide to water?

Answer 13

superoxide dismutase

catalase

Question 14

What is the mechanism of superoxide dismutase?

Answer 14

converts 2 molecules of superoxide into one molecule of O2 and one H2O2 (less toxic)

Question 15

What does the cytosolic SOD (superoxide dismutase) use as a ligand? Mutation of this enzyme causes what?

Answer 15

Cu and Zn

mutation in Sod1 causes amyotrophic lateral sclerosis (ALS/Lou Gehrig’s disease)

Question 16

What does mitochondrial SOD use as a ligand?

Answer 16

Mn

Question 17

What are the three different enzymes/pathways to decompose hydrogen peroxide?

Answer 17

1. glutathione peroxidase - H2O2 to water, consuming two molecules of reduced glutathione
2. peroxiredoxin pathway - has production of oxidized peroxiredoxin with a disulfide bond - must be reduced 2X more
3. catalase - heme containing - located in peroxisome

Question 18

What does deficiency in glucose-6 phosphate dehydrogenase cause? Why?

Answer 18

oxidative stress and hemolytic anemia because NADPH (created by PPP by glucose-6 phosphate dehydrogenase) is needed by glutathione reductase (to regenerate reduced glutathione for the glutathione peroxidase)

Question 19

What are non-enzymatic ROS defenses?

Answer 19

Anti-oxidant therapy:

• coenzyme Q10 = dietary supplemetn, scavenge RO2. radicals and inhibit lipid peroxidation
• glutathione = also co-factor for glutathione peroxidase - keeps sulfhydryls of proteins reduced and maintains their biological activity
• vitamin E = protects membrane lipids and lipoproteins
• vitamin C = reacts with wide spectrum of radicals
• plant phenols = inhibits LDL oxidation
• Flavonoids = from tea, fruit skins, veggies - reduce coronary artery diseases and stroke

Question 20

What are some physiological roles of ROS/RNOS?

Answer 20

moderate levels are require for redox signaling that regulate cell growth, differentiation, and apoptosis
-also: innate immunity, anti-cancer, body weight control, wound healing

Question 21

How is redox homeostasis maintained?

Answer 21

glutathione (GSH) and thioredoxin (TRX)

Question 22

How are ROS used in innate immunity?

Answer 22

neutrophils undergo respiratory burst - potent antimicrobial agents and also participate as redox signaling molecules (modulate transcription factors such as NF-kappa B which regulates expression of key cytokines and chemokines that further regulate the inflammatory response)

Question 23

How do ROS cause apoptosis?

Answer 23

catalyze cardiolipin peroxidation which facilitates the detachment of cytochrome c from mitochondrial - release into cytoplasm

Question 24

Why is lowering ROS by antioxidant therapy bad in cancer patients?

Answer 24

might stimulate growth of tumors by suppressing apoptosis (needs ROS)

Question 25

How do ROS control food intake and body weight control?

Answer 25

ROS is signal for activating POMC neurons to release anorexigenic hormones and decreasing AgRP’s activity in secreting orexigenic hormones

= Reduced food intake