Reactive Oxygen Species and Oxidative Stress Flashcards
How are ROS formed? What defines are free radical and what are the two main ones we focus on that are related to oxygen? Which of the oxygen free radicals is more unstable? Trace out how one can go from O2 to water?
Incomplete reduction of oxygen; ROS containing an unpaired electron; superoxide and hydroxyl radical; Add e- to O2 to make superoxide, then add 1 e- and 2H to make H2O2, then add 1 e- and 1H to make hydroxyl radical, then add 1e and 1 H to hydroxyl radical to make water, but also oxidizing the substrate that hydroxyl radical had reduced
Which of the ROS has the highest level of activity? Which is the only ROS that is a primary ROS? Where is most superoxide made? How many electrons can leak to make superoxide? Where can you have these leaks to make superoxide?
Hydroxyl radical; superoxide; In the mito; 1-5% of all electrons can leak; Complex III (semiquinone at Q0 site to deliver electron to intermembrane space) and complex I (reduced flavin group delivers electron to O2 and superoxide released into matrix)
How can you generate ROS made by non-mito oxidases?
NADPH oxidases (requires NADPH and 2O2 to make superoxide); Xanthine oxidase and monoamine oxidase can make O2- and H2O2, and DOPAC, NH3, and H2O2 respectively
What is the Fenton reaction? What helps catalyze this reaction?
Superoxide + H2O2 –> OH- + O2 + hydroxyl radical; Iron (Fe2+ reacts with H2O2, while Fe3+ can react with superoxide)
What is the reaction for making NO? What is this categorized as? What generates the NO? What can NO potentially give rise to that’s even more of an oxidant? What can this reactive oxidant give rise to?
Arg + O2 + NADPH –> citrulline + NO + NADP+; reactive nitrogen/oxygen species (RNOS); Nitric oxide synthase; Peroxynitrite; give rise to OH and NO2
What is an indicator for the extent of DNA damage and a biomarker of oxidative stress? What is happening at the nucleotide level?
8-hydroxy-2’-deoxyguanosine, resulting from guanosine oxidation; this marker can mispair with deoxyadenosine, leading to G-to-T transversion
Describe the steps involved in lipid peroxidation: IPT; What does non-enzymatic breakdown of lipid peroxidation products lead to?
- Initiation: unsaturated lipid (PUFA) combines with hydroxyl radical to make lipid radical 2. Lipid radical + O2 –> lipid peroxyl radical and free fatty acid 3. Lipid peroxyl radical and free fatty acid –> lipid radical (cycle) and lipid peroxide 4. Otherwise termination: two radicals collide to make two non-radical products, or Vit E (antioxidant) reacts with free radical to make two non-radical molecules; malondialdehyde (MDA) and 4-hydroxy-(2E)-nonenal (4-HNE), which are highly reactive aldehydes
What is the most reactive carbonyl group? What other molecules can cause protein damage? What process do these two mediate on proteins?
4-HNE, reacting with side chain of cysteine, histidine, and lysine residues; hydroxyl radicals; carbonylation, or addition of reactive carbonyl functional groups on proteins
Is H2O2 a free radical? What can it do with respect to some proteins?
It is not; can oxidize some protein cysteinyl residues to make disulfide cross-links
How can you destroy superoxide (first step)? Where can you find the enzymes that participate in this process?
SOD: 2 superoxides + 2 H –> O2 and H2O2; Cu,Zn-SOD (Sod1) in the cytosol, while Mn-SOD (Sod2) in intermembrane space cannot cross inner membrane
What is the second step in getting rid of superoxides and what enzymes are involved? What are the reactions? Where are the enzymes located? How do you regen GSH?
Degradation; catalase and glutathione peroxidase; H2O2 + H2O2 –> H2O + H2O + O2; 2 GSH + H2O2 –> GSSG + 2H2O; catalase in cytosol, GPx in the mito matrix; Glutathione reductase
How else can you dispose of H2O2? What helps make GSSG? What else do you need to keep reaction going? What are sources of NADPH?
Prx (peroxiredoxin) being oxidized, with Trx (thioredoxin being oxidized), and thioredoxin reductase continuing to reduce Trx so Trx can reduce Prx, while NADPH can be made by isocitrate dehydrogenase, or by glucose-6-phosphate dehydrogenase in RBC’s
What do antioxidants do? What are two examples? What does the GSH/GSSG ratio serve as?
Scavenge free radicals and protect proteins against sulfhydryl oxidation; Glutathione (help take free rads to make non-rads to generate GSSG), and coenzyme Q10 or ubiquinone in its reduced form; Indicator for the redox state of the cell
Give examples of antioxidants in the diet
- Vitamin E (alpha-tocopherol) - water-insoluble and protects membrane lipids and lipoproteins 2. Vitamin C (ascorbate) - water-soluble and protects other molecules 3. Plant phenols - cardioprotective by inhibiting LDL oxidation 4. Flavonoids (e.g. green tea) - reduce coronary heart disease and stroke 5. N-acetylcysteine and CoQ10 (ubiquinone)
From reducing to strong oxidizing, what would you expect to see on behalf of cells?
Reducing - cell proliferation Mild oxidizing - cell differentiation Moderate oxidizing - apoptosis Strong oxidizing - necrosis (pathogen destruction!!!)
