Reactions with target moleculesCellular deregulationRepair mechanisms

“Essentials of Toxicology”

by Klaassen Curtis D. and Watkins John B Chapter 3

Stages of toxicity…See Figure 3.1

1. Delivery

2a. Interaction with target molecule

2b. Alteration of biological environment

3. Cellular dysfunction

4. Repair or repair failure

Toxicity

No ToxicityNo/inadequate repair

Successful repair

1. Delivery

Delivery to target site

Concentration at target site

AbsorptionDistribution toward target

Re-absorptionActivation

EliminationDistribution away from target

ExcretionDe-activation

Toxicity

No Toxicity

Stages of toxicity…See Figure 3.1

1. Delivery

2a. Interaction with target molecule2b. Alteration of biological environment3. Cellular dysfunction4. Repair or repair failure

Mechanisms of toxicityMolecular targets are usually proteins, lipids, coenzymes, or

nucleic acids, but rarely carbohydrates

Three basic mechanisms– Formation of a stable non-covalent complex with receptor,

enzyme, cofactor– Induction of a physicochemical change, e.g. pH, pO2,

solvation, physical damage– Formation of reactive intermediate that binds covalently to

macromolecules and/or triggers immune response

Mechanism of action

Effect on specific biochemical process that leads to disruption/alteration of cellular function that eventually results in impaired physiological function (health effect)

(Transient or permanent…)

Symptom is the observed manifestation of a health effect (outward, macroscopic)

Mechanisms of action

• Disruption or destruction of cell membrane (oxidative species, e.g. radical species)

• Direct binding to cell molecule (CO+Hb; adducts, lead)

• Enzyme inhibition– Cofactor

• Inactivation (sequestration of cofactor)• Competition (replacement)

– Binding to active site • Directly (classic enzyme inhibitors)• Indirectly: toxic metabolite binds

See also Chapter 3 of Casarett and Doull’s “Toxicology”

• Secondary action: release of endogenous substance that causes damage (histamine, neuropeptides, metals displacement)

• Free-radical cascade reactions (damage to proteins, DNA, lipids, mitochondria)

• Structural analogue properties – Neuroendocrine context– Receptor involvement– Agonists (mimic action of endogenous substance)– Antagonists (block action of endogenous substance)

Mechanisms of action

Cytochrome oxidase inhibition by cyanide stops mitochondrial respiration

Mechanism of action - dioxin

http://www.stanford.edu/group/whitlock/research.html

Metabolism of bromobenzene to reactive

epoxide intermediates which deplete glutathione

and cause liver toxicity

Metabolism of halothane leads to direct and indirect (immune) toxicity

Carbon tetrachloride toxicity via free radical formation

Redox cycling of herbicide Paraquat produces reactive oxygen species

2GSH

HOOH(H2O2)2H2O

GSSG

HOOH(H2O2)

O2

CAT

HOOH

2H2O

2H+

O2- .

O2- .

SOD

O2

HOOH

GPX

Coupling reactions:

Effects of oxidative species on proteins:

Oxidation of:

• sulphydryls• amines• alcohols• aldehydes

Inactivation/inhibition of enzymes in cellular compartments

Aminoacids targets:

• cystein• methionine• tryptophan• tyrosine

Effects of oxidative species on lipids:

• Polyunsaturated fatty acids (PUFA): primary target of O3 peroxidation of membrane lipids• Most important mechanism of O3-induced injury

O3 + PUFA carbonyl oxideH2O

Hydroxyhydroperoxy compound

HO.

H2O2Lipid peroxidation cascade

aldehydes

Lipid fragmentationMalondialdehyde (MDA)8-isoprostaneLTB4 (PMN chemotractant)

Lipid peroxidation

cascade

Effects on nucleic acids

Electrophiles react with strong nucleophilic atoms of nucleic acids

DNA + HO. Imidazole ring-opened purines or ring-contracted pyrimidines

Strand breaksBlocked DNA replication

Formation of adducts depurination (apurinic sites: mutagenic)

Reactions with target molecules• Non-covalent

– Receptors– Ion channels– Enzymes– Co-factor depletion

• Covalent binding– DNA– Proteins

• H removal (neutral radicals)– Amino acid CH2

– Proteins

• e- transfer– Hemoglobin Fe2+ hemoglobin Fe3+ (methemoglobin)

• Enzymatic reactions– Protein toxins (diphtheria, cholera)

Effects on target molecules

• Dysfunction– Mimics endogenous molecule– Inhibition, blocking (receptors, ion channels)– Conformational change– DNA mis-pairing

• Destruction– Cross linking– Fragmentation– Oxidation/degradation (lipids)

Effects on target molecules

• Antigenicity Immune responseUnchanged– Dinitrobenzene– Nickel– Penicillin

Following change– Quinones – Biotransformation products

Hapten formation and immune reaction: penicillin G

Stages of toxicity…See Figure 3.1

1. Delivery

2a. Interaction with target molecule

2b. Alteration of biological environment3. Cellular dysfunction

4. Repair or repair failure

Alteration of biological environment

• Alter pH (methanol, ethylene glycol, 2,4-dinitrophenol)

• Solvents and detergents

• Direct chemical effect (phosgene, sulfuric acid)

• Physical space occupation (silica, asbestos, ethylene glycol, CO2)

Ethylene glycol toxic metabolites

Stages of toxicity…See Figure 3.1

1. Delivery2a. Interaction with target molecule2b. Alteration of biological environment

3. Cellular dysfunction4. Repair or repair failure

3. Cellular impairment

1. Cell regulation (fig. 3.6)

A. Gene expressiona. Transcription

b. Signal transduction (fig. 3.7)

c. Extracellular signal (hormone)

B. Cellular activity (table 3.1)

a. Excitable cells - neurotransmission

b. Other cells (Kupffer, exocrine, pancreatic)

2. Internal maintenancea. ATP depletion (Fig. 3.8, table 3.2)

Oxidative phosphorylationb. Intracellular Ca+ increase (Table 3.3)

Influx to cytosol» from outside (channels, membrane)» from mitochondria/ER

Efflux out of cytosol» Ca+ transporters» ATPase inhibition

c. ROS, RNS, radicals ATP

Cellular impairment

Effects of increased cytosolic Ca+• Inhibition of ATPase

– Mito loading with Ca2+– Dissipation of membrane potential– Reduced ATP synthesis, oxidative phosphorylation and Ca2+

cycling

• Microfilament dissociation– Membrane rupture

• Hydrolysis - enzyme increase– Protein, phospholipids, DNA

• ROS, RNS production– Ca2+ activates dehydrogenases in citric acid cycle --> e- transport

increase --> ROS, RNS

Inter-relationships

ATP Ca2+ in cytosol

Ca2+ channels that control cytosolic Ca2+ need ATP

Mito potential

Ca2+ ROS, RNS

Inactivated pump

Inter-relationships

ROS, RNS ATP

DNA damage PARP NAD+

Enzyme inhibition

ONOO-

Mito Permeability Transition

Ca2+ uptakeMembrane potentialROS, RNSATP

MPT

Mitochondrial damage leads to cell death

Pores open (1500 Da)

Influx of protons, negative potentialCa2+ from mito to cytosol

ATP synthesis

Osmotic H20 influx

Mito swelling

ATP hydrolysisBurst

Glycolysis

Energy

Two options for cell death

http://www.roche-applied-science.com/prodinfo_fst.htm?/apoptosis

Robertson JD & Orrenius S.

Critical Rev. Toxicology 2000, Sep; 30(5):609-27

“Molecular mechanisms of apoptosis induced by cytotoxic chemicals”

MTP - cell death

Necrosis

• Extensive damage

• All mito

• Multiple metabolic defects

• Random sequence

• ATP severely depleted

• Cell swelling and lysis

Apoptosis

• Less extensive

• Some/many mito

• Some metabolic defects

• Ordered sequence

• Some ATP available

• Cell shrinkage, membrane bound fragments

Stages of toxicity…See Figure 3.1

1. Delivery2a. Interaction with target molecule2b. Alteration of biological environment3. Cellular dysfunction

4. Repair or repair failure

Levels of repair

Molecular repair

• Proteins reduction (re-activation) NADPH

• Protein refolding (heat-shock proteins)

• Protein degradation and re-synthesis

• Lipid reduction (GPO, GR, NADPH)

• DNA repair

DNA damage repair

• Direct: photolyase (UV-dimers, O6-methyl-G removal)

• Excision

DNA glycosylase (removes AP site)

AP endonuclease (PO3 bond)

DNA polymerase (replicates sequence)

Ligase (ties the ends)

PARP (multiple ADP ribose - unfolds/facilitates repair)

• Recombination

Sister chromatid exchange

Cellular/Tissue repair

• Single cell - regeneration (neurons)

• Tissue – Apoptosis

– Proliferation • Chemokine priming (G0-G1) :TNFa, IL-6

• Chemokine progression (G1-GM) :HGF, TGFa

– Migration

– ECM (Stellate cells, PDGF, TGFb)

InflammationMacro’s

IL-1, TNFa

endothelia, fibroblastsVascular dilation

Leukocyte infiltrationRelease of PAF, LTB4, cytokines

Leuko-endo adhesion

Side reactions - Inflammatory oxidative burst

Three pathways of HO. generation:

• NAD(P)H oxidase (macro’s and granulo’s)• Nitric oxide synthase (NOS) (macro’s)• Myeloperoxidase (MPO) (granulo’s)

HO.

NAD(P)H + O2 O2.

NAD(P)+H+

Fenton

HOOH + H+ +Cl- HOClMPO

Oxidase

L-arginine + O2 NO.NOS

H+

NO2

.

O2

Cl-

L-citruline

H20

More side reactions

• Gene expression – Cytokines IL-6, IL-1, TNFa– Acute phase proteins

• Minimize injury• Facilitate repair (inhibit lysosomal proteases)• Plasma proteins • CYP450, GSTs (detox)

• Generalized reactions– Fever (IL-1, IL-6, TNFa) hypothalamus– Pituitary (ACTH --> cortisol) (negative feedback)

Repair failure

• Tissue necrosis– No apoptosis, no proliferation, dose matters

• Fibrosis– ECM deposition– TGFb matrix synthesis, autocrine control

Repair failure cont.

• Carcinogenesis– Failure to repair DNA in:

• Proto-oncogenes - activating mutation (GF, recept., TF, signal transduction proteins)

• Tumor suppressor genes - inactivating mutation(p53, protein kinase inhibitors, TF)

– Failure of apoptosis– Failure to stop proliferation

• Mutation accumulation• Repair is less likely• Neoplastic transformation (reduced methylation)• Reduced cell-cell contact• Inhibition of cell-matrix contact

Factors determining specificity• Sensitivity

– Neurons and heart require high levels of O2 to make ATP via mitochondrial respiration; CO and CN are therefore very toxic to these organs.

– Bone marrow and gut epithelium contain rapidly dividing cells; mitotic substances damage these tissues.

• Distribution– Inorganic mercury (Hg) cannot cross the blood-brain

barrier; methylmercury can.

• Selective uptake– Strontium 90 into bone instead of calcium– Paraquat mistaken for polyamines in lung cells

• Metabolism– Localized activation in Clara cells of lung– Absence of detoxification: eye lacks

formaldehyde dehydrogenase (methanol blindness)

• Lack of repair mechanism– Liver has high capacity to remove O6-

alkylguanine but brain capacity is low

Factors determining specificity