Unit 7 - Reactive O2 Species and Oxidative Stress Flashcards
how are ROS defined? free radical VS oxidant?
collective term that describes chemically reactive molecular species formed upon incomplete reduction of O2
-FR only if have unpaired electron
are the following free radicals or oxidants, primary or secondary ROS, and have low, moderate, or high activity?
- superoxide
- hydrogen peroxide
- hydroxyl radical
.O2- is free radical and oxidant, primary ROS, and moderate reactivity
H2O2 is oxidant, secondary ROS, and low reactivity
.OH is free radical, secondary ROS, and highest reactivity (very short halflife)
–steals an electron to make water and oxidized substrate
where is most superoxide production located? what does this mean for electrons?
> 90% within mitochondria as byproduct of ATP synthesis
-1-5% of all electrons “leak” to O2 prematurely to make superoxide
what free radicals do complexes I, III, and IV make in the ETC?
I: single e- leak makes .O2- to matrix
III: single e- leak makes .O2- to intermembrane space
IV: 2 electrons from cyt c make H2O (no superoxide) to matrix
when is superoxide production increased?
- high membrane potential (increased reduction level of e- carriers at Qo site, increasing e- leak to O2)
- high NADH/NAD+ ratio (over-reduction of ETC)
- ETC damage (alters e- flow accuracy and increases e- leak)
- hypoxia (mech not fully understood)
- xenobiotics (block ETC)
what are 3 non-mitochondrial oxidases that can generate ROS?
- NADPH oxidase (Nox) - in phagocytes to kill pathogens, and non-phagocytes for cell signaling
- NADPH + 2O2 –> NADP+ + 2 .O2- + H+ - xanthine oxidase - purine catabolism
- xanthine + O2 –> uric acid + .O2- + H2O2 - monoamine oxidase - dopamine catabolism
- dopamine –> DOPAC + NH3 + H2O2
- -contributes to hypersensitivity of dopaminergic neurons to oxidative stress
how are ROS interconverted non-enzymatically?
- superoxide spontaneously dismutes to O2 and H2O2 at a slow rate, especially at low concentrations
- H2O2 converted to .OH by Fenton RXN
- transfers e- to H2O2 from free metal ions (Fe, Cu), which are recycled back to reduced forms by reacting with .O2- - .OH made by ionizing radiations (homolytic fission of H2O2)
why does increased free Fe cause oxidative stress?
presence of Fe is catalytic in Fenton reaction
why is free radical production used in cancer radiation therapy?
kills actively proliferating tumor cells
what is the primary reactive nitrogen/oxygen species and how is it made?
nitric oxide is made by specific nitric oxide synthase
- arg + O2 + NADPH –> citriulline + .NO + NADP+
- NO is mild radical, but can react with SO to make peroxynitrite (ONOO-)
what is peroxynitrite?
ONOO- is very reactive oxidant, that can give rise to .OH
where is there irreversible damage by .OH and reversible modifications by H2O2?
.OH –> DNA, PRO, lipid
H2O2 –> thiol groups in PRO
what are biomarkers for DNA damage and lipid peroxidation?
DNA damage: formation of 8-hydroxy-2’-deoxyguanosine from guanosine oxidation
-mispairs with deoxyadenosine, causing G-to-T transversion
Lipid peroxidation: malondialdehyde (MDA) and 4-hydroxy-2E-nonenal (4-HNE)
-react with side chain of systeine, histidine, and lysine, affecting activity or increasing degradation
what is protein carbonylation and what mediates this?
.OH radicals add reactive carbonyl functional groups o n proteins
-affects activity or increases degradation
what does H2O2 do to cysteinyl residues?
oxidizes them to form disulfide X-linkes with other cysteines
-has role in cell signaling, and needs mediator like peroxiredoxin
what are the 3 enzymatic defense mechanisms against ROS?
- superoxide dismutase: .O2- –> H2O2
- catalase, glutathione peroxidase, or peroxiredoxin: H2O2 –> H2O
- if above fail, removal of .OH by antioxidant scavenging
what are the types of superoxide dismutase?
2 .O2- + 2H+ –> O2 + H2O2
- SOD1 - very abundant in cytosol; has 1 Cu and 1 Zn (Cu,Zn-SOD)
- specific missense and point dominant mutations cause ALS (high oxidative stress)
- -mutant SOD1 is misfolded and becomes cytotoxic, but doesn’t necessarily lose enzymatic activity - SOD2 - mitochondrial PRO that uses Mn as ligand (Mn-SOD)
- freely crosses mitochondrial outer, but not inner membrane
- responsible for degrading .O2- in matrix
what are the 3 pathways to decompose H2O2?
- glutathione peroxidase (has Se): 2 GSH + H2O2 –> GSSG + 2 H2O
- peroxiredoxin pathway: peroxiredoxin is small sulfhydryl-containing PRO
- catalase (has heme): 2 H2O2 2 H2O + O2
what is glutathione?
tripeptide (yGlu - Cys - Gly) with e-rich sulfhydryl group
- forms dipeptide (GSSG) with another GSH when exposed to oxidizing agent via glutathione peroxidase
- reduced GSH is regenerated by glutathione reductase (NADPH-dependent)
how is NADPH produced in
- mitochondria
- erythrocytes
- isocitrate dehydrogenase
- pentose phosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase)
- -deficiency in these cause oxidative stress and hemolytic anemia
where is the peroxiredoxin important to detox H2O2?
in mitochondria and erythrocytes
-reacts with H2O2 to form water concomitant with oxidized peroxiredoxin with disulfide bond, which is then reduced by thioredoxin by thioredoxin reductase (NADPH dependent)
where is catalase usually found?
in peroxisome (H2O2 from mitochondria can freely cross membrane)
what is acatalasia?
AR disease with low levels of catalase
- relatively benign, but causes more periodontal infections
- suggests predominant H2O2 in normal mammalian cells may be scavenged by glutathione peroxidase and peroxiredoxin pathways
what are some in vivo antioxidants? what do they do and where are they abundant?
- glutathione - directly scavenges .OH by making thiyl radicals (GS.) and .O2-
- -keeps sulfhydryls or cycteines of PRO reduced to maintain activity
- -in cytosol, nuclei, and mitochondria in high GSH:GSSG ratio
- ubiquinone - in ETC, other membranes, and in lipoproteins
- -reduced form (CoQ10/ubiquinol/CoQH2) has antioxidative function
- scavenges RO2. radicals and inhibits lipid peroxidation
what is GSH:GSSG ratio used for?
indicator for redox state of the cell
- if high, more reduced PRO (maintains activity)
- if low, more oxidized PRO
what is the preferred reaction of ascorbyl radicals?
disproportionation (simultaneously reduced and oxidized) after collision to give nonradical ascorbate and dehydroascorbate
how do ROS and RNOS have physiological roles? (as in, what can reduction and oxidation do?)
reduction: cell proliferation
mild oxidation: cell differentiation
moderate oxidation: apoptosis
strong oxidation: necrosis
what is “redox signaling”?
describes a regulatory process in which the signal is delivered through redox reactions
-requires that “redox balance” (GSH/GSSG ratio) is disturbed by either increase in ROS/RNOS formation, or decrease in antioxidant systems
how can ROS and RNOS be considered second messengers?
increased H2O2 production causes oxidation of specific reactive cysteine residues within regulatory proteins with concomitant modulation of function
- regulation of cellular processes
- signal proteins are poorly competitive, and oxidation involves sensors
how are ROS related to innate immunity?
“respiratory burst” of neutrophils
-NADPH oxidase creates superoxide-related ROS to be antimicrobial and modulate transcription factors to further the inflammatory response
how are ROS related to apoptosis and cancer?
ROS catalyze cardiolipin peroxidation (CL is specific phospholipid on IMM)
- facilitates detachment of cyt c from outer surface for apoptosis
- must be careful w/ cancer patients b/c lowering ROS might stimulate tumor growth by suppressing apoptosis (IOW: antioxidants protect healthy people from cancer, but promote growth of pre-initiated tumor cells)
- ROS production is critical mediator of ionizing-radiation therapy of cancer, so excess antioxidants contribute to tumor radioresistance
ROS, food intake, and body weight control
ROS production is increased in POMC and AgRP neurons in response to feeding and leptin
-play important roles in negatively and positively regulating food intake, respectively
how are ROS related to diabetes?
beta cells are susceptible to oxidation damage
-folding insulin properly needs O2, so cannot stay in reduced environment very long
