1. Explain how O2 is both essential to life and toxic
Chapt. 24 Oxygen toxicity
Ch. 24 Oxygen toxicity,
free-radical injury
Student Learning Outcomes:
Explain how O2 is both essential to life and toxic
Oxidative phosphorylation, Reactive Oxygen radicals
Explain formation of the major ROS
Enzymatic by products, non-enzymatic
Describe toxic effects of ROS on cells
Describe nitrogen NO and RNOS radicals
Explain cell protective mechanisms
Enzymes, antioxidants, compartmentalization
Describe association between ROS and diseases

2. Table 1 diseases associated with free-radical injury
Amyotrophoic lateral sclerosis (ALS)
Ischemia/reperfusion injury
OXPHOS diseases (mitochondria)
Alzheimer’s disease
Parkinson’s disease
Diabetes
Aging
Fig. 2; Cell has defenses against damage by ROS and RNOS (Reactive oxygen species, Reactive nitrogen-oxygen species):
Antioxidants, enzymes

3. Reactive Oxygen Species
Reactive Oxygen Species (ROS) are a natural occurrence:
Accidental products of nonenzymatic
and enzymatic processes
Deliberate production by immune
cells killing pathogens
UV irradiation, pollutants
Cells have many defenses
Fig. 1 O2 has 2 antibonding e- with parallel spins; tendency to form toxic ROS

4. Radical nature of Oxygen
Radical is an unpaired e-
Free radical has independent existence (not bound to enzyme)
Free radical extracts e- from other molecules
O2- accepts e- from strong reducer
such as CoQH• in ETC
Fig. 3, O2 can accept total of 4 e- to form H2O

5. Table 2 Some Reactive Oxygen species (ROS)
Table 2 ROS species
Table 2 Some Reactive Oxygen species (ROS)
O2- Superoxide anion Produced by ETC and other sites;
does not diffuse far, generates other ROS, such
as by reaction with H2O2 in Haber-Weiss reaction
H2O2 Hydrogen peroxide Not a free radical, but generates free
radicals by reaction with transition metal (e.g. Fe2+);
diffuses into and through cell membranes
OH• Hydroxyl radical The most reactive in attacking biological molecules; produced from H2O2 in Fenton reaction in presence of Fe2+ or Cu+
HOCl Hypochlorous acid Produced from H2O2 by neutrophils
to destroy invading organisms (OCl-)

6. Formation of very reactive oxygen species OH•
Hydroxyl radical
Formation of very reactive oxygen species OH•
Two nonenzymatic reactions can form OH• by transfer of single e-
Metals Fe2+ or Cu+ are kept sequestered
Fig. 4

7. COQH• in electron transport chain accidental interaction with O2
C. Major sources of ROS
C. Major source of ROS:
COQH• in electron transport chain
accidental interaction with O2
(COQH• is free in membrane)
Fig. 5; ETC
Fe-H is Fe-heme of cytochromes

8. Oxidases, oxygenases, peroxidases generate ROS:
Sources of ROS
Oxidases, oxygenases, peroxidases generate ROS:
Enzymes bind O2, transfer 1 e- via metals;
Accidental leakage of free-radical intermediates
Ex. Cyt P450 mono-oxygenases detoxify many organic compounds (alcohol, drugs, toxic chemicals like CCl4)
Peroxidases generate H2O2 (ex. VLCFA in peroxisomes)
Fig , cytochrome P450 mono-oxygenase

9. Ionizing Radiation generates OH• Cosmic rays Radioactive chemicals
Sources of ROS
Ionizing Radiation generates OH•
Cosmic rays
Radioactive chemicals
X-rays
May also generate organic radicals
from contact biomolecules
Fig. 6

10. Oxygen radicals react with cell components
Lipid peroxidation of membranes
Increased permeability → influx Ca2+ → mitochondrial damage
Cys SH and other aa of proteins oxidized and degraded
DNA oxidized → breakage
Fig. 7*

11. Lipid peroxidation: free-radical chain reaction:
Lipid peroxy radicals
Lipid peroxidation: free-radical chain reaction:
A. Initiation by OH• attack of poylunsaturated lipid → lipid•
B. free-radical chain reaction by reaction with O2
C. Lipid peroxy radical propagates, lipid peroxide degrades
D. Terminate by vitE or lipid-soluble antioxidants
Major contribution to
ROS-induced injury
Fig. 8

12. ROS attack proteins, peptides, DNA
ROS attack proteins, peptides and DNA
Pro, his, arg, cys, met most susceptible
Protein fragment, cross-link, may aggregate, also will be degraded
Glutathionine (g-glu-cys-gly) is
anti-oxidant, cell defense
DNA oxidized bases mispair
at replication (G-C → T-A)
DNA backbone broken
repair mechanisms exist
Fig. 9

13. Reactive Nitrogen-oxygen species
Nitric Oxide and Reactive Nitrogen-oxygen species
NO is both essential for life and toxic:
Gas, diffuses through membranes
Low concentrations: neurotransmitter, hormone (vasodilation)
Nitroglycerin tablets release NO, vasodilator for heart
Binds Fe-heme in receptor guanylyl cyclase,
cGMP activates signal cascade
NO works short distance from source
nNOS and eNOS regulated by Ca2+
iNOS inducible in immune cells,
prouces high levels of NO
Fig. 10

14. At high concentrations, NO is toxic, RNOS form
RNOS can cause as much damage as ROS, plus also do nitrating, nitrosylating
RNOS damage proteins, cause lipid peroxidation, DNA breaks
RNOS are involved in:
neuro-degenerative diseases like Parkinson’s,
chronic inflammation like rheumatoid arthritis (RA)
Fig. 11

15. Phagocytosis uses free radicals
Phagocytic cells of immune system do respiratory burst: O2 → ROS, RNOS
Part of antimicrobial defense, also anti-tumor (~ min)
NADPH oxidase forms O2- → H2O2 and OH•
Myeloperoxidase forms HOCl → OCl-
iNOS activated, makes NO → RNOS
Free-radical release in
some disease states
contributes to injury;
chronic inflammation
Fig. 12

16. V. Cells have defenses against oxygen toxicity:
V. Cellular Defenses
V. Cells have defenses against oxygen toxicity:
Antioxidant scavenging enzymes (red)
Nonenzymatic antioxidants (free radical scavengers)
Compartmentalization
Repair of damaged components
Metal sequestration
Fig. 13* compartmentalization
SOD = superoxide dismutase
GSH = glutathione

17. Antioxidant scavenging enzymes
Superoxide dismutase (SOD)
Converts O2- to H2O2
3 isoforms:
Cytosol, mitochondria, extracellular
Catalase
Reduces H2O2 to H2O
Prevents OH• formation
mostly peroxisome
Fig. 14

18. Glutathione peroxidase, glutathione reductase:
Antioxidant enzymes
Glutathione peroxidase, glutathione reductase:
GSH = glutathione (g-glu-cys-gly)
Peroxidase reduces H2O2, oxidizes two SH groups → GSSG
Peroxidase in cytosol, mitochondria, have selenium
Reductase recycles the glutathione, reduces with NADPH
Fig. 15

19. Nonenzymatic antioxidants
Vitamin E (a-tocopherol) is antioxidant:
Lipid-soluble, protects against lipid peroxidation in membranes
Nonenzymatic terminator of free-radical chain reaction
Lipid fraction of vegetable oils,
liver, egg yolks, cereals
Lipoprotein particles in blood
Fig. 16

20. Vitamin C (ascorbate) is antioxidant:
Antioxidants
Vitamin C (ascorbate) is antioxidant:
Can donate e- to vitamin E to regenerate Vitamin E
Water-soluble, circulates blood and fluids to access membranes
Vitamin C is also redox coenzyme for collagen synthesis, other reactions
Fig. 17

21. Carotenoids (b-carotene, precursor of vitamin A):
Antioxidants
Carotenoids (b-carotene, precursor of vitamin A):
Are antioxidants, found in fruits and vegetables
May slow cancer, atherosclerosis
Lutein and zeoxanthin in macula of eye (may help protect against macular degeneration of retina)
Fig. 18

22. Flavonoids are antioxidants:
May inhibit enzymes responsible for ROS (xanthing oxidase)
May chelate Fe and Cu; free-radical scavengers
Endogenous antioxidants: melatonin and uric acid
Fig. 19,20

23. Parkinson’s disease and neuronal degeneration
Model for ROS and RNOS in neuronal degradation in Parkinson’s disease:
Dopamine levels are reduced because degenerated dopaminergic neurons
Unknown trigger, model:
MAO (monoamine oxidase) generates H2O2 → (also SOD in Mt)
Damaged Mt leak Fe2+ → OH•
NO forms RNOS
Radical chain reactions
Fig. 21

24. Protection against ozone in lung epithelium
Protection against ozone in lung lining fluid:
Many pathways to protect from ozone, which can form ROS
Fig. 22
AA = vit C
GSH-Px = perosidase
Neutrophil activation can worsen problem

25. Key concepts: Key Concepts
Oxygen radical generation contributes to cell death and degeneration in a variety of diseases
Radical damage occurs via e- extraction from biologic molecules
ROS include superoxide, hydrogen peroxide, hydroxyl radical
ROS can damage DNA, proteins, lipids, lead to cell death
Other radical species are NO and HOCl
NO reacts with oxygen or superoxide to form RNOS
The immune response produces radical species to destroy microorganisms (superoxide, HOCl, NO)
Cell defenses against radical damage include defense enzymes, antioxidants and compartmentalization.

26. Review question
5. The level of oxidative damage to mitochondrial DNA is 10 times greater than that to nuclear DNA. This could be due, in part, to which of the following?
Superoxide dismutase is present in the mitochondria
The nucleus lacks glutathione
The nuclear membrane presents a barrier to ROS
The mitochondrial membrane is permeable to ROS
Mitochondrial DNA lacks histones