Transcript Reactions with target molecules Cellular deregulation Repair

Reactions with target molecules
Cellular deregulation
Repair 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
Successful repair
No Toxicity
No/inadequate repair
Toxicity
1. Delivery
Delivery to target site
Concentration at target site
Absorption
Distribution toward target
Re-absorption
Activation
Toxicity
Elimination
Distribution away from target
Excretion
De-activation
No Toxicity
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
Mechanisms of toxicity
Molecular 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”
Mechanisms of action
• 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
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–
–
–
Neuroendocrine context
Receptor involvement
Agonists (mimic action of endogenous substance)
Antagonists (block action of endogenous substance)
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
Coupling reactions:
(H2O2)
HOOH
CAT
2H2O
HOOH
O2
2H+
O2- .
SOD
HOOH
O2- .
O2
GSSG
2GSH
(H2O2)
2H2O
HOOH
GPX
Effects of oxidative species on proteins:
Oxidation of:
• sulphydryls
• amines
• alcohols
• aldehydes
Aminoacids targets:
• cystein
• methionine
• tryptophan
• tyrosine
Inactivation/inhibition of enzymes in cellular compartments
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 oxide
aldehydes
H2O
Hydroxyhydroperoxy compound
.
HO
H2O2
Lipid peroxidation cascade
Lipid fragmentation
Malondialdehyde (MDA)
8-isoprostane
LTB4 (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 breaks
Blocked DNA replication
Formation of adducts
depurination (apurinic sites: mutagenic)
Reactions with target molecules
• Non-covalent
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–
–
–
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
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–
–
–
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 response
Unchanged
– 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 environment
3. Cellular dysfunction
4. Repair or repair failure
Alteration of biological
environment
• Alter pH (methanol, ethylene glycol, 2,4dinitrophenol)
• 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. Delivery
2a. Interaction with target molecule
2b. Alteration of biological environment
3. Cellular dysfunction
4. Repair or repair failure
3. Cellular impairment
1. Cell regulation
(fig. 3.6)
A. Gene expression
a. 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)
Cellular impairment
2. Internal maintenance
a. ATP depletion
(Fig. 3.8, table 3.2)
Oxidative phosphorylation
b. 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
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
Ca2+ channels that control cytosolic Ca2+ need ATP
Ca2+ in cytosol
ATP
Mito potential
Ca2+
ROS, RNS
Inactivated pump
Inter-relationships
Enzyme inhibition
ROS, RNS
ATP
ONOO-
DNA damage
PARP
NAD+
Mito Permeability Transition
Ca2+ uptake
Membrane potential
ROS, RNS
ATP
MPT
Mitochondrial damage leads to cell death
Pores open (1500 Da)
Ca2+ from mito
to cytosol
Influx of protons, negative potential
ATP synthesis
Osmotic H20 influx
Glycolysis
Energy
Mito swelling
ATP hydrolysis
Burst
Two options
for cell death
Robertson JD & Orrenius S.
Critical Rev. Toxicology 2000, Sep;
30(5):609-27
“Molecular mechanisms of apoptosis
induced by cytotoxic chemicals”
http://www.roche-applied-science.com/prodinfo_fst.htm?/apoptosis
MTP - cell death
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•
•
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Necrosis
Extensive damage
All mito
Multiple metabolic defects
Random sequence
ATP severely depleted
Cell swelling and lysis
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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. Delivery
2a. Interaction with target molecule
2b. Alteration of biological environment
3. Cellular dysfunction
4. Repair or repair failure
Levels of repair
Molecular repair
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•
•
•
•
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)
Inflammation
Macro’s
IL-1, TNFa
endothelia, fibroblasts
Vascular dilation
Leukocyte infiltration
Release of PAF, LTB4, cytokines
Leuko-endo adhesion
Side reactions - Inflammatory oxidative burst
.
Three pathways of HO generation:
• NAD(P)H oxidase
• Nitric oxide synthase (NOS)
• Myeloperoxidase (MPO)
L-arginine + O2
L-citruline
NOS
H+
.
.
O
2
NAD(P)+
H+
HOOH + H+ +Cl-
NO2
.
NO
Oxidase
NAD(P)H + O2
(macro’s and granulo’s)
(macro’s)
(granulo’s)
MPO
H20
HOCl
Fenton
HO
O2
Cl-
.
More side reactions
• Gene expression
– Cytokines IL-6, IL-1, TNFa
– Acute phase proteins
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•
•
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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
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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
Factors determining specificity
• 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 O6alkylguanine but brain capacity is low