SYLLABUS 13: Reactive Oxygen Intermediates in Biology & Medicine

Question 1

free radical is?

Answer 1

a species which contains an unpaired e- in its outer orbital

b/c e- want to pair up, free radicals are very reactive

Question 2

charge of free radicals?

Answer 2

1. negative charged, i.e. superoxide anion radical
2. positively charged, i.e. methonium radical
3. neutral, i.e. glutathione radical

Question 3

why is molecular oxygen important?

Answer 3

it is used in aerobic metabolism, which produces more ATP than anaerobic metabolism does, 32 vs. 2 for glucose oxidation

Question 4

net of the e- respiratory chain?

how does this occur?

what is the result?

Answer 4

O2 is reduced by 4 e- to 2 molecules of water

small amounts of O2 can be reduced by 1 or 2 e- transfers to raective oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals

dangerous b/c these react w/ celllarmacromolecules and disrupt their function or structure

Question 5

what diseases are ROS implicated in?

Answer 5

toxicity of oxygen, inflammation, sepsis, carcinogenesis, cardiovascular diseases/atherosclerosis (LDLs and foam cells), aging (free radical theory of aging: we rust w/ age), ischemia/stroke/infarction, reperfusion/transplantation injury, neurodegenerative diseases like Parkinson and Alzheimer, radiation injury, smoking injury xenobiotic and drug toxicity, alcohol toxicity, iron and asbestos toxicity, vitamin deficiency g vitamin E and C, mineral deficiency eg selenium, zing, manganese, cataracts & retinopathies, others

all b/c oxygen radicals react w/ macromolecules and disrupt their structure and/or function

Question 6

what is oxidative stress?

Answer 6

imbalance btwn oxidants and antioxidants either b/c of increased production of ROS or decreased levels of antioxidants or both

Question 7

prooxidant is?

Answer 7

ROS which can cause tissue damage, & whose levels may be increased by certain drugs, infection, external exposures, tissue injury

Question 8

antioxidants are?

Answer 8

inhibit either formation of ROS or remove/scavenge the generated ROS

Question 9

what are RNS?

Answer 9

reactive nitrogen species

derived from nitric oxide, which can be produced from arginine by nitric oxide synthases

can also cause tissue injury, like ROS

Question 10

what is the state of molecular oxygen in the ground state?

Answer 10

O2 is a diradical

6 e- surrounding each of the O atoms that make up O2, so 1 unpaired e- on each oxygen atom and 2 unparied e- in outer orbital

although a radical, is unreactive b/c the unpaired e- are spinning in the same direction, and to make a covalent bond w/ another atom, they have to spin in opposite directions

Question 11

what is required of unpaired e- for covalent bond formation?

Answer 11

unpaired e- must be spinning in opposite directions

Question 12

what produces the singlet ground state of oxygen?

what can it do?

Answer 12

absorption of energy, eg UV light or radiation, by triplet oxygen

this inverts the spin of 1 of the e- to produce the very reactive singlet ground state of oxygen

singlet oxygen can insert into double bonds or aromatic amino acids of proteins (Phe, Tyr, Try), of unsautrated fatty acids, or purine and pyrimidine bases of DNA and RNA

Question 13

what causes skin lesions?

Answer 13

sunlight-induced singlet oxygen formation

Question 14

what blocks singlet oxygen formation?

Answer 14

B-carotene, a vitamin A derivative

it is a potent scavenger of singlet oxygen, and common ingredient in sunscreens & skin blister treatments

Question 15

how is the superoxide anion radical formed?

what is its pKa?

how is it at physiological pH?

Answer 15

triplet oxygen is reduced by a single e-

has a negative charge, = anion

its pK = 4.8

it is unprotonanted at physiological pH

Question 16

what forms the perhydroxyl radical?

Answer 16

if superoxide anion radical is protonated

Question 17

what forms peroxide?

how is peroxide at physiological pH?

Answer 17

if 2 e- are added to triplet oxygen

done by enzymes, mostly peroxisomal enzymes like urate oxidase, glycolate oxidase, D-amino acid oxidase, fatty acyl oxidase

is protonated and in the form of H2O2, hydrogen peroxide, at physiological pH

Question 18

is H2O2 a radical?

Answer 18

no - has no unpaired e-

however, it’s a potent oxidizing agent, and therefore reactive and toxic

Question 19

what is a dismutation rxn?

ex of it w/ superoxide radical?

Answer 19

dismutation: when 2 identical compounds interact, 1 becoming reduced & the other oxidized - 1 loses an e- and the other gains the e- lost by the first superoxide

occurs non-enzymatically or w/ enzymes w/ superoxide

Question 20

what are SODs, superoxide dismutases?

why do they matter?

Answer 20

enzymes that can catalyze superoxide dismutation reaction

important antioxidant enzymes b/c they remove superoxide

Question 21

what is the most powerful ROS? why?

Answer 21

hydroxyl radical

can react w/ any biochemical or macromolecule

reacts and inactivates or disrupts proteins, lipids, DNA and RNA

Question 22

what is the fenton reaction?

what must occur for it to continue?

Answer 22

explains dominant way that hydroxyl radical is produced in cells

H2O2 reacts w/ Ferric iron (Fe2+), which is reduced by superoide to Ferrous iron (Fe3+)

for it to continue, Fe3+ must be reduced back to Fe2+ which occurs by superoxide which is good at reducing metals:

Fe3+ + O2- radical -> Fe2+ + O2

Question 23

what is the Haber-Weiss reaction?

what can catalyze it?

Answer 23

way that it was believed OH radical was produced, from direct reaction between hydroxyl radical and hydrogen peroxide

iron or copper can catalyze it, but iron’s more plentiful and more reactive

Question 24

how can toxicity associated w/ ROS be treated? think haber-weiss and fenton rxns

Answer 24

toxicity of ROS and its role in many diseases can be mitigated in part by removal or chelation of iron since they are involved in generated hydroxyl radicals

Question 25

what biological systems produce ROS?

Answer 25

1. Mito respiratory chain: Complex I, III 2. Heme enzymes which react w/ molecular oxygen, like hemoglobin & myoglobin 3. Cytochrome P450 mixed function oxidase electron transport chain - CYP2E1, which oxidizes drugs & xenobiotics 4. Autooxidation, direct oxidation by O2, of cellular biochemicals like catecholamines (epinephrine, norepinephrine, dopamine), serotonin, flavins, tetrahydrobiopetrin

Question 26

what enzymes produce ROS?

Answer 26

superoxide and H2O2 produced by: aldehyde oxidases xanthine oxidase peroxisomal peroxidases cyclooxygenases - produce prostaglandins, leukotrines

Question 27

what's a major source of ROS in the brain?

Answer 27

monamine oxidase uses O2 to oxidize important neurotransmitters like epinephrine, norepinephrine, dopamine, serotonin this all produces H2O2

Question 28

major source of ROS in most tissues? how specifically?

Answer 28

mito electron transfer chain produces superoxide from the oxidation of reduced flavin in Complex I and autooxidation of reduced ubisemiquinone

Question 29

how does the autodoxidation of quinones form oxygen radicals?

Answer 29

Oxidized Q, Ubiquinone + 1 e- from either complex 1 or 2 forms Ubisemiqunone or Semiquinone radical Semiquinone radical + 1 e- forms reduced Q or ubiquinol. This radical passes into Complex 3, reduces. OR can directly react w/ O2 to give Oxidized Q plus Superoxide This regenerates Oxidized Q, which can again be reduced by 1 e- to Ubisemiquinone, and reaction can repeat, producing lots of superoxide

Question 30

what inner source can form oxygen radical? (think enzymes)

Answer 30

in heme enzymes such as cytochrome P450s, hemoglobin, myoglobin, etc., Ferric iron (Fe3+) of heme is reduced to Ferrous iron (Fe2+) Fe2+ can bind O2 to form heme ferrous-O2 complex resonance structur for this is obtained by transferring just 1 e- from Fe2+ to O2, and producing Fe3+-O₂^-* some of this decays to yield ferric heme enzyme and superoxide radical

Question 31

external sources of ROS?

Answer 31

drugs and xenobiotics alcohol infection radiatino smoke ozone

Question 32

in what ways are ROS toxic to proteins?

Answer 32

1) esp. hydroxyl radical **oxidize SH group of cysteine residues** of **proteins** to the disulfides or to sulfoxide (SO) or sulfonic acid (SOOH) - lose enzymatic activity dependent on cysteine, protein tertiary structure's disrupted 2) **CH₃S of methionine is readily oxidized to methionine sulfoxide, CH₃SO,** which interferes w/ methionine fxn like **methylation reactions ** 3) **Oxidize aromatic rings of Phe and Tyr or indole ring of Try,** open up the rings, form protein **carbonyls** & cause loss of enzymatic activity and structure 4) disrupt **peptide bonds**, covalent and noncovalent interactions

Question 33

how are ROS toxic to nucleic acids?

Answer 33

1) cleave phosphodiester bonds, break chain structure of RNA and DNA 2) cause base excision 3) oxidize bases eg dG -\> 8OHdG - 8C position is v. sensitive 4) cause base mispairing mutations via **deaminations** from adenine or guanine to form hypoxanthine or xanthine, or remove amino group from cytosine to form uracil

Question 34

what occurs to nucleic acid base pairing if ROS deaminate them?

Answer 34

H, hypoxanthine, pairs w/ G, so A:T base pair changes to H:G this is major cause of mutations

Question 35

what is lipid peroxidation, when and why does it occur

Answer 35

ROS oxidation of polyunsaturated fatty acids = lipid peroxidation it disrupts normal membrane structure rancidity of foods is due to lipi peroxidation as they age

Question 36

steps of lipid peroxidation?

Answer 36

1) initiation: unsaturated fatty acids in lipid become lipid radical 2) propagation: lipid can be peroxidized by oxygen to make a lipid peroxyl radical; can extract a H from another lipid 3) degredation: if lipid radical takes on another H, it becomes unstable, v. reactive 4) termination: propagation steps eventually are halted by Vitamin E

Question 37

what do lipid radicals formed during propagation step bind to?

Answer 37

amino groups of amino acids, purines and pyrimidines, and SH groups of cysteines this inactivates enzymes and disrupts protein, RNA, and DNA structure

Question 38

what does fragmentation of LOOH do?

Answer 38

destroys the phopholipid bilayer holding membranes together is major reason for toxicity of ROS - i.e. mitochondria cannot generate a protein motor force, permeability of plasma membrane's altered, ligand interactiosn w/ receptors are lost, etc

Question 39

what is vitamin E's role in lipid peroxidation?

Answer 39

reacts w/ lipid and lipid alkoxyl radicals to restore lipid and form a Vitamin E radical Vitamin E then is regerated from E radical by ascorbic acid (Vitamin C)

Question 40

when are ROS beneficial?

Answer 40

1) phagocytosis 2) cell signaling 3) enzymatic reactions

Question 41

how are ROS important re: signaling agents in biological systems?

Answer 41

H2O2 can catalyze"mild" oxidations which can activate or inactivate enzymes or transcription factors, including Hrf2, NF-kB, Ap-1 these are catalyzed by H2O2-dependent oxidation of Cysteine SH to Cystine Disulfied These txn factors subsequently activate many genes, some of which code for cellular antioxidants SO low levels of ROS can protect vs. High levels of ROS

Question 42

what are phagocytes? how do they contribute to ROS?

Answer 42

a type of WBC contain the enzyme NADPH oxidase which is activated when phagocytes come into contact w/ bacteria and other cells, ie tumor cells **NADPH oxidase catalyzes a respiratory burst **b/c oxygen uptake is increased

Question 43

what occurs in a respiratory burst?

Answer 43

rapid, large production of superoxide and other ROS kills invaders activates the PPP pathway to regenerate NADPH

Question 44

what is myeloperoxidase? its action?

Answer 44

enzyme in phagocytes catalzyues interaction of H2O2 w/ chloride anion, produces the powerful oxidant **hypochlorite, HCLO** HCLO is active oxidant in bleach, destroys bacteria & other organisms

Question 45

what causes chronic granulomatous disease?

Answer 45

if WBC phagocytes are deficient in some of the subunits which make up the NADPH oxidase which is crucial to a respiratory burst if cannot mount a burst, cannot deal w/ invading organisms this makes individuals very sensitive to infection often is **fatal b/c of sepsis**

Question 46

what does superoxide dismutase do?

Answer 46

catalyzes the removal of 2 moles of superoxide via a dismutation reaction - oxidizes 1 mole to O2 and 1 mole to H2O2

Question 47

what superoxide dismutases do humans have?

Answer 47

1) copper/zinc SOD in the cytosol and space between the outer and inner mito membranes 2) a manganese SOD in the matrix of the mito

Question 48

what kind of SOD do bacteria have?

Answer 48

iron-dependent SOD

Question 49

what metals are considered antioxidants, why?

Answer 49

manganese, zinc they are needed for SOD activity

Question 50

effect of low amounts of ROS on SODs?

Answer 50

increases levels of SODs to protec against large levels of ROS

Question 51

what does chemical inhibition of SOD or siRNA against SOD cause?

Answer 51

increases the sensitivity of cells to ROS proves that SOD protect cell from ROS

Question 52

function of catalase?

Answer 52

in peroxisome heme-containing enzyme that catalyzes removal of 2 moles of H2O2 has a high Km for H2O2 and very high Vmax

Question 53

function of glutahion peroxidase? location?

Answer 53

in cytosol and mito functions in conjunction w/ GSH, NADPH and glutathione reductase to get rid of ROS

Question 54

what is GSH

Answer 54

most important antioxidant in cells tripeptide made of glutamate + cysteine + glycine *glutamate and cystine linked by gamma carboxyl * contains selenium at its active site, so low amounts of selenium are important for antioxidant protection

Question 55

peroxiredoxins are?

Answer 55

family of enzymes found to have potent H2O2 removal activity heme oxygenase, Vitamins E and C, Carotenoids, melatonin, uric acid, ...

Question 56

how is iron usually in cells? result re: antioxidant action?

Answer 56

there's little free iron available in cells - most is stored in ferritin iron stored in ferritin cannot catalyze Haber-Weiss or Fenton reactions

Question 57

cellular repair enzymes' fxn re: oxidative stress?

Answer 57

some can repair or remove oxidized amcromolecules from the cell, such as DNA repair enzymes and proteasome complex

Question 58

what foods have antioxidants?

Answer 58

plant extracts - flavonoids, polyphenols, catechinds, curcumin

Question 59

how do ROS play a role in neurodegenerative diseases?

Answer 59

brain is a major target for ROS Parkinson, Alzheimer, other nd diseases thought to have ROS involvement 1. brain is v enriched in polyunsaturated fatty acids and iron - perfect for LP 2. monoamine neurotransmitters are substrates for oxidation by mono amine oxidase, with formation of ROS, mainly H2O2

Question 60

how does smoke cause oxidant stress

Answer 60

smoke has many oxidants these inactive anti-proteases like tripsin inhibitor found in high amounts in lung tissue this inactivation allows proteases to be highly active, cause damage to lung tissue results in emphysema

Question 61

how can drugs cause ROS-induced issues

Answer 61

drugs w/ a quinone-like structure can cause ROS-induced tissue injury b/c high levels of such drugs can easily undergo a 1 e- reduction by cellular reductases in the mito and microsomal e- transfer chains this forms the semiquinone drug, which can react w/ oxygen to regenerate the drug and superoxide this is **redox-cycling **results in ROS-induced tissue injury

Question 62

what is adriamycin? action re: ROS?

Answer 62

one of most commonly prescribe anti-cancer agents can only be used for short periods of time b/c it causes signfiicant heart injury due to its ability to redux cycle

Question 63

what is free radical theory of aging

Answer 63

posits that our cells fill up w/ damaged oxidized molecules, which accumulate over a lifetime of metabolism and oxidation some antioxidants may decrease w/ age posits taking extra Vitamins E and C would help slow aging ramifications

Question 64

what does increased metabolic rate cause in lower organisms

Answer 64

increased metabolic rate = increased oxygen uptake = lessens life span whereas decreased metabolic rate = decreased oxygen uptake = enhanced life span

Question 65

what is ischemia

Answer 65

lack of oxygen occurs in heart tissue during an MI or in brain tissues during storke b/c of prevention of blood flow have immediate need to restore oxygen by moreval of the blockage

Question 66

what is a reperfusion injury

Answer 66

when resumption of blood flow and delivery of oxygen to ischemic tissue causes tissue damage due to an oxygen paradox by which ROS are produced during reperfusion

Question 67

reasons for reperfusion injury?

Answer 67

1) during ischemia, mito etc is in a highly reduced state b/c of lack of oxygen; when reperfusion's initiated & oxygen's re-introduced, there's a burst of ROS from eg reduced ubisemiquinone, which may overwhelm tissue antioxidant defense 2) XDH normally oxidizes purine bases to uric acid for excretion, uses NAD+ for this oxidation and produces NADH; if XDH is converted to oxygenase form, XO, it uses oxygen instead of NAD to oxidize, making a superoxide product instead of NADH

Question 68

why does a XDH conversion occur during ischemia

Answer 68

XDH -\> XO b/c certain Ca-activated proteases are increased, since calcium floos ischemic tissue due to lack of ATP production that prevents calcium from being pumped out of the cell ATP also isn't made due to lack of oxygen, so it breaks down to hypoxanthine, and GTP breaks to xanthine when reperfusion provides Oxygen, the tissue is flooded w/ a brust of ROS via the XO reaction

Question 69

how can reperfusion or organ transplant occur w/o issue?

Answer 69

to avoid a reperfusion injury, load the blood or soak the organ in fluid w/ : antioxidants, esp. GSH, and Vitamins E and C, and chelators of Iron such as desferrioxamine, and an inhibitor of Xo and XDH called **allopurinol **

Question 70

what is a free radical? why is it reactive?

Answer 70

an atom with an unpaired electron in its outer orbital. It’s reactive because of that unpaired e-, which wants to pair up. it tends to make very destructive molecules in the process as well as messes up proteins

Question 71

what is the state of molecular oxygen at ground state? what would happen if added 1 e-? 2 e-? 3 e-?

Answer 71

Molecular oxygen is a diradical triplet in the ground state – it has 2 unpaired e-, but their spin is the same, so it’s unreactive. In the singlet ground state, the spin of 1 of these e- on molecular oxygen is inverted, so it becomes very reactive. This is formed if 1 e- is added to the triplet state, forming the superoxide anion radical. If another e- is added to this, the perhydroxyl radical forms. If another e- is added, then it’s peroxide.

Question 72

is ROS production always destructive?

Answer 72

no - protective sometimes ie low levels of ROS initiate production of some antioxidants, so these can mount a larger response in the presence of even more ROS Also a really important part of the immune system. Useful for destroying bacteria when engaged in a controlled fashion.

Question 73

what do these metals do re: ROS? pro-oxidant or antioxidant? Iron, Copper, Selenium, Zinc, Manganese

Answer 73

**Iron and copper** catalyze the formation of the OH radical. **Selenium** is in the active site of GSH, so it’s an important antioxidant. **Zinc** **SOD** is in the cytosol and space between the outer and inner mitochondrial membranes in humans – it’s an important antioxidant. **Manganese** **SOD** is in the matrix of the mitochondria in humans – also thus an important antioxidant.

Question 74

what are redox cycling agents re: drugs? what does this cause?

Answer 74

drugs w/ a quinone-like structure that can cause ROS-induced tissue injury because high levels of the drug can easily undergo a 1 e- reduction that makes them into the semiquinone-like structure, which can react with oxygen to regenerate the drug and superoxide. This causes tissue injury. eg: adriamycin and heart tissue injury

Question 75

what is ROS impact on: nucleic acids, enzymes, lipids, proteins

Answer 75

ROS cause mutations by breaking purine rings and causing base mismatch (A:H instead of A:T). ROS inhibit enzyme activity by inhibiting SOD, which are important in protecting cells from ROS. The chain reaction of Lipid Peroxidation is how ROS oxidation of polyunsaturated fatty acids occurs – this causes membrane structure disruption, for example, which can be very dangerous for the cell. Proteins: ROS oxidize cysteine’s SH groups, disrupting any enzyme dependent on cysteine.

Question 76

what is ischemic reperfusion injury? how can it be avoided?

Answer 76

when ischemic tissue is reperfused with oxygen and this causes ROS production injury is minimizable by putting antioxidants in the blood used for transfusion, or the solution in which an organ for transplantation is soaked, to minimize the ROS production This can occur via GSH, Vitamin E and C addition, chelators of Iron like desferrioxamine (since Iron is important in ROS formation), or an inhibitor of XO and XDH, allopurinol