09 OXYGEN METABOLISM AND TOXICITY

What is oxygen at room/physiologic conditions?

Diatomic molecule O2; colorless, odorless; symbol O; bond order 2; protons 8, electrons 8, neutrons 8; valency 2.

What is special about oxygen's electron configuration?

O2 has two unpaired electrons in two different orbitals with parallel spins (biradical).

How does oxygen's biradical nature affect reactivity?

The two unpaired parallel-spin electrons make O2 thermodynamically reactive but spin-restricted kinetically — it needs an electron spin flip to oxidize covalent bonds.

What is “spin restriction” and why is it biologically important?

Spin restriction is the kinetic barrier from parallel spins; it prevents uncontrolled oxidation of organic molecules and allowed life to evolve.

Define a radical.

A molecule with a single unpaired electron in an orbital; highly reactive and capable of chain reactions.

What is a free radical?

A radical that can exist independently and readily oxidize other molecules to become stable.

Why are radicals destructive?

They have low chemical specificity and steal electrons (often hydrogen atoms) from nearby molecules, initiating damaging chain reactions.

Is every molecule with an unpaired electron a harmful free radical?

No—some radicals cannot exist independently and are not free radicals capable of widespread damage.

What is a biradical?

A molecule with two unpaired electrons (like O2) occupying different orbitals; highly reactive.

List the 4-electron reduction sequence of oxygen (reactant→final product).

O2 → O2•− (superoxide) → H2O2 (hydrogen peroxide) → OH• (hydroxyl radical + H2O) → H2O (water).

Is hydrogen peroxide (H2O2) a free radical?

No — H2O2 has no unpaired electron but is classed as ROS because it can generate free radicals.

Why are reactive oxygen species (ROS) biologically important?

ROS mediate most damaging effects of oxygen; they react indiscriminately and can damage lipids, proteins, and DNA.

What is the superoxide anion (O2•−)?

Superoxide anion is O2 with one added electron (one unpaired electron, negatively charged); reactive but poorly lipid soluble so it stays near its site of production.

Why is limited lipid solubility of superoxide biologically relevant?

It restricts diffusion, keeping damage localized to organelle/site of production (e.g., mitochondria).

How is hydrogen peroxide formed?

By two-electron reduction of O2 or by dismutation of superoxide (via SOD); it has no unpaired electrons.

What are properties of hydrogen peroxide?

Not a radical, weak oxidizing agent, can diffuse through membranes, and is a precursor to hydroxyl radical and HOCl.

How is the hydroxyl radical (OH•) formed?

Via Fenton reaction (H2O2 + Fe2+/Cu+ → OH• + OH− + Fe3+) or Haber-Weiss (O2•− + H2O2 → OH• + O2 + OH−).

Why is the hydroxyl radical especially dangerous?

OH• is the most reactive ROS; it initiates lipid peroxidation and causes severe damage to membranes, proteins, and DNA.

What is an organic radical (R•)?

A radical formed on organic moieties when RH (e.g., PUFA double bond or RSH) loses an electron/hydrogen via OH• or O2•− attack.

Where do organic peroxyl radicals (ROO• or LOO•) come from?

They form during lipid peroxidation of polyunsaturated fatty acids (PUFAs) and propagate chain reactions damaging membranes.

What is hypochlorous acid (HOCl) and how is it produced?

HOCl is produced in neutrophils by myeloperoxidase using H2O2 + Cl− → HOCl; HOCl dissociates to OCl− (hypochlorite) — a strong oxidant.

Is HOCl a radical?

No, HOCl is not a free radical but can be converted to reactive species (e.g., OCl−) and lead to OH• formation.

List some direct actions of HOCl on biomolecules.

Oxidizes Fe- and S-containing groups, decarboxylates and deaminates proteins, cleaves peptide bonds; contributes to microbial killing.

What is singlet oxygen (¹O2)?

A non-radical high-energy form of O2 with an empty orbital that reacts with conjugated double bonds; produced under high O2 tension or UV exposure.

Is singlet oxygen long-lived in cells?

No — it decays rapidly and is usually of limited significance biologically.

How do carotenoids interact with singlet oxygen?

Carotenoids (e.g., beta-carotene, lycopene) “quench” singlet O2 and protect against singlet-oxygen-mediated damage.

What is nitric oxide (NO•) and how is it produced?

NO• is produced by nitric oxide synthase (NOS) from arginine; iNOS (inducible NOS) produces NO during inflammation.

What happens when NO• meets superoxide (O2•−)?

They react to form peroxynitrite (ONOO−), a strong oxidizing RNOS that causes nitration and oxidative damage.

Name the major endogenous sources of ROS in cells.

Mitochondrial electron transport (Coenzyme Q leak), oxidases/oxygenases/peroxidases (peroxisomes), and phagocytic respiratory burst.

How does Coenzyme Q (CoQ) generate ROS?

The one-electron reduced form CoQH• can escape in membranes and transfer an electron to O2, forming superoxide (O2•−).

Why are mitochondria major ROS producers?

High rates of oxidative phosphorylation and electron transfer increase the probability of single-electron leak to O2 forming O2•−.

Give examples of physiologic enzymes that produce ROS as by-products.

Monoamine oxidase (makes H2O2), peroxisomal fatty acid oxidase (H2O2), and xanthine oxidase (O2•− or H2O2).

How can monoamine oxidase contribute to neurodegeneration?

MAO generates H2O2 during dopamine metabolism in substantia nigra; accumulated ROS can damage dopaminergic neurons (Parkinson’s link).

Why is xanthine oxidase important in ischemia-reperfusion injury?

During reperfusion xanthine oxidase reduces O2 to O2•−/H2O2 causing oxidative damage in reperfused tissues.

What is the respiratory burst?

A phagocyte process producing large amounts of ROS to kill microbes during phagocytosis.

What enzyme initiates the respiratory burst and what does it make?

NADPH oxidase transfers electrons from NADPH to O2 to generate superoxide (O2•−) in the phagosomal membrane.

What is the role of superoxide dismutase (SOD) in respiratory burst?

SOD catalyzes dismutation of superoxide to H2O2 and O2, converting O2•− into H2O2.

How does myeloperoxidase (MPO) contribute to microbial killing?

MPO in neutrophils converts H2O2 + Cl− → HOCl, which directly kills microbes via oxidation/halogenation.

What toxic ROS cascade follows MPO activity?

HOCl produced can convert to hypochlorite and react with O2 and metals to form highly reactive species including OH•.

What gives pus its green color in infections?

Myeloperoxidase (heme-containing enzyme) contributes to the greenish color of pus.

List the 6 steps outlined in respiratory burst (short form).

1) NADPH oxidase → O2•−, 2) SOD → H2O2, 3) MPO → HOCl, 4) Fenton → OH•, 5) NOS → NO•, 6) NO• + O2•− → ONOO−.

How is NO• produced during respiratory burst?

By inducible nitric oxide synthase (iNOS) using arginine as substrate; NO reacts to form RNOS.

Name major exogenous sources of ROS.

Ionizing radiation, certain drugs (anticancer, antibiotics), tobacco smoke, alcohol (MEOS), inorganic particles (asbestos/silica), and gases like ozone.

How does ionizing radiation produce ROS?

High-energy radiation ionizes water producing OH• and H• radicals that damage cellular components (basis of radiotherapy side effects).

What skin-protection advice was highlighted against UV-induced ROS?

Use sunblock minimum SPF 30 and reapply every two hours; avoid 10am–4pm sun exposure.

How do some drugs increase ROS?

Anticancer agents (e.g., bleomycin, methotrexate) and some antibiotics produce radicals during metabolism, increasing oxidative stress.

Why does tobacco smoke enhance lung oxidative damage?

Smoke contains oxidants and semiquinones; micro-hemorrhages deposit iron which via Fenton reaction generates OH•; increased neutrophils augment ROS.

How does excessive alcohol increase ROS?

High alcohol shifts metabolism to MEOS (microsomal ethanol oxidizing system) in microsomes, increasing oxidase/peroxidase activity and ROS production.

Define a “standard drink” as given in the notes?

One standard drink ≈13.7 g pure alcohol (0.6 oz): 12 oz beer (360 mL), 8 oz malt liquor (240 mL), 5 oz wine (150 mL), 1.5 oz spirit (45 mL).

How do inorganic particles (asbestos, silica) cause ROS-related lung injury?

Inhaled particles are phagocytosed, trigger respiratory bursts and chronic inflammation → sustained ROS release and lung tissue damage.

What is ozone’s oxidative property?

Ozone is a potent oxidizing agent (not a free radical) that degrades to OH• under physiological conditions and causes airway injury.

What cellular components are targeted by ROS?

Cell membrane lipids, organelle membranes, proteins/peptides, and DNA.

What is the initiation step of lipid peroxidation?

A hydroxyl radical (or other radical) abstracts a hydrogen from a PUFA (LH) forming lipid radical (L•).

What happens during propagation of lipid peroxidation?

L• reacts with O2 → LOO• (lipid peroxyl radical) which abstracts H from another lipid → continues chain reaction producing LOOH and new L•.

What are common degradation products of lipid peroxidation?

Malondialdehyde (MDA) and other aldehydes; MDA is soluble and appears in blood/urine as a marker.

How is lipid peroxidation terminated?

Lipid-soluble antioxidants (vitamin E) donate electrons to lipid radicals; two subsequent reductions convert vit E to oxidized form and stop the chain.

How many electrons can vitamin E donate in termination reactions?

Vitamin E can donate up to 2 electrons, neutralizing two lipid radicals before becoming fully oxidized.

What are the effects of membrane lipid peroxidation?

Disrupts bilayer structure, cross-links proteins, increases membrane permeability, and damages organelle membranes (e.g., mitochondria).

How can mitochondrial membrane damage escalate ROS production?

Mitochondrial membrane disruption allows escape of superoxide and impairs electron transport, increasing ROS generation.

Which amino acids are most susceptible to oxidative protein damage?

Histidine, arginine, cysteine, and methionine are particularly susceptible to OH• attack and oxidative modification.

What are consequences of oxidative protein damage?

Protein fragmentation, cross-linking, aggregate formation, increased susceptibility to proteolysis, and loss of function.

How does ROS cause DNA damage?

OH• generated near DNA causes strand breaks and base modification (e.g., guanine → 8-hydroxyguanine), causing mutations and replication errors.

What is 8-hydroxyguanine and its significance?

An oxidized base resulting from OH• attack on guanine that causes base mispairing and DNA mutations.

Name diseases associated with ROS-mediated injury (short list).

Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, aging (free radical theory), atherosclerosis, ischemia–reperfusion injury, cytokine storm in COVID-19.

How do ROS contribute to Parkinson’s disease?

ROS from dopamine metabolism (MAO-generated H2O2) and mitochondrial dysfunction lead to substantia nigra neuron loss and reduced dopamine.

What role do ROS play in Alzheimer’s disease?

Oxidative damage to lipids, proteins, and nucleic acids contributes to neurodegeneration and dementia.

Why is multiple sclerosis linked to ROS?

MS involves autoimmune inflammation and respiratory burst activities in CNS leading to ROS-mediated myelin and axonal damage.

What is the free radical theory of aging?

Aging results from cumulative oxidative damage to cellular macromolecules from aerobic metabolism and environmental oxidants.

How do ROS participate in atherosclerosis?

ROS-driven endothelial injury, macrophage infiltration and oxidation of LDL drive plaque formation and smooth muscle proliferation.

Explain ischemia–reperfusion injury in terms of ROS?

Reperfusion delivers O2 to ischemic tissue; xanthine oxidase and recruited phagocytes generate ROS causing further cell death and inflammation.

How do cytokine storms in SARS-CoV-2 (COVID-19) relate to ROS?

Cytokine-driven recruitment of neutrophils/macrophages → intense respiratory burst → ROS release that damages lung tissue and worsens respiratory failure.

Define oxidative stress?

A state where ROS production exceeds the cell’s antioxidant/repair capacity leading to net oxidative damage.

What is compartmentation as a cellular defense?

Segregation of ROS-producing reactions and antioxidants in organelles (e.g., CoQ in mitochondria, H2O2 enzymes in peroxisomes) to limit damage.

How does metal sequestration protect against ROS?

Proteins (ferritin, transferrin, ceruloplasmin, albumin) bind Fe/Cu to prevent participation in Fenton reactions that produce OH•.

Name major metal-binding proteins and their roles.

Ferritin binds cellular Fe3+ (storage), transferrin transports Fe3+ in plasma, ceruloplasmin binds copper, albumin binds Fe2+/Cu2+ weakly.

What cellular repair mechanisms counteract oxidative damage?

Removal of oxidized fatty acids, proteolytic degradation and resynthesis of damaged proteins, and DNA repair enzymes for strand/bas e repair.

What is the primary enzymatic defense against superoxide?

Superoxide dismutase (SOD) — it catalyzes O2•− → H2O2 + O2 and is the primary defense versus O2•−.

List SOD isoenzymes and localization (tabulated).

Cu/Zn SOD — cytosol; Mn SOD — mitochondria; Cu/Zn SOD — extracellular.

Mnemonic to remember SOD isoforms and locations?

“CuZn in the Cytosol, Mn Means Mitochondria, CuZn Outside” → Cu/Zn (cytosol & extracellular), Mn (mitochondria).

What is the biochemical reaction catalyzed by catalase?

2 H2O2 → 2 H2O + O2 (dismutation of hydrogen peroxide into water and oxygen).

Where is catalase mainly found and what tissues have highest activity?

Found in peroxisomes, cytosol, microsomes; highest in organs with many peroxisomes like liver and kidney.

Why is catalase important in phagocytes?

Catalase helps protect phagocytes from damage by H2O2 produced during their own respiratory burst.

What is glutathione (GSH)?

A tripeptide antioxidant (γ-glutamyl-cysteinyl-glycine) whose sulfhydryl (-SH) group donates electrons to reduce H2O2 and lipid peroxides.

What reaction does glutathione peroxidase catalyze?

2 GSH + H2O2 → GSSG + 2 H2O (reduces H2O2 and lipid peroxides to non-toxic forms).

Why is glutathione peroxidase a selenium enzyme?

Active site contains selenocysteine; selenium is essential for enzyme activity, linking dietary selenium to antioxidant defenses.

What does glutathione reductase do?

Uses NADPH (via FAD) to convert oxidized glutathione (GSSG) back to reduced GSH, restoring antioxidant capacity.

Why is NADPH important in antioxidant defense?

NADPH provides the reducing equivalents for glutathione reductase (and other reductive reactions) that regenerate antioxidants.

What metabolic pathway is a major source of NADPH?

The Pentose Phosphate Pathway (Hexose Monophosphate Shunt, HMP) — crucial for producing NADPH for antioxidant defense.

What structural feature is common to many nonenzymatic antioxidants?

A conjugated double bond system (alternating single and double bonds) that delocalizes electrons and stabilizes donated electrons.

Name important endogenous nonenzymatic antioxidants.

Uric acid and melatonin (both produced endogenously and contribute to antioxidant capacity).

How does uric acid act as an antioxidant?

Soluble plasma antioxidant that scavenges OH• and peroxy radicals and contributes major free-radical-trapping capacity of plasma.

What is melatonin’s antioxidant significance?

Melatonin (pineal hormone) is both hydrophilic and lipophilic, crosses membranes/BBB, and neutralizes ROS — linked to “beauty sleep” benefits.

Name key dietary (exogenous) antioxidants.

Vitamin E (alpha-tocopherol), Vitamin C (ascorbate), carotenoids (β-carotene, lycopene, lutein/zeaxanthin), and flavonoids (quercetin, EGCG, resveratrol).

What is Vitamin E’s primary antioxidant role?

Alpha-tocopherol is a lipid-soluble chain-breaking antioxidant that terminates lipid peroxidation by donating electrons to lipid peroxyl radicals.

How does Vitamin E get regenerated?

Vitamin C (ascorbate) can reduce oxidized tocopherol back to its active form, linking aqueous and lipid antioxidant systems.

Describe Vitamin C’s antioxidant mechanism?

Ascorbate donates one electron to radicals (becoming ascorbyl radical) and a second to become dehydroascorbate; it scavenges superoxide, H2O2, HOCl, OH•, and NO2.

Why is vitamin C unstable in aqueous solutions?

It oxidizes easily in solution and with light; more stable in dry/powder form; juices diluted/exposed to air lose antioxidant potency.

What are carotenoids and what do they do?

Terpenoid-derived lipophilic antioxidants with conjugated double bonds that quench singlet oxygen and scavenge lipid peroxyl radicals (e.g., β-carotene, lycopene).

What is β-carotene’s nutritional role?

A major precursor of vitamin A (retinol): 1 mol β-carotene can yield up to 2 mol retinol in intestine.

Name macular carotenoids and their function.

Lutein and zeaxanthin — concentrated in retina; support macular health and improve eyesight.