Post on 12-Jan-2016
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