Lecture 2: Cell Injury & Oxidative Stress

Reversible vs. Irreversible Cell Damage

Cell function → death → ultrastructural changes → light microscopic changes → gross morphological changes

Vulnerable Systems

Membrane integrity

ATP generation/mitochondrial function

Protein synthesis/enzyme function

Genetic integrity

General Mechanisms

ATP depletion (decreased oxidative phosphorylation or glycolysis)

Ischemia/hypoxia

ROS

Loss of Ca²⁺ homeostasis

Loss of plasma membrane integrity

Mitochondrial damage

Oxygen Balance

Hypoxia (ischemia): Too little

Hyperoxia (oxidative stress, ROS): Too much

Ischemic + Hypoxic Cell Injury

Decreased oxidative phosphorylation → decreased ATP

• Decreased ATP-dependent pump function → increased intracellular Na⁺, H₂O, Ca²⁺, decreased intracellular K⁺ → ER/cell swelling, microvilli loss, membrane blebs

• Increased anaerobic glycolysis

  • Decreased glycogen

  • Increased lactic acid → decreased pH → nuclear chromatic clumping

• Ribosome detachment → decreased protein synthesis

Loss of Ca²⁺ Homeostasis

Influx of Ca²⁺ → increased Ca²⁺ release from mitochondria, smooth ER → increased cytosolic Ca²⁺

• Activation of cellular enzymes

  • Phospholipase → decreased phospholipids → membrane damage

  • Protease → disruption of membrane and cytoskeletal proteins → membrane damage

  • Endonuclease → nuclear damage

  • ATPase → decreased ATP

• Increased mitochondrial permeability transition → decreased ATP

HIF1

Hypoxia-inducble factor I (HIF1a–HIF1B heterodimer)

• Transcription regulator for 40+ genes, many glycolytic enzymes

• Binds hypoxia response elements in promoter regions of target genes

HIF1a low at normoxic conditions, HIF1B constitutively expressed

• Oxygen-dependent prolyl hydroxylases modify P402, P564 to allow VHL binding → recognition signal for ubiquitination → proteasome degradation

O₂^-’

Superoxide

ETC complex: I/III electron leak, increased when O₂ decreases (chain backup)

• Rapidly neutralized by SOD1/2 in cytosol/mitochondria

Xanthine oxidase: Xanthine + 2O₂ → uric acid + 2O₂^-’ + H⁺

NADPH oxidase (neutrophil respiratory burst): NADPH + 2O² → NADP + 2O₂^-’ + H⁺

Chronic Granulomatosis Disease

Failure to mount respiratory burst:

Inability to fight infections → granulomas, organ strictures, decreased lung tissue

Death from sepsis (eg. Staph aureus), fungal infection (eg. aspergillus)

H₂O₂

Hydrogen Peroxide

Superoxide dismutase (SOD): 2O₂^-’ + 2H⁺ → H₂O₂ + OH^-

Peroxisome oxidases + fatty acid metabolism

Catalase: 2H₂O₂ → 2H₂O + O₂

‘OH

Hydroxyl

Fenton reaction: H₂O₂ + Fe²⁺ → Fe³⁺ + ‘OH + OH^-

Haber-Weiss reaction: H₂O₂ + O₂^-’ → ‘OH + OH^- + O₂

Ionizing radiation: H₂O → ‘H + ‘OH

No endogenous defenses, reacts at diffusion control

Fenton catalysis: O₂^-’ + H₂O₂ + H⁺ → O₂ + H₂O + ‘OH

1. O₂^-’ + Fe³⁺ → O₂ + Fe²⁺

2. Fe²⁺ + H₂O₂ + H⁺ → Fe³⁺ + H₂O + ‘OH

B^o

Beta thalessemia major

Deficient/mutated hemoglobin B chain, relative excess of a-globin → insoluble a-globin aggregate

• Most erythroblasts die in bone marrow → anemia

• Few abnormal cells leave → destroyed in spleen → anemia

Anemia → tissue anoxia, increased EPO → marrow expansion → skeletal deformities

Increased dietary iron absorption, blood transfusions → deposition in heart + liver → systemic iron overload

• Transfusions monthly beginning early in life → Fe deposits life-threatening → cirrhosis, endocrine dysfunction, cardiomyopathy, diabetes

• Iron cannot be excreted, must be removed by chelation → deferoxamine (bacterial siderofore) given via infusion nightly (short t_1/2, no po efficacy)

Oxidative damage

DNA: 2-deoxyguanosine + ‘OH → 8-hydroxy-2-deoxyguanosine

Lipid peroxidation: LH → L’ → LOO’ → LOOH → low MW aldehydes (termination)

• Abstraction of H from another lipid chain → self-propagation

Unsaturated phospholipid R₂ chain can be oxidated to form an epoxide → peroxide → susceptibility to phospholipase A₂

Nrf2-Keap1

Transcription regulator for antioxidants, drug-metabolizing enzymes

• GSTs, NADPH-quinone oxidoreductase, haem oxygenase-1, thioredoxin, y-glutamyl cysteine transferase (regulates glutathione synthesis)

Keap1 binds Nrf2, localizes in cytosol for proteasome degradation

• ROS, electrophiles, sulfhydryls interact with Keap1 cysteine, freeing Nrf2 → Nrf2 translocates to nucleus with concomitant stabilization to activate genes for cytoprotective elements through cis-acting ARE or ERE

NF-kB

Oxygen-sensitive dimeric transcription regulator for >300 genes

• Stress response genes, acute phase proteins, regulators of apoptosis, cytokines and their modulators, growth factors, etc.

Activation mediated by ROS, potentially one major signaling molecule involved in oxidative stress

• Activated by H₂O₂, xanthine-oxidase-derived oxidative stress

• Activated alongside AP-1 in cells enriched with polyunsaturated fatty acids

• Activated alongside AP-1 by reactive nitrogen species (RNS)

• Blocked by antioxidants in some system

• May be inhibited by ROS as well?!

ROS/RNS not necessarily exclusive regulators, NF-kB not necessarily only messenger for ROS/RNS-mediated cellular effects