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 eliminationInfluencing 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

Specialized transport across the plasma membrane

Accumulation in cell organelles (lysosomes and mitochondria)

Reversible intracellular binding

in the hepatic sinusoids

in the renal peritubular capillaries

ion channels

protein transporters

endocytosis-toxicant-protein complex

membrane recycling

amphipathic xenobiotics with a protonable

amino group and lipophilic character

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 targetBinding 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 transporters-

peptide 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

DetoxicationNo functional groups

add a functional group (OH,COO) by cytP450

then endogenous acid (glucuronic acid, sulfuric acid) by trans

ferase

Nucleophiles

Conjugation at the nucleophilic functional group (OH, SH)

Electrophiles (Metal ion, etc)

conjugated with the SH of glutathione

specific mechanism:

epoxide hydrolase-epoxidediols, 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, se

lenoprotein P, oxyhemoglobin, heme-containig peroxida

se, albumin

peroxidase-generated free radical-electron transfer from

glutathione

Protein toxin-extra- and intracellular protease

toxins with disulfide bond are inactivated by

thioredoxin

2HOHPrx(SH)2 PrxS2

chlopromazine

peroxidase

Homework p54

Describe at least 3 ways to prevent peroxynitrite (ONOO-) buildup.

When detoxication failsToxicants 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 stru

ctures

Critical function-not all targets for chemicals

contribute to the harmful effects

ex. CO for Hb but not cytP450

Types of reactionsNoncovalent binding

Covalent bindingcovalent adduct formation

Hydrogen abstractionR-SH, RSOH

Electron transfer

enzymatic reactions

Hydrogen bond, ionic bond ex. Interaction of toxicants with receptors, ion channels,

and some enzymes

ADP ribosylation-diphthera toxin, cholera toxin

Fe(II)Fe(III)

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 DNAaflatoxinbind to G: GC GA

HO.

8-hydroxyguanine and 8-hydroxyadenine mispairing

Destruction of target molecules

Cross-linking

Fragmentation spontaneous degradation after chemical attackhydrolytic degradation

Neoantigen formation

Covalent binding altered protein evoke immune responsedrug-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 spaceethylene glycol form water insoluble precipitates in the renal tubulessulfomides 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 t

he extracellular signaling molecules

Systemic lupus erythemathosusInduced by ProcainamideHydrolazineInhibit DNA methylation in CD4+T lymphocyteOverexpression 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

ExtracellulrSignalingmolecules

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 maintenanceImpairment of internal cellular maintenance:

mechanism of cell death

ATP depletion (Table 3-6)

Ca accumulation (Table 3-7)

ROS/RNS generation.

tcu

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 mechanisms1. 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, myotoxicity

DNA repair

Direct repairDNA photolyase-cleavge dimerized pyrimidineO6-alkylguanine-DNA-alkyltransferase-remove minor adducts

Excision repairBase excision-DNA glycosylaseNucleotide excision-ATP dependent nuclease

poly(ADP-ribose) polymerase (PARP)poly(ADP-ribose) glycohydrolase

Recombination (or postreplication) repairWhen excision repair fail to occur before DNA repli

cation 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 Necrosis

cell shrinks cell and organelles swell

apoptotic bodies membrane lysis

phagocytosed

orderly process disorderly process

without inflammation induce inflammation

Proliferation : Regeneration of tissueReplacement of lost cells by mitosis

After injury, intracellular signaling turns on

§ Activation of protein kinase and TF

§ Immediately early genes-transcription factors and cytokine-l

ike 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 proteogly

can glycoconjugates

Matrix metalloproteinase

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 proteinspositive acute-phase proteins minimize tissue injury and facilatating repair ex. 2-macroglobulin, 1-antiprotease inhibit lysosomal

protease released from the injured cellmetallothionein 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 adaptationAdaptation 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 growt

h

Failue to terminate proliferation:promotion of mutation,

protooncogene overexpression, and clonal growth

Nongenotoxic carcinogens:promotors of mitosis and inhibit

ors of apoptosis

Conclusions

An organism has mechanisms that

1. Counteract the delivery of toxicant, such as detoxication2. 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.

Homework1. Describe the types of ultimate toxicant .

2. How detoxication of free radical exert?

3. What will occur following reaction of ultimate toxicant with endog

enous 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?