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Mechanisms of Toxicity
To understand
 how a toxicant enters an organism
 how it interacts with target molecules
 how the organism deal with the insult
To provide a rational basis for
 interpreting descriptive toxicity data
 estimating the probability that a chemical will cause
harmful effects
 establishing procedures to prevent or antagonize
the toxic effects
 designing drugs and industrial chemicals that are
less hazardous
 developing pesticides that are more selectively
toxic for their target organisms
Example for better understanding of
fundamental physiologic and biochemical process
 Cancer and carcinogen
 Parkinson’s disease and MPTP
Step 1-Delivery:from the site of exposure to the target
Step 2a-Reaction of the ultimate toxicant with the target
molecule
Step 2b- Alteration of biological environment
Step 3-Cellular dysfunction, injury
Step 4-Inappropriate repair or adaptation
Ultimate toxicant is the chemical species that reacts
with the endogenous target molecule or critically
alter the biological environment, initiating structural
and /or functional alteration that result in toxicity.
 Parent compounds
 Metabolites of parent compounds
 Reactive oxygen or nitrogen species
 Endogenous molecules
Absorption vs. presystemic elimination
Influencing factors for absorption
 concentration of the chemical at the absorbing
surface
 the area of the exposed site
 the characteristics of the epithelial layer
 the intensity of the subepithelial microcirculation
 physicochemical properties of the toxicant-lipid
solubility
Presystemic elimination
 Usually for chemicals absorbed from GI tract
first pass through GI mucosal cells, liver, and lung
Mechanisms facilitating distribution to a target
Porosity of the capillary endothelium
in the hepatic sinusoids
in the renal peritubular capillaries
Specialized transport across the plasma membrane
ion channels
protein transporters
endocytosis-toxicant-protein complex
membrane recycling
Accumulation in cell organelles (lysosomes and mitochondria)
amphipathic xenobiotics with a protonable
amino group and lipophilic character
Reversible intracellular binding
organic and inorganic cations and PAH bind /release to
melanin (polyanionic aromatic polymer)
Homework p48
1. Explain the mechanism of cardiac toxicity
of lipophilic local anethetics ( e.g. tetracaine,
bupivacaine).
2. Why amine ( e.g. amiodarone) can cause
phospholipidosis?
3. Why melanin-containing cells are more
sensitive to cations and polycyclic
aromatics?
Mechanisms opposing distribution to a target
Binding to plasma protein
DDT and TCDD are bound to high M.W. protein
or lipoprotein
Specialized barriers (for hydrophilic toxicants)
blood-brain barrier
reproductive cells
Distribution to storage sites (where they do not exert effects)
Association with intracellular binding proteins
metallothionein
Export from cells by ATP dependent transports
multidrug-resistance protein (P-glycoprotein)
in brain cappilary endothelial cell, oocyte
stem cell, and tumor cell
Excretion
 Hydrophilic, ionized chemicals
Renal glomeruli-hydrostatically filter
Proximal renal tubular cells-active transport
Hepatocyte
 Nonvolatile, highly lipophilic chemicals
Excretion by the mammary gland
Excretion in bile in association with biliary micelles
and /or phospholipid vesicles
Intestinal excretion
 Volatile, nonreactive toxicant
Pulmonary capillaries into the alveoli
Reabsorption
•Renal tubule
diffusion-lipid solubility, ionization (pH)
carriers and transporterspeptide transporter sulfate transporter (chromate & molybdate),
phosphate transporter (arsenate)
•Intestinal mucosa
Biliary, gastric, and intestinal excretion
secretion by salivary glands and exocrine pancreas
lipid solubility
Toxication (metabolic activation)
•Formation of electrophilic Metabolites (table3-2)
molecules containing an electron-deficient atom with
partial or full positive charge
insertion of an oxygen atom
conjugated double bonds are formed
Heterolytic bond cleavage, C-O
•Free radials
accepting an electron from reductases (fig.3.3)
losing an electron and form free radical by peroxidase
homolytic fission of a covalent bond
(CCl4
CCl3. , HO., Fenton reaction)
•Nucleophiles (relatively uncommon)
HCN from amygdalin, CO
•Redox-active reactants
Detoxication
No functional groups
add a functional group (OH,COO) by cytP450
then endogenous acid (glucuronic acid, sulfuric acid) by
transferase
Nucleophiles
Conjugation at the nucleophilic functional group (OH, SH)
Electrophiles (Metal ion, etc)
conjugated with the SH of glutathione
specific mechanism:
epoxide hydrolase-epoxidediols, arene dihydrodiols
carboxylesterase
DT-diaphorase
alcohol dehydrogenase
Free radicals
O2. - -.superoxide dismutase
HOOH-glutathione peroxidase, catalase
peroxyl radical-glutathione, -tocopherol, ascorbic acid
ONOO--selenocysteine-containing glutathione peroxidase,
selenoprotein P, oxyhemoglobin, heme-containig
peroxidase, albumin
peroxidase-generated free radical-electron transfer from
glutathione
Protein toxin-extra- and intracellular protease
toxins with disulfide bond are inactivated by
thioredoxin
Prx(SH)2
PrxS2
2HOH
chlopromazine
peroxidase
Homework p54
Describe at least 3 ways to prevent
peroxynitrite (ONOO-) buildup.
When detoxication fails
Toxicants may overwhelm detoxication process
 exhaustion of the detoxication enzymes
 consumption of the cosubstrates
 depletion of cellular antioxidants
Toxicant inactivates a detoxicating enzyme
ONOO-incapacitates Mn-SOD
Some conjugation reactions reversed
Sometimes detoxication generates potentially harmful
byproducts
ex. glutathione thiyl radical (GS.)
glutathione disulfide (GSSG)
Attributes of target molecules
DNA, protein, membrane lipids, cofactor
 Appropriate reactivity and/or configuration
 Accessibility-endogenous molecules that are in
the vicinity of reactive chemicals or are
adjacent to sites where they are formed
ex. enzyme responsible for production of reactive
metabolites or the adjacent intracellular
structures
 Critical function-not all targets for chemicals
contribute to the harmful effects
ex. CO for Hb but not cytP450
Types of reactions
Noncovalent binding
Hydrogen bond, ionic bond
ex. Interaction of toxicants with receptors, ion channels,
and some enzymes
Covalent binding
covalent adduct formation
Hydrogen abstraction
R-SH, RSOH
Electron transfer
Fe(II)Fe(III)
enzymatic reactions
ADP ribosylation-diphthera toxin, cholera toxin
Hydrogen abstraction
Dysfunction of target molecules
Activation- agonist, activator
Inhibition- antagonist
Alteration in conformation or structure of protein- thiol group
Interference with template with the function of DNA
aflatoxin bind to G: GC GA
.
HO  8-hydroxyguanine and 8-hydroxyadenine 
mispairing
Destruction of target molecules
Cross-linking
Fragmentation
spontaneous degradation after chemical attack
hydrolytic degradation
Neoantigen formation
Covalent binding altered protein evoke immune response
drug-protein adduct
Toxicity not initiated by reaction with
target molecules
1. Chemicals that alter H ion concentrations
Acids and substance biotransformed to acids
protonophoric uncoupler
2. Solvents and detergents alter the lipid phase of cell
membrane and destroy transmembrane solute gradients
3. Occupying a site or a space
ethylene glycol form water insoluble precipitates in the
renal tubules
sulfomides occupy bilirubin binding sites of albumin
Dysregulation of gene expression
 Dysregulation of transcription
Promoter region of the gene
Transcription factors (TFs)
ligand-activated (Table 3-4)
altering the regulatory region of the genes
direct chemical interaction –thalidomide/GCbox
methylation of cytosine
Dysregulation of signal transduction
 Dysregulation of the synthesis, storage, or release of
the extracellular signaling molecules
Systemic lupus erythemathosus
Induced by
Procainamide
Hydrolazine
Inhibit DNA methylation
in CD4+T lymphocyte
Overexpression of protein for
inflammation
TCDD hypermethylation in
Insulin-like growth factor-2 gene
Dysregulation of signal transduction
*sinaling molecules to activate TFs ( c-FOS,
c-JUN, c-Myc) that control transcriptional
activity of genes that influence cell cycle
 Altering protein phosphorylation
 by kinases,  by phosphatases
 Interfering with the GTPase activity of G protein
 Disrupting normal protein-protein interaction
 Altering the synthesis or degradation of the
signaling proteins
Extracellulr
Signaling
molecules
Chemically altered signal transduction with proliferative effect
Chemically altered signal transduction with antiproliferative effect
Dysregulation of ongoing cellular activity
dysregulation of electrically excitable cells (Table 3-5)
due to an alteration in
 the concentration of neurotransmitters
 receptor function
 intracellular signal transduction
 the signal terminating process
dysregulation of the activity of other cells
ex.liver cells possess -1 adrenergic receptors
exocrine secretory cells controlled by Ach
receptor
Toxic alteration of cellular maintenance
Impairment of internal cellular maintenance:
mechanism of cell death
 ATP depletion (Table 3-6)
Ca accumulation (Table 3-7)
ROS/RNS generation
.
Sustained elevation of intracellular Ca2+
can result in :
1. Depletion of energy reserve
 mitochondria Ca2+ uptake dissipate membrane
potential
 continuous Ca2+ uptake and export causing
oxidative injury to inner membrane
 impair ATP synthesis
  ATP consumption by the Ca2+ -ATPase (eliminate
the excess Ca2+
2. Dysfunction of microfilaments
dissociation of actin filaments from -actinin
and fodrin (anchor proteins)  membrane blebbing
3. Activation of hydrolytic enzymes
calpains
phospholipases
Ca2+ -Mg2+ dependent endonuclease
4. Generation of ROS and RNS
Mitochondrial permeability transition (MPT)
Mitochondrial inner-membrane permeability caused
by opening of a proteinaceous pore (megachannel)
free influx into the matrix space of protons
rapid and dissipation of membrane potential and
cessation of ATP synthesis
osmotic influx of water mitochondrial swelling
 apoptosis or necrosis
Induction of cell death by unknown mechanisms
1. Chemicals directly damage the plasma membrane
lipid solvents, detergents, venom-drived hydrolytic
enzymes
2. Xenobiotics that damage the lysosomeal membrane
aminoglycoside, hydrocarbons binding to a2u-globulin
3. Toxins that destroy the cytoskeleton
microfilament toxins-phalloidin and cytochalasins
microtubular toxins-colchicine, 2,5-hexanedione
4. Protein phosphatase inhibitor cause hyperphosphorylation
mycrocystin
5. Toxins that disrupt protein synthesis--amaitin and ricin
6.Cholesterol lowing drug statin –inhibit HMG coenzyme, myotoxi
DNA repair
Direct repair
DNA photolyase-cleavge dimerized pyrimidine
O6-alkylguanine-DNA-alkyltransferase-remove minor
adducts
Excision repair
Base excision-DNA glycosylase
Nucleotide excision-ATP dependent nuclease
poly(ADP-ribose) polymerase (PARP)
poly(ADP-ribose) glycohydrolase
Recombination (or postreplication) repair
When excision repair fail to occur before DNA
replication begins
Cellular repair: A strategy in peripheral neurons
Macrophages-remove debris and produce cytokine and
growth factors
Schwann cells-proliferate and transdifferentiate from
myelinating operation mode into a growth-supporting
mode
  synthesis of cell adhension molecules (N-CAM)
 Elaborating excellular matrix protein for base
membrane construction
 Producing neurotrophic factors and their receptors
 Comigrating with the regrowing axon, physically guide
and chemically lure the axon to reinnervate the target
cell
Tissue repair
Apoptosis: an active deletion of damaged cells
Proliferation: regneration of tissue
Side reactions to tissue injury
Apoptosis
cell shrinks
apoptotic bodies
phagocytosed
orderly process
without inflammation
Necrosis
cell and organelles swell
membrane lysis
disorderly process
induce inflammation
Proliferation : Regeneration of tissue
Replacement of lost cells by mitosis
After injury, intracellular signaling turns on
§ Activation of protein kinase and TF
§ Immediately early genes-transcription factors and
like secreted protein
§ Delayed early genes-antiapoptotic protein
§ Cell cycle accelerators (cyclin D)
§ Cell cycle decelerators (p53, p21)
§ Mediators of tissue repair and side reactions
Replacement of the extracellular matrix
Proteins, glycosamineoglycans, glycoprotein and
proteoglycan glycoconjugates
Matrix metalloproteinase
cytokine-
IEG
Growth factors
Side reaction to tissue injury
•Inflammation
Cells and mediators
tissue damage resident M secreting cytokines
endothelial cells and fibroblasts release mediator
Alteration of the microcirculation
Accumulation of inflammatory cells (leukocyte)
chemoattractant
selectins on the membrane of endothelial cells
ligand on the surface of leukocyte
adhesion
ICAM on endothelial cells
integrins on the membrane of leukocyte
Production of ROS and NOS
M and leukocytes
•Altered protein synthesis: acute-phase proteins
positive acute-phase proteins
minimize tissue injury and facilatating repair
ex. 2-macroglobulin, 1-antiprotease inhibit lysosomal
protease released from the injured cell
metallothionein complexes metals
Negative acute-phase proteins
plasma proteins-albumin, transthyretin, transferrin
Cytochrome P450
Glutathione S-transferase
•Generalized reaction
Cytokines evoke neurohormonal responses
ex. IL-1
sickness behavior
ACTH release
Mechanisms of adaptation
Adaptation by decreasing delivery to the target
-Repression of iron absorption
-Induction of ferritin and metallothionein
-Induction of detoxication
Adaptation by decreasing the target density or responsiveness
-induction of opioid tolerance
Adaptation by increasing repair
-induction of enzymes repairing oxidized proteins (Fig 3-23)
-induction of chaperones repairing misfolded proteins
heatshock response, ER stress response
-induction of enzymes repairing DNA (p53)
-adaptive increase in tissue repair (NF-κB)
Mechanisms of adaptation
Adaptation by compensating dysfunction
Adaptation to hypoxia –the hypoxia response
(HIF-1)
Adaptation to energy depletion-energy stress
response (AMPK)
Adaptation by neurohormonal mechanisms
Toxicity resulting from dysrepair
 Tissue Necrosis
 Fibrosis-excessive disposition of an extracellular
matrix of abnormal composition
 Carcinogenesis
Failure of DNA repair: mutation, the initiating event in
carcinogenesis
Failure of apoptosis:promotion of mutation and clonal
growth
Failue to terminate proliferation:promotion of mutation,
protooncogene overexpression, and clonal growth
Nongenotoxic carcinogens:promotors of mitosis and
inhibitors of apoptosis
Conclusions
An organism has mechanisms that
1. Counteract the delivery of toxicant, such as detoxication
2. Reverse the toxic injury, such as repair mechanisms
3. Offset some dysfunctions, such as adaptive responses
 Toxicity is not an inevitable consequence of toxicant
exposure.
 Toxicity develops if the toxicant exhausts or impairs the
protective mechanisms and/or overrides the adaptability
of biological systems.
Homework
1. Describe the types of ultimate toxicant .
2. How detoxication of free radical exert?
3. What will occur following reaction of ultimate toxicant with
endogenous molecule?
A. at molecular level
B. at cellular level
4. What are the three major processes to impair the internal cellular
maintenance and cause cell death?
5. Why is the sustained rise of intracellular calcium level harmful?
6. Describe how a cell to repair proteins, lipids, and DNA?
7. Explain the heatshock response and ER stress response.
8. What are the possible outcomes when repair fails?