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1. Define oxidative stress.
2. What is such an unstable cellular environment linked to?
3. Name 4 ROS that are free radicals. Name 2 ROS that are nonradicals
4. Same for NOS (2, 2)
1. Oxidative stress: imbalance b/t pro-oxidants and anti-oxidants in favor of the pro-oxidant leading to cell injury or death. Anti-oxidants can't neutralize stress.
2. Linked to tissue damage, acceleration of aging, and degenerative disease
3. ROS (free radicals) .O2- (superoxide), .OH (hydroxyl), .RO2- (peroxyl), and .HO2- (hydroxperoxyl); hydrogen peroxide (H2O2) and hypochlorous acid (HOCl)
4. .NO (nitric oxide), .NO2- (nitrigen dioxide)
Also peroxynitrate OONO- and nitrous oxide (HNO2)
1. What rxn forms free radical?
2. Define free radical
3. What can a free radical lead to?
4. What do antioxidants do?
5. Can free radicals be produced by cells under normal conditions?!??!?!
6. How much of inhaled O2 metabolized yield to ROS?
7. Which is the most reactive ROS?
- 1 Redox by nonradical
- 2. Free radical: highly chemically reactive ion; molecule/fragment of a molecule with at least one unpaired electron
3. Can lead to chain rxn forming lots more free radicals
- 4. Donate electrons, neutralizing free radical
- 5 Yes.
- 6. 5%
- 7. Hydroxyl radical (.OH)
1. What enzyme catalyzes formation of superoxide? What is the substrate?
2. What enzyme catalyzes formation of H2O2 from superoxide?
3. What enzyme catalyzes rn from RNH2 --> nitric oxide?
- 1. NADPH oxidase
- 2. SOD
- 3. NOS
1. What are the main endogenous sources we need to know? 5 (very broadly)
2. What are examples of exogenous sources? (5)
1. ETC, respiratory burst, cytochrome p450, hydroxyl radical from fenton rxn, amino acids like arginine
2. UV/light radiation, pollution, smoking, foods, and toxins
1. What is a major source of free radicals in the cell?
2. Describe how ETC contributes to ROS. What is the resulting ROS?
3. Describe specific places (3)
4. Can TCA contribute to free radical formation?
1. ROS are products of normal oxidative metabolism --> major source of free radicals in the cell
2. Leaky mitochondrial ETC: flow of electrons from NADH and succinate through series of electron carriers is leaky, resulting in superoxide anion
3. Iron-sulfur clusters of complex I, coenzyme Q, and a-ketoglutarate dehydrogenase
4. yes look above.
1. Describe how superoxide radical is turned into H2O2
2. What are possible cofactors? (5) two of them go together.
3. Where is it located? (for two of them)
1. Via superoxide dismutase!!!! (HUGELY IMPORTANT ANTIOXIDANT ENZYME), reduces .O2- to H2O2 in attempts to neutralize it.
2. Cu & Zn, Mn, Ni, or Fe
3. Mn-SOD (SOD2) is located inside mitochondria, while Cn/Zn-SOD (SOD1) is located on the outer mitochondrial membrane
1. In what cells does respiratory burst occur in?
2. What is the purpose of respiratory burst? What does it lead to?
3. What enzyme does it require? **
4. What happens to the bad things produced?
5. What is chronic granulomatous disease?*
6. What are commerical uses of this? 1
- 1. Phagocytic cells (i.e., macrophages & PMNs)
- 2. These phagocytic cells need to generate ROS (superoxide radical and H2o2) to kill bacteria
- 3. NADPH oxidase OR myeloperoxidase(MPO) which converts H2O2 (made from superoxide anion) into HYPOCHLORIC ACID and water.
- 4. Superoxide radial (.O2-) is converted into H2O2 using SOD 3 (EC Cu/Zn SOD).
- 5. Defect in NADPH oxidase!!!! that leads to failure of phagocytic cells to kill organisms they have engulfed b/c they CAN'T produce free radicals
6. HOCl- is used to disinfect swimming pools
1. How does cytochrome p450 create ROS? Which one is created? What does it have to react /w first?
2. How is hydroxyl radical created in human body? What is it responsible for?
2.5 What are normal functions of NO? (5)
3. How is .NO produced? (enzyme, what is NO, what are the 3 types of enzymes involved?
4. Which enzyme is highly regulated? What cofactors are needed (5)
1. By reacting with oxygen to create superoxide radical. Xenobiotic agents can be metaebolized to radical species (i.e., aflatoxin & other foods)
2. Fenton rxn. SIGNIFICANT PART OF DAMAGE ASSOCIATED w/ OXIDATIVE STRESS
2.5 Vascular tone, insulin secretion, airway tone, cardiac function, and angiogenesis
3. By nitric oxide synthase (nNOS - neuronal, iNOS - inducible, and eNOS - endothelial) NO is a signaling molcule
4. NOS and requires 5 co-factors (FAD, FMN, heme bound O2, NADPH, and BH4).
1. What bombards the pig's house? (N:3, 3)
2. What reinforces the pig's house? (3)
1. .NO, OONO, NO2- AND superoxide radical from ETC, burst NADPH oxidase --> supueroxide, and xenobiotics from cytochrome p450
2. SOD --> H2O2, MPO: H2O2 --> HOCl-, and H2O2 Fe2+ --> superoxide radical
1. Describe exogenous sources of prooxidants in further detail:
- Why is cigarette smoke bad for you (3)?
- What are metals in the air that are contained in air pollutants by coal burning? (2)
- What are two other important air pollutants? (2)
- What essential metals are oxidants in foods? (2)
- B/c it contains oxidants
, inducing NOS, xanthine oxidase, ONOO- synthesis
- - Zn, As (also Cd, Pd) and other organic oxidants contained in fly ash
- - SO2 (sulfur dioxide) and Ozone (O3)
1. What are the effects of oxidative stress on the cell? (5)
2. Where does radical damage occur in? (4) - give teensy explanation for each one
1. Damage to DNA, proteins, lipids; disruption in cellular homeostasis, accumulation of damaged molecules
2. Unsaturated bonds in lipids (found in all cell membranes --> loss of membrane fluidity, problems w/ receptor alignment/signaling and potentially cellular lysis)
Sulfur containing enzymes (inactivation, cross-linking, denaturation
DNA --> mutations (cancer)
1. How are thiols (i.e., glutathione or cysteine) affected by free radicals? Draw rxn w/o compounds
2. HOw do essential ions react w/ ROS to form bad stuff? Draw rxn.
3. How about nitric oxide?
4. What are clinical uses of NO
1. GSH (glutathione) + ROS/RNS --> GSSG (Glutathione disulfide) + ROH/RNH.
2. For example iron. Fe2+ + H2O2 --> Fe3+ + .OH + -OH
- 3. .NO + .O2- (superoxide) --> OONO-
- 4. Viagra
1. What is specifically targeted in the damage to lipid membranes?
2. What is so bad about this?! !!
3. What else can lipid radicals interact w/?
4. What is MDA? What is it used for?
5. What are additional important products? 2
1. Lipid peroxidation on PUFAs
2. Lipid peroxidation is a radical-initiated chain rxn that is self-propagating!!!
3. Can also interact w/ things like DNA and proteins
4. MDA!!!!! is a marker of oxidative stress. It is the product of pathologic lipid oxidation found in rancid food.
5. 4-hydroxynonenal (marks lipid damage!!!) and 8-isoprostane
1. Which amino acids are most susceptible to oxidation by ROS/RNS? (3)
2. How do they affect each one?
1. Cysteine, methionine, and tryptophan
2. SH groups of cysteine residues are essential for structural/catalytical functions. ROS/RNS oxidize -SH groups to form inactive disulfides
- Methionine residue oxidation yields methionine-sulfoxide impairing the function of proteins
3. Tryptophan residues are readily oxidized impairing protein structure & function
BASICALLY FOR ALL OF THEM, THE RESIDUES ARE OXIDIZED AND THIS IMPAIRS STRUCTURE & FUNCTION.
What are the 2 ways that proteins are affected? What does this ultimately cause??***
Describe the second one. What is it linked to?
What affects this second one? (2)
- 1. Amino acid modification via oxidation
- 2. Protein carbonylation triggers degration of proteins
2. Direct ROS/RNS attack OR metal-catalyzed oxidative attack on amino acid side chains
of lysine, proline, arginine, and threonine TRIGGERS DEGRADATION OF PROTEINS!!!!
Linked to diseases such as Parkinson's, Alzheimer's, cancer, cataracts, diabetes etc.
Aging increases this rate and caloric restriction decreases this rate.
How is DNA affected?
Describe each in detail
- 1. Oxidative damage to DNA
- 2. Formation of DNA adducts
1. Oxidative damage to DNA - reaction of DNA w/ hydroxyl radical (.OH-)
1. Which ROS normally reacts with DNA? (1) Which ones usually don't (2)
2. What does ROS react with?
3. What is the most important rxn? What is the most important product?
3.5 What does this do?
4. What is the final product of the above?
1. (.OH-), superoxide radical and H2O2 generally are unreative towards DNA!
2. All 5 (deoxy)nucleotides (AGCTU)
3. Interaction with the purine G, which generates 8-hydroxy-deoxyguanosine (8OHdG)
3.5 8OHdG mispairs with adenine (usually thymine does this)
4. This causes single or double-strand breaks because it's a basic site in the DNA strand.
1. How else do oxidants affect DNA?
2. Describe mechanism? What can it bind to (3)
3. What does it form?
1. By covalently binding to DNA forming DNA adducts
2. DNA can covalently bidn to oxidants or products of oxidative degradation (MDA or 4-HNE) to form DNA adducts
3. DNA adducts.
A DNA adduct is a piece of DNA covalently bonded to a (cancer-causing) chemical.
1. What is oxidative stress linked to in terms of dsiease? (2)
2. Describe mechanism (probs not important);
1. Metabolic disease & diabetes; along w/ parkinson's/alzheimer's from protein carbonylation --> disease)
2. Obesity increases NADPH oxidase and decreases antioxidant enzyme activity leading to ROS that lead to oxidative stress in remote tissues + dysregulation of adipokines --> systemic inflammation --> endothelial cell dysfunction --> metabolic syndrome (insulin resistance, diabetes, and atherosclerosis)
Oxidative stress & T2D:
1. What stimulus leads to production of ROS? (2)
2. What do they activate? ** What happens next?
3. Which are the two signaling molecules activated by ROS? What do they specifically do? (2)
4. What are the effects of these signaling molecules?
5. What happens after this?
1. Hyperglycemia (affects redox balance thru polyolpathway in ETC decreasing levels of NADPH & glutathione) and increased circulating FFAs (increases oxidative stress via B-oxidation phosphorylation in mitochondria)
2. They activate stress-related kinases that activate pro-inflammatory cytokines
3. NFkB and Ap-1 activated by ROS will upregulate immune response and inhibit IRS-1 blocking insulin signaling
4. Blunts insulin signaling through dysregulation of insulin receptor substrate 1 (IRS1)
5. Ensuing inflammation leads to further production of oxidative stress, tipping balance towards inflammatory pathways.
1. What are 3 main ways to measure markers of oxidative stress?
1. Measuring products of rxns of ROS/RNS with endogenous compounds, direct ROS/RNS levels, or total antioxidant capacity.
Describe which products of ROS/RNS oxidation w/ endogenous compounds you'd study (7) which are the 5 most important ones?
- 1. GSH:GSSG ratio - GSSG is bad.
- 2. Nitrotyrosine (product of rxn of ONOO- w/ proteins)
- 3. MDA, 4HNE or 8-isopranes: products of lipid peroxidation
- 4. Lipid peroxidases
- 5. Ethane, pentane (gaseous products of lipid peroxidatoin)
- 6. DNA adducts (from DNA isolated in blood)
- 7. Oxidized nucleotides (8OHdG)
How would you directly measure ROS or RNS levels? 4)
- 1. Peroxidases - very unstable
- 2. Fe2+
- 3. Nitric oxide
- 4. Free radicals (ELISA)
How would you measure total antioxidant capacity?
What are the two groups of antioxidants we'll discuss?
Using plasma, serum, urine, saliva or cell lysates to provide general info. NOTHING SPECIFIC.
It relies on the ability of antioxidants in a sample to inhibit oxidation of a provided substrate by an added oxidant.
Low MW antioxidants and antioxidant enzymes
1. What are the low molecular weight antioxidants? (very broadly) - 5
2. What are the 3 major ones?
3. What ist he major IC and EC antioxidant?
1. GSH, Vitamin E, vitamin C, carotenoids, and flavinoids
2. the first 3
1. Describe mechanism of GSH. What is the end product?
2. Why is GSH so effective? (2)
3. How do GSH levels indicate oxidative stress?
4. In healthy tissues/cells, how much of total glutathione pool is GSH?
- 1. GSH's cysteine's thiol group can donate reducing equivalent (H+ +e-) to unstbale molecules (ROS/RNS).
- 2. By donating, glutathione becomes reactive and readily pairs up with another reactive glutathione to form glutathionedisulfide (GSSG)
2. (1) There is relatively high glutathione levels in the cell - up to 5 mM in liver and (2) GSH can be regenerated from GSSG by the enzyme glutathione reductase
3. if GSSG: GSH ratio is high
4. 90% (only 10% is GSSG)
1. What is another name for vit E?
2. What are its main two functions? (2) Where? (1)
3. What does it require for reactivation?
4. At phys concentrations, what is the strongest reductant/radical scavenger?
5. Where is the above found? What does it form (2) when interacting w/ vit E?
6. What does the above form when it reacts directly w/ ROS or RNS?
7. How does it reform?
- 1. a-tocopherol
- 2. To donate electrons to free radicals thereby stabilizing them and preventing lipid peroxidation chain rxn in the CELL MEMBRANE
- 3. Vit C
- 4. Vitamin C
- 5. Aq solutions. It forms DHA and H2O2 and reduced a-tocopherol
- 6. When it reacts w/ thigns like H2O2, it forms 2H2O and DHA.
- 7. Using glutathione ,which is then reactivated using glutathione reductase.
1. What are the two types of low MW antioxidants that are found mainly in foods that haven't been mentioned?
2. Where are they found?
1. Carotenoids (B-carotene lutein, lycopene); lipid-soluble pigments found in plants and act as radical scavengers in membranes
2. Flavinoids - polyphenolic compound in fruits & veggies
Name the 7 antioxidant enzymes
Genes encoding these enzymes have what element in their sequence?
What is the most important transcription factor for regulating the above?
1. Glutathione peroxidase (Gpx), thioredoxin reductase (TR), superoxide dismutase (SOD1-3), Catalase, Bilirubin, and Heme oxygenase and biliverdin reductase
2. Antioxidant response element (ARE)
3. Nrf2 is most important for regulating ARE
1. How many Gpxs are there?
2. What do they contain that's so important?
3. What is main function of Gpx? Draw rxn
4. Draw it in action
5. What does it require? 2
- 1. 5 (1-4,6)
- 2. Se
- 3. To regenerate glutathione
- 5. It requires riboflavin (B2) and NAPDH
1. What are the 3 Thiroedoxin REductases? What do they contain?
2. Describe mechanism
3. Why is maintenace of TR in its reduced form important? (2)
4. What are other substrates for TRs? (3)
5. Draw mech. Is it water-sol?
- 1. TR1-3. Se
- 2. TRs donates reducing equivalents through its disulfides in thioredoxin. These reducing equivalents are from NADPH
- 3. B/c it's important for cell growth & viability
- 4. Selenite (SeO32-), Vit C, and possibly vit E
1. What does catalase depend on?
2. What is its substrate? What does it convert it to?
3. Where is it found? (2)
4. In which organisms is it expressed in?
5. What are the two major enzymes in antioxidant defense?
- 1. Heme(fe)
- 2. H2O2 --> H2O + O2
- 3. Found in blood plasma & peroxisomes of all living cells
- 4. All organisms exposed to oxygen
- 5. SOD and catalase
1. Where is bilirubin found?
2. What is it from?
3. What it's green form?
4. What is it better at scavenging peroxyl radicals than?
- 1. In serum
- 2. End products of metabolism
- 3. Biliverdin
- 4. vit E
1. What does heme oxygenase do? Where is it found? What is its mechanism?
2. What does biliverdin reductase (BR) do?
3. Where does this rxn occur?
4. What is a classic example of this?
5. Where is activity of HO highest?
6. How is this related to jaundice?
1. HO is a membrane-boudn enzyme that oxidizes heme to release CO, Fe2+, and biliverdin
2. BR reduces biliverdin to a potentially toxic & insoluble bilirubin
3. Almost every cell
4. Bruising: red(heme) --> green (biliverdin) --> yellow (bilirubin)
5. In the spleen where old RBCs are sequestered and destroyed
6. Excess RBC lysis ---> bilirubin (yellow color)
1. How is superoxide radical eliminated? (2)
2. How about hydrogen peroxide (4)
3. How about hydroxyl radicals? (4)
4. Carbon-centered peroxyl, hydroperoxyl, and lipid peroxides? 6
- 1. By converting it into something else via vitamin C or SOD (Cu, Zn, Mn)
- 2. Vitamin C interacts w/ H2O2 readily, or GSH/GPx, Catalase (CAT), or MPO, neutrophils.
- 3. Vitamin C, uric acid, thiols (i.e., glutahtione), E is less effective
- 4. Vitamin E, carotenoids, Mn, Vitamin C, glutathione, Gpx