Lecture 2: Toxicity, ROS and Oxidative Stress

What are Reactive Oxygen Species?

• Chemically reactive molecules containing oxygen

• Highly reactive and short-lived in living systems → rapidly react in the cell/tissue components in which they are generated

• Generated as byproducts of metabolism, e.g. respiration during ATP production

• React with and damage cellular components

  • Impair cellular function,

  • Causing DNA mutations

  • Cause cell death

• Physiological roles, e.g., the immune system. cell signalling

• Toxicants can lead to their excessive generation and oxidative stress

What are the main ROS Species?

• Superoxide (O₂•⁻)

  • O₂ molecule gains an extra electron → free radical

  • Reactive, can initiate damage

• Hydroxyl radical (HO•)

  • Highly reactive & extremely damaging

  • No cellular defenses

• Hydrogen peroxide (H₂O₂)

  • Not a radical (no unpaired electron)→ less reactive and damaging

  • Can undergo Fenton reaction (non-physiological) with Fe²⁺/Cu⁺ producing HO• formation, causing damage

• Singlet oxygen (¹O₂)

  • Molecular O₂ in a high-energy unstable state

  • Highly damaging → produced as a consequence of toxicity

  • No physiological defenses

What is Oxidative Stress and How is it Normally Controlled

• The imbalance between ROS production and removal

• Normally, antioxidants in cells/ tissues:

  • Neutralise ROS

  • Prevent ROS accumulation

  • Minimise cellular damage

What happens when ROS overwhelm antioxidants in cells?

• When ROS are generated at high levels (and overcome antioxidants), this gives rise to oxidative damage

• Oxidative damage is a frequent manifestation of disease and toxicant-induced cell damage, causing:

  • DNA breakage/mutagenesis

  • Protein aggregation, fragmentation, or inactivation

  • Lipid peroxidation

  • Destruction of small molecules → impaired cell function

  • May promote cell death

What are the Enzyme-Free Antioxidant Defence Mechanisms?

• One of two cellular mechanisms that aim to protect against oxidative damage

• This includes:

  • Reduced Glutathione

  • Vitamin C (Ascorbate): a hydrophilic compound present in the aqueous compartment of the cell

  • Vitamin E (a-tocopherol): hydrophobic compound → antioxidant defence mechanism in membranes

What are the Enzyme-Dependent Antioxidant Defence Mechanisms?

• One of two cellular mechanisms that aim to protect against oxidative damage

• This includes:

  • Catalase

  • Glutathione peroxidase

  • Peroxiredoxin

  • Glutaredoxin

  • Thioredoxin

  • Superoxide dismutase

How does antioxidant depletion affect cells’ sensitivity to oxidative stress?

• Depletion or impairment of antioxidants (by toxicants or poor nutrition) can make cells more vulnerable to ROS

• Removal of protective mechanisms increases oxidative damage

• Cellular defences are critical; without them, even normal ROS levels can cause harm

Why are cellular antioxidants limited in ROS detoxification:

• Role: Scavenge free radicals to reduce oxidative damage

• But

  • HO• inefficiently scavenged

  • H₂O₂ not removed

• Some ROS need enzyme detoxification for removal

What is Reduced Glutathione and how does it participate in redox cycling?

• Major cellular antioxidant

  • Present at high levels at physiological conditions (~10–14 mM)

• Composed of 3 amino acids (central residue: Cys)

• Key functional group: sulfhydryl (-SH) on Cys → can be (readily) oxidised to GSSG

• GSSG = 2 GSH molecules dimerised and linked via a disulfide bridge (S–S bond)

• GSSG can be:

  • Reduced back to 2 GSH by Glutathione reductase

  • Exported (lost) from cells

• Normal ratio: GSH:GSSG ≈ 100:1

How does GSH function in oxidative stress and Phase II detoxification?

• In oxidative stress, GSH:GSSG drops to ~1:1 (GSSG levels increase)

• In Phase II detoxification, GSH is conjugated and covalently attached to xenobiotics, where it is destroyed and no longer participates in antioxidant defence

• Use as a cofactor for enzyme-dependent oxidative defences:

  • Needed for glutathione-dependent antioxidant enzymes

• GSH Synthesis is slow, rate-limited by cysteine availability (~10 µM)

What happens if GSH removal exceeds replenishment?

• Glutathione depletion → reduced antioxidant capacity

• Prevents direct ROS removal

• Blocks glutathione-dependent enzyme defenses

• Reduces Phase II detoxification of xenobiotics

• Increases cellular vulnerability to oxidative damage

How do enzyme-dependent ROS detoxification mechanisms work, and why do some cells remain sensitive to ROS?

• Enzymes have evolved to specifically remove ROS or reduce oxidised molecules

• Do not all rely on GSH

• Their expression varies between tissues/cell-types → differences in ROS sensitivity :

  • Pancreatic beta cells: poor oxidative defense → sensitive to ROS

  • Kidney & liver: high enzyme expression → less ROS sensitive

• Limitations: ineffective for HO• and ¹O₂

What cofactors are needed for enzyme-dependent ROS detoxification, and what happens if they are deficient?

• Metal cofactors essential for redox reactions

  • Many enzymes rely on Selenium (Se)

    • Incorporated in selenoproteins via selenocysteine (amino acid)

    • Se deficiency → impaired antioxidant defence

• Some enzyme detoxifications also require GSH → GSH depletion impairs enzyme-dependent ROS removal

• Glutathione peroxidases requires both Se and GSH

How Does Superoxide Dismutase (SOD) Act As An Enzyme-Dependent ROS Detoxification Mechanism?

• Substrate: Superoxide (O₂•⁻)

• Product: Hydrogen peroxide (H₂O₂)

• Notes:

  • Only route for removing superoxide

  • Does not require GSH or Se

  • Hydrogen peroxide must then be detoxified by catalase

How Does Catalse (CAT) Act As A Enzyme Dependent ROS Detoxification Mechanism?

• Substrate: Hydrogen peroxide (H₂O₂)

• Product: H₂O + O₂

• Notes:

  • High levels in liver

  • Low levels in pancreatic beta cells, brain, and endothelium

  • Does not require GSH or Se

How Does Peroiredoxin Act As A Enzyme Dependent ROS Detoxification Mechanism?

• Substrate: H₂O₂ and organic peroxides

• Product: Reduced peroxide

• Cofactor: Requires thioredoxin reductase (TrxR, selenoprotein) to regenerate the active form

• Notes:

  • PRX6 is an exception: it uses GSH instead of TrxR

How Does Gutahione Peroxidase (GPX) Act As An Enzyme-Dependent ROS Detoxification Mechanism?

• Substrate: Peroxides (H₂O₂); GPX4: lipid hydroperoxides in membranes

• Product: Reduced peroxide

• Cofactors: Most require GSH & Se; GPX5 requires TrxR

• Notes:

  • GPX4 Detoxifies lipid hydroperoxides in membranes

  • Most are selenoproteins

  • Se deficiency → impaired antioxidant defence

How Does Glutaredoxin (GRX) Act As An Enzyme-Dependent ROS Detoxification Mechanism?

• Substrate: Oxidised cysteine residues in proteins

• Product: Reduced cysteine (-SH)

• Cofactor: Requires GSH

• Notes: Repairs proteins oxidized by ROS at Cystine residues

How Does Thioredoxin (Trx) Act As A Enzyme Dependent ROS Detoxification Mechanism?

• Substrate: Oxidized cysteine residues in proteins

• Product: Reduced cysteine (-SH)

• Cofactor: Requires thioredoxin reductase (TrxR, selenoprotein) to regenerate the active form

• Notes:

  • Protective effect on proteins oxidised by ROS, reducing and regenerating the SH group on cystine side chain

  • Works in tandem with PRX for peroxide detoxification

What happens to ROS defense if both GSH and Se are depleted?

• Only SOD and Catalase remain functional

• Other enzyme-dependent defenses (PRX, GPX, GRX, Trx) fail

• Antioxidant defense is severely compromised

What is Lipid Peroxidation?

• Occurs in cells and membranes rich in polyunsaturated fatty acids (PUFAs)

• Highly reactive ROS (e.g. hydroxyl radical) attacks polyunsaturated fatty acids, destroying itself

• Generates an unstable carbon-centred radical from fatty acid structure

• Radical reacts with oxygen to produce peroxyl radical

• Peroxyl radical attacks an adjacent polyunsaturated fatty acid

• Produces:

  • Lipid hydroperoxide (Generated by first ROS attack)

  • New carbon-centred radical (Generated by second ROS attack)

How is Lipid Peroxidation Propagated and How Is it Stopped?

• Forms a chain reaction:

  • Peroxyl radicals attack neighbouring lipids

  • Generates more carbon-centred radicals + lipid hydroperoxides

  • Damage spreads across the membrane

• Stops when:

  • Antioxidants neutralise radicals (e.g. vitamin E)

  • Two radicals react together and ablate each other

What are the damaging effects of lipid peroxidation?

• Lipid hydroperoxides break down and decay to reactive aldehydes (e.g. MDA, 4-HNE)

• These react with cellular components, causing:

  • Altered membrane fluidity

  • Loss of membrane integrity

  • Altered membrane protein function

  • DNA/protein adduct formation

  • Immune response to modified proteins

  • Cell damage or death

How Do ROS Damage Proteins

• Amino acid oxidation (especially cysteine)

  • Alters protein function → location dependent

  • Can cause enzyme activation/inactivation if located in the active site

• Protein cross-linking / aggregation

  • Disulfide bond formation

• Metal ion release (e.g. Fe²⁺)

  • Promotes Fenton reaction with H2O2 → more ROS

• Adduct formation

  • Reaction with aldehydes (formed from lipid peroxidation)

  • Can trigger immune response

• Carbon-centred radical formation

  • Decay produces aldehydes → DNA/protein adduct formation → immune response and damage

• Peptide bond cleavage

  • Protein fragmentation → impaired function

How Does ROS Damage DNA?

• Base oxidation (especially guanine)

• Abasic sites (complete removal of DNA bases)

• Single- and double-strand breaks of sugar phosphate backbone)

• Altered DNA methylation (epigenetic effects)

• DNA adduct formation from lipid/protein oxidation products (e.g. MDA, 4-HNE)

• This can result in cell death, mutation or increased cancer risk (may develop years after exposure to oxidative insult)

Why is Mitochondrial DNA More Vulnerable to ROS Mediated DNA Damage?

• No histones → less protection

• Mitochondria are major sites of ROS generation

How Do ROS Damage The Small Molecule Nitric Oxide (NO)?

• NO, an important mediator generated by vascular endothelium, reacts with superoxide to produce peroxynitrite (ONOO⁻)

• This results in:

  • Depletion of NO → impaired VSMC contraction and neurotransmission

  • Cardiovascular damage from excess superoxide

  • Peroxynitrite damages proteins → altered function

• NO normally regulates VSMC contraction, Neurotransmission and Cardiovascular health

How Do ROS Damage The Small Molecule Reduced Glutathione (GSH)?

• Vulnerable while scavenging ROS (protective mechanism) → can be oxidised

• Excess oxidation → GSH depletion

• This results in:

  • Reduced antioxidant defence

  • Impaired Phase II detoxification

  • Increased oxidative stress

  • Promotes cell death

How Do ROS Damage The Small Molecule dGTP?

• ROS oxidises free dGTP

• Oxidised dGTP incorporated into newly synthesised DNA

• Leads to mutations during DNA replication

How Do Toxicant Species Exert Harmful Effects Via ROS?

• Via

  • Direct features of the molcule

  • Indirect interactions with features of the cell

How do toxicants generate ROS via Redox Cycling?

• The toxicant does not target a specific molecule but a modification in the body generates ROS through cycling reactions

• Mechanism:

1. Electron donated to Toxicant (e.g. via Cytochrome P450 metabolism) → generates unstable radical

2. Radical donates an electron to molecular O₂ → generates Superoxide

3. The original toxicant is regenerated

4. Process repeats continuously, undergoing further cycles → large ROS (superoxide) production without removing original toxicant

• One toxicant molecule can generate many ROS molecules without being consumed

How does Paraquat (Week Killer) cause toxicity?

• Mechanism: Redox cycling → ROS generation

  • Metabolised by Cytochrome P450 allows the addition of an electron and generates an unstable radical

  • Radical donates an electron to O₂ → generates superoxide

  • Original paraquat regenerated → continuous ROS production

• This leads to toxic effects of:

  • Lung fibrosis (enters via polyamine uptake system in lung cells)

  • Nephrotoxicity

  • Long-term exposure linked to Parkinson's disease

  • Hearing loss

How Do Toxicants Generate ROS via Phototoxicty?

• Toxicant does not directly target molecules → Light converts it into an unstable form that causes ROS generation

  • Toxicant is inactive in the dark (ground state)

  • Light exposure converts the toxicant into an excited unstable form

  • Excited toxicant generates ROS

What is the Mechanism of ROS Generation Via Phototoxicity

• Toxicant in ground state (inert; inactive in the dark)

• Light exposure at a given wavelength excites the toxicant → enters the singlet excited state

• Converted to the triplet excited state

• Decays via two pathways

  • Type 1 reaction:

    • Interacts with various cellular molecules and molecular substrates

    • Generates superoxide and hydroxyl radicals

    • Toxicant/photosensitiser destroyed or returns to ground state and regenerates → repeated ROS production in presence of light (continual excitation and decay)

  • Type 2 reaction:

    • Converts O₂ → Singlet Oxygen

    • Highly reactive and damaging ROS

What are examples of Phototoxicants?

• Hypericin

  • A plant alkaloid from St John's Wort

  • Herbal remedy used for low mood

  • Causes phototoxicity in humans and grazing animals

• Chlorpromazine

  • Antipsychotic

  • Causes hyperpigmentation of sun-exposed skin (face, neck, and hands most affected)

How is Phototoxicity Used Therapeutically?

• Phototoxicity is used to selectively destroy diseased tissue using light-activated ‘photosensitiser’ drugs

  • Light activates photosensitiser leading to ROS generation and selective cell destruction

• Used for:

  • Skin lesions

  • Some cancers

  • Macular degeneration of retina

  • Pathogen destruction (alternative to antimicrobials)

How does phototoxic (photodynamic) therapy work?

• Photosensitiser drug applied in the dark (inactive)

• Drugs accumulate in diseased tissue

• Controlled light exposure (a directed beam of light) is applied and activates the drug

• ROS generation and selective destruction of abnormal cells, e.g. tumour cells, abnormal blood vessels

What are the Ideal Properties for a photosensitiser?

• Low (negligible) toxicity in dark

• Accumulates in target tissue

• Rapid clearance from healthy tissue

• Activated by controlled light pulses (wavelength, intensity, duration) applied to tissue

Why is Type 1 phototoxicity preferred in tumours? Give an example drug.

• Tumour cores are hypoxic (low oxygen)

• Type 2 reactions require O₂ → less effective in tumours

• Type 1 reactions preferred (do not require O₂)

• Example: 5‑Aminolevulinic Acid

  • Pro-drug, metabolised to protoporphyrin IX

  • Used in cancer treatment

How do Mitochondria Contribute to ROS Generation in Cells?

• Mitochondria are a major physiological source of ROS.

• During ETC, electrons are normally transferred to acceptors to generate a proton gradient to produce ATP.

• Sometimes, electron leakage/donation to O₂ produces superoxide (O₂•⁻).

• Normally, ~2% of O₂ is converted to superoxide during respiration

• As a result, mitochondria have antioxidant defences: SOD, GPX, PRX3.

How Do Toxicants Increase ROS Via Mitochondria Damage

• Mitochondria contain many iron-rich proteins leading to oxidative damage and the release of Fe²⁺ ,

• This leads to the Fenton reaction with H₂O₂ producing more ROS.

• This damages Complex I or III, increasing superoxide generation.

Which toxicants generate ROS by interfering with the mitochondrial ETC?

• Rotenone (plant insecticide): inhibits Complex I → Parkinson’s in rats, long-term use in humans correlates with PD

• Barbiturates, haloperidol, chlorpromazine, and some local anaesthetics: inhibit Complex I

• Cadmium (heavy metal): inhibits Complex III, and can displaces Fe²⁺ promoting Fenton reaction and ROS

• Mitochondria are a major ROS source, and toxicants can amplify oxidative damage

Where is the microsomal electron transport system found and what is its role in ROS generation?

• Present in the ER (microsomes) of most tissues, e.g. liver and kidney; enriched in tissues exposed to xenobiotics.

• Involved in Phase I metabolism, which aims to mono-oxygenate xenobiotics to facilitate removal or inactivation via oxidation of reactive groups.

• Key components:

  • Cytochrome P450 (~50+ isoforms in humans)

  • Flavoprotein reductase → forms a complex with associated proteins

• Normally oxidises xenobiotics, but can generate ROS (superoxide;O₂•⁻) if reactions are “leaky” and electrons are donated directly to oxygen

How Do CYP450 Enzymes Contribute to ROS Production?

• Poorly coupled (“leaky”) CYP450 isoforms donate electrons directly to O₂, generating superoxide (O₂•⁻) instead of completing xenobiotic oxidation.

• Reaction: NADPH + O₂ + H⁺ + xenobiotic → NADP⁺ + H₂O + oxidized xenobiotic

• Amount of ROS varies depending on:

  • Substrate type

  • Isoform of CYP450

How Does Xenobiotic Exposure Influence ROS Generation via CYP450?

• Xenobiotics may induce CYP450 expression in some tissues, increasing the number of metabolising enzymes.

• If induced CYP450 is “leaky,” superoxide generation increases.

• Example: Chronic ethanol exposure leads to more CYP450 and more ROS production.

What substrates does CYP2E1 metabolize and how does it generate ROS?

• Substrates: Ethanol and paracetamol

• Mainly converts ethanol to acetaldehyde

• Also generates high levels of superoxide (O₂•⁻) and a reactive radical from ethanol, hydroxyethyl radical (HER)

• Usually, only 10% of ethanol is metabolised by CYP2E1, with low ethanol consumption still producing ROS

How does ethanol consumption affect CYP2E1 activity and ROS production?

• Inducible isoform: activity increases 10–20-fold in ethanol-fed rats, and shows a marked increase following moderate ethanol consumption in humans

• Higher blood ethanol concentration (increased consumption or taken over a longer period) → more substrate and enzyme present with more ethanol metabolised via CYP2E1 (~40%)

• Saturable enzyme: at high ethanol concentrations, CYP2E1 contributes more to ethanol metabolism

• Results in increased superoxide and HER production

What is the role of CYP2E1-mediated ROS in alcoholic liver disease?

• Superoxide from CYP2E1 is strongly implicated in alcoholic liver disease and contributes to oxidative liver damage

  • ALD responsible for 70–80% of UK alcohol-related deaths

• Evidence from rodent models:

  • CYP2E1 knockout: less oxidative DNA damage after ethanol vs WT following ethanol consumption

  • SOD1 knockout: moderate ethanol → liver necrosis & inflammation

  • SOD overexpression (Tg rodents): protection against ethanol-induced liver injury

How do enzymes contribute to ROS production in response to toxicants?

• Many enzymes generate superoxide (O₂•⁻) and/ or H₂O₂ as by-products in response to toxicants

• Some enzymes have evolved and are physiologically designed to produce ROS for normal functions.

• Toxicants can perturb these enzymes, causing excess ROS and cellular damage.

What is the role of NADPH oxidases (NOX) in ROS production, and how do toxicants affect them?

• Physiological Function: Specifically produce ROS (e.g., NOX2 → oxidative burst in neutrophils to kill pathogens via superoxide)

• Toxicant effect: Cigarette smoke or other toxicants may activate NOX, generating excess ROS (via activation of upstream signalling pathways)

• Shows how toxicants hijack normal enzyme functions to cause oxidative damage

How Can Toxicants Cause NOS to Procue ROS instead of NOS?

• Physiological function: normally generates nitric oxide (NO); requires BH4 (tetrahydrobiopterin) as a cofactor

• BH4 is sensitive to oxidative damage → loss of BH4 “uncouples” NOS

• Uncoupled NOS produces superoxide instead of NO

• Toxicants (e.g., diesel fumes, cigarette smoke) can trigger this uncoupling, causing ROS-mediated damage

How does xanthine oxidase contribute to ROS production, and how can toxicants exacerbate this?

• A specific form of xanthine oxidoreductase generates superoxide and H₂O₂ during purine metabolism

• Toxicants (e.g., cigarette smoke) can increase ROS production, especially in vascular endothelium

• Example of a normal physiological enzyme subverted by toxicants

How is ROS exploited therapeutically in medicinal honey?

• Used for thousands of years in traditional remedies for its antimicrobial properties.

• Contains glucose oxidase (added by bees to honeycomb) → converts glucose to H₂O₂, generating ROS that suppresses microbial growth.

• Renewed interest due to antibiotic resistance: used in wound gels and creams to reduce post-surgical infections and kill microorganisms.