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

Table 1 diseases associated with free-radical injury

Fig. 2; Cell has defenses against damage by ROS and RNOS (Reactive oxygen species, Reactive nitrogen-oxygen species):Antioxidants, enzymes

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

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

Radical nature of Oxygen

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

Table 2 ROS speciesTable 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-)

Hydroxyl radical

Fig. 4

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

C. Major sources of ROS

Fig. 5; ETCFe-H is Fe-heme of cytochromes

C. Major source of ROS:

• COQH• in electron transport chainaccidental interaction with O2

(COQH• is free in membrane)

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. 19.12, 19.13 cytochrome P450 mono-oxygenase

Sources of ROS

Fig. 6

Ionizing Radiation generates OH•

• Cosmic rays

• Radioactive chemicals

• X-rays

May also generate organic radicalsfrom contact biomolecules

Oxygen radicals react with cell components

Fig. 7*

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

Lipid peroxy radicals

Fig. 8

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

ROS attack proteins, peptides, DNA

Fig. 9

ROS attack proteins, peptides and DNA• Pro, his, arg, cys, met most susceptible• Protein fragment, cross-link, may aggregate, also will be degraded• Glutathionine (-glu-cys-gly) is anti-oxidant, cell defense

• DNA oxidized bases mispair at replication (G-C → T-A)

• DNA backbone broken• repair mechanisms exist

Reactive Nitrogen-oxygen species

Fig. 10

Nitric Oxide and Reactive Nitrogen-oxygen speciesNO 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 cascadeNO works short distance from source• nNOS and eNOS regulated by Ca2+

• iNOS inducible in immune cells, prouces high levels of NO

NO is toxic

Fig. 11

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)

Phagocytosis uses free radicals

Fig. 12

Phagocytic cells of immune system do respiratory burst: O2 → ROS, RNOS• Part of antimicrobial defense, also anti-tumor (~ 30-60 min)• NADPH oxidase forms O2

- → H2O2 and OH•• Myeloperoxidase forms HOCl → OCl- • iNOS activated, makes NO → RNOS

Free-radical release insome disease states contributes to injury; chronic inflammation

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* compartmentalizationSOD = superoxide dismutaseGSH = glutathione

Antioxidant scavenging enzymes

Fig. 14

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

Antioxidant enzymes

• Glutathione peroxidase, glutathione reductase:• GSH = glutathione (-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

Nonenzymatic antioxidants

Fig. 16

Vitamin E (-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

Antioxidants

Fig. 17

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

Antioxidants

Fig. 18

Carotenoids (-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)

Antioxidants

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

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

Protection against ozone in lung epithelium

Protection against ozone in lung lining fluid:• Many pathways to protect from ozone, which can form

ROS

Fig. 22AA = vit CGSH-Px = perosidaseNeutrophil activation can worsen problem

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.

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?

A.Superoxide dismutase is present in the mitochondria

B.The nucleus lacks glutathione

C.The nuclear membrane presents a barrier to ROS

D.The mitochondrial membrane is permeable to ROS

E.Mitochondrial DNA lacks histones