Toxicology I PHTX 831 Spring 2023

Mechanism of Toxicity II

Tutorial 4

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Course ILOs

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 Tutorial Intended learning outcomes

⮚ Assess & select medicines based on the mechanism of toxicity

⮚ Understand the principles of body function in disease states as well as

basis of genomic pathways underlying the mechanism of toxicity.
⮚ Identify different types of Toxicant-induced cellular Dysregulation
⮚ Apply various principles to determine the toxicity mechanism of
biopharmaceutical products.
⮚ Identifying the different mechanisms of toxicity repair and dysrepair.

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 Tutorial Outline

⮚ Potential stages in the development of toxicity after chemical exposure

⮚ Step 3: Cellular dysfunction and resultant toxicities
Toxicant-induced cellular Dysregulation:
I) Impairment in Cell Regulation (signaling):
1) Dysregulation of gene expression:
i) Dysregulation of Transcription
ii) Dysregulation of signal Transduction
iii) Dysregulation of Transcription through altering regulatory regions of genes
2) Dysregulation of Ongoing cellular activity
II) Impairment of internal cellular maintenance (i.e. mechanism of toxic cell death)

⮚ Step 4: Repair or Dysrepair

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 Potential stages in the development of toxicity
after chemical exposure

⮚ Step 1:Delivery from the site of exposure to the target

⮚ Step 2:Reaction of the ultimate toxicant with the target
molecule
⮚ Step 3: Cellular dysfunction and resultant toxicities
⮚ Step 4: Repair or Dysrepair

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Step 3: Cellular dysfunction and resultant
toxicities Dysregulation of Transcription

Dysregulation of gene Dysregulation of signal

expression Transduction
Impairment in Cell
Regulation (signaling):
Dysregulation of Transcription
through altering regulatory
regions of genes

Dysregulation of
Ongoing cell function
molecule
Role of
target

ATP depletion

Impaired internal Sustained rise in

maintenance
intracellular Ca2+

Impairment of cellular Impaired external

Overproduction of ROS
maintenance maintenance and RNS
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Cell Signaling → Regulation of Gene expression
Signaling
Molecules

P
Signal transduction

TF
Transcription

mRNA

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Step 3: Cellular dysfunction and resultant
toxicities Dysregulation of Transcription

Dysregulation of gene
expression
Impairment in Cell
Regulation (signaling):

Transcription factors
For Ex:
molecule
Role of
target

▪ Estrogens (Diethylsterbesterol & Ethynyl estradiol)

▪ Glucocorticoids (Dexamethasone)
▪ Retinoic acids (13-cis-Retinoic acid)

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Step 3: Cellular dysfunction and resultant
toxicities Dysregulation of Transcription

Dysregulation of gene Dysregulation of signal

expression Transduction
Impairment in Cell
Regulation (signaling):
Dysregulation of Transcription
through altering regulatory
regions of genes

Dysregulation of
Ongoing cell function
molecule
Role of
target

ATP depletion

Impaired internal Sustained rise in

maintenance
intracellular Ca2+

Impairment of cellular Impaired external Overproduction of ROS

maintenance maintenance and RNS
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Step 3: Cellular dysfunction and resultant
toxicities
Dysregulation of gene Dysregulation of signal
expression Transduction
Impairment in Cell
Regulation (signaling):

Phosphorylation De-phosphorylation

⮚ Activation of PKC by For Ex: ⮚ Inhibition of Phosphatases

molecule

⮚ For Ex:
Role of
target

▪ Phorbol ester & Fumonisin B

(Mimics DAG) ▪ Inhibition of PP2A by
▪ Pb2+ (Mimics Ca2+) ▪ Oxidative stress, UV
⮚ Phosphorylation of inhibitory radiation & Arsenite
binding proteins eg.:
▪ ROS stimulates IkB kinase (IKK)
causing IkB phosphorylation
and dissociation from NF-kB
(enter nucleus as TF) 10
Step 3: Cellular dysfunction and resultant
toxicities Dysregulation of Transcription

Dysregulation of gene Dysregulation of signal

expression Transduction
Impairment in Cell
Regulation (signaling):
Dysregulation of Transcription
through altering regulatory
regions of genes

Dysregulation of
Ongoing cell function
molecule
Role of
target

ATP depletion

Impaired internal Sustained rise in

maintenance
intracellular Ca2+

Impairment of cellular Overproduction of ROS

Impaired external
maintenance
maintenance
and RNS
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Step 3: Cellular dysfunction and resultant
toxicities
Dysregulation of gene
expression
Impairment in Cell
Regulation (signaling):
Dysregulation of Transcription
through altering regulatory
regions of genes

Regulatory regions
molecule
Role of
target

⮚ By direct interaction
For Ex:
▪ Thalidomide that intercalates GC boxes impairing
IGF-1 & FGF-2 → Phocomelia
⮚ By changing methylation pattern
For Ex:
▪ Procainamide & Hydralazine that inhibits DNA
methylation in CD4+ T lymphocytes → SLE
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Q1. Dysregulation of transcription can be due
to interaction of …………….with……………:
a. Exogenous molecule, Transcription initiation complex

b. Endogenous compound, Transcription factors

c. Exogenous compound, Promoter region

d. All of the above

e. All of the above except b

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Q2. Match each toxicant with the correct receptor
from Column A and effect from Column B

Toxicant Column A Column B

1. 13-cis Retinoic acid B , D a. Estrogen receptor d. Craniofacial
malformation in fetus

2. Dexamethasone C,F b. RAR e. Mammary and

hepatic carcinogenesis

c. Glucocorticoid
3. Ethynylestradiol A, E
receptor f. Cleft palate &
apoptosis of
lymphocytes

4. Diethylstilbsterol A, E 14
Q3. Alteration of retinoic acid receptor (RAR)
function has been associated with ……….

a) Cardiac and vascular toxicity

b) CNS and peripheral nerve toxicity

c) Hepatic and renal toxicity

d) Embryo toxicity

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Q4. Alteration of regulatory regions of genes,
dysregulates transcription by which of the
following:

A. Direct interaction of thalidomide with GC boxes

B. Development of phocomelia by thalidomide

C. Inhibition of DNA methylation by procainamide

D. Induction of SLE in Hydralazine

E. All of the above

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Q5. Dysregulation of gene expression can be
due to dysregulation of signal transduction by:

A. Increasing phosphorylation of transcription factors through

activation of kinases
B. Activation of dephosphorylation of transcription factors
leading to mitosis and tumor formation
C. Inhibition of dephosphorylation of transcription factors by
protein phosphatases
D. A & B
E. A & C

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Q6. MAPK is
a) A gene encoding the protein Cyclins D and E that
accelerate cell cycle

b) A protein kinase signal molecule

c) A dephosphatase enzyme

d) A signal-activated TF

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Q7. Activation of TFs control the transcriptional
activity of the genes, determining the fate of
cells by:

A. Leading to cell cycle progression (mitosis) only.

B. Leading to cell cycle arrest (apoptosis) only.

C. May lead to apoptosis or/and mitosis

D. None of the above

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 Q8. Complete the following sentences

Pb2+ Fumonisin B UV Arsenate NF-KB Phorbolester PP2A

1. Protein kinase C (PKC) can by activated by xenobiotics such
as……………… & ……………………..which mimic the endogenous
molecule DAG and………….. Which mimics the Ca2+

2. Oxidative stress,……….. And………………… Inhibit the

dephosphorylation of transcription factors by
inhibiting……………….

Phosphorylation Dissociation of Increased

3. . ROS of IKB IKB from ……….. proliferation

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Step 3: Cellular dysfunction and resultant
toxicities Dysregulation of Transcription

Dysregulation of gene Dysregulation of signal

expression Transduction
Impairment in Cell
Regulation (signaling):
Dysregulation of Transcription
through altering regulatory
regions of genes

Dysregulation of
Ongoing cell function
molecule
Role of
target

ATP depletion

Impaired internal Sustained rise in

maintenance
intracellular Ca2+

Impairment of cellular Impaired external Overproduction of ROS

maintenance maintenance and RNS
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Step 3: Cellular dysfunction and resultant
toxicities
ATP depletion

Impaired internal
maintenance

Impairment of cellular
maintenance
1-ATP depletion
molecule
Role of
target

by For ex:
▪ P-benzoquinone & Ethanol inhibit H+ delivery
to ETC
▪ CCL4 inhibit transport of e- to ETC
▪ Cocaine & CO inhibit O2 delivery to ETC
▪ DDT inhibits delivery of ADP & P to ATP
synthase

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Step 3: Cellular dysfunction and resultant
toxicities Dysregulation of Transcription

Dysregulation of gene Dysregulation of signal

expression Transduction
Impairment in Cell
Regulation (signaling):
Dysregulation of Transcription
through altering regulatory
regions of genes

Dysregulation of
Ongoing cell function
molecule
Role of
target

ATP depletion

Impaired internal Sustained rise in

maintenance
intracellular Ca2+

Impairment of cellular Impaired external Overproduction of ROS

maintenance maintenance and RNS
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Step 3: Cellular dysfunction and resultant
toxicities
Impaired internal Sustained rise in
maintenance
intracellular Ca2+

Impairment of cellular
maintenance

2-Inc. in intracellular Ca2+ by:

molecule
Role of

⮚ Inc. Ca 2+ influx to Cytoplasm by Glutamate receptor

target

agonists for ex:

▪ Glutamate
⮚ Inhibition of Ca 2+ export to EXC & ER by inhibition of
Ca 2+ -ATPase by for ex:
▪ Acetaminophen & Bromobenzene

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Step 3: Cellular dysfunction and resultant
toxicities Dysregulation of Transcription

Dysregulation of gene Dysregulation of signal

expression Transduction
Impairment in Cell
Regulation (signaling):
Dysregulation of Transcription
through altering regulatory
regions of genes

Dysregulation of
Ongoing cell function
molecule
Role of
target

ATP depletion

Impaired internal Sustained rise in

maintenance
intracellular Ca2+

Impairment of cellular Impaired external Overproduction of ROS

maintenance maintenance and RNS
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Step 3: Cellular dysfunction and resultant
toxicities Overproduction of ROS
and RNS

Impaired internal
maintenance

Impairment of cellular
maintenance
molecule
Role of
target

3-Overproduction of ROS & RNS

⮚ As they inactivate respiratory chain complexes
I,II &III → ATP production
⮚ ONOO- : Can induce DNA single strand breaks

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Q9. Impairment of internal cell maintenance:

A. It is a toxic cell death

B. Can be caused by ATP depletion

C. Happens due to increased extracellular Calcium

D. Can be caused by ROS and RNS

E. All of the above except c

F. B and D

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Q10. Chemicals cause major cell death by all of
the following mechanisms except:

a.Rise in intracellular calcium

b.ATP depletion

c.Inhibition of the drug metabolizing enzymes

d.Overproduction of reactive oxygen species

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Q11. Match each toxicant with the mechanism
by which it causes ATP depletion
1. P-benzoquinone C A. Impair transport of electrons
to ETC

2. CCl4 ( electron acceptor) A B. Impair delivery of ADP & P to

ATP synthase

3. Cocaine (Ischemia) D C. Impair delivery of hydrogen to

ETC

4. DDT B D. Impair delivery of oxygen to

ETC 29
Q12. The agents that directly interfere with the
cellular energy production are as follows Except:

a. CO

b. Cocaine

c. Vitamin A

d. Ethanol

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Q13. A rise in the intracellular calcium
leads to toxic cell death through:

a. Inhibition of ATP synthesis by decreasing

mitochondrial membrane potential
b. Increasing consumption of ATP by Ca2+ ATPase
c. Activation of phospholipases, proteases and
endonucleases
d. Activation of xanthine oxidase and NOS, forming
ROS and RNS
e. All of the above

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Q14. Activation of hydrolytic enzymes ,
proteases, endonucleases and Phospholipases
leads to all except

a.Overproduction of ATP molecule

b.Aggravates hypercalcemia

c.Damage to the plasma membrane

d.Overproduction of ROS

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Q15. Mention the mechanism of toxic cell death
1. Inhibition of Ca2+ ATPase by acetaminophen
⮚ Rise in intracellular Ca2+
2. Inactivation of respiratory chain complexes I, II, III by superoxide
anion radical
⮚ Overproduction of ROS
3. Impairment of ADP and inorganic phosphate delivery to ATP synthase
⮚ ATP depletion
4. Induction of DNA single Strand breaks by peroxy nitrite
⮚ Overproduction of RNS
5. Impairment of oxygen delivery to the electron transport chain by
carbon monoxide
⮚ ATP depletion
6. Activation of ligand-gated channels in neurons by Glutamate receptor
agonists
⮚ Rise in intracellular Ca2+
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Step 4: Repair or Dys-repair:

Repair of proteins

Molecular Repair of lipids

Repair Mechanism

Repair of DNA

Autophagy of damaged
cell organelles
Cellular
Regeneration of
damaged axons

Apoptosis
Replacement of lost
Tissue
cells
Proliferation
Replacement of
Extracellular matrix
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 Tissue repair (Regeneration of lost cells)
• Release chemical mediators
Damaged cells

• e.g. resident macrophages and endothelial cells

Non-parenchymal
cells

• + dormant cell to move from G0 to G1 phase

TNF-α, IL-6
(priming stage)

• drive primed cells into mitosis

HGF, TGF-α

Tissue repair (Regeneration of ECM)

Stellate cells Collagen
Damaged TGF-β and ECM
& Fibronectin
platelets PDGF components
Fibroblasts Proteoglycans
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Q16. For the following examples; mention the enzyme or the growth
factor that is responsible for the repair and the type of the repair
mechanism

1. Conversion of Methemoglobin to hemoglobin

⮚ NADH cytochrome b5 (Molecular repair)
2. Activation of stellate cells and fibroblasts to synthesize collagen
and fibronectin
⮚ TGF-B & PDGF ( Tissue repair)
3. Cleavage of the O6-methyl that is attached to guanine
⮚ O6-methyl guanine DNA methyl transferase; MGMT (Molecular repair)
4. Enhancement of the cell to move from G0 to G1 phase by non-
parenchymal cells
⮚ TNF-a & IL-6 (Tissue Repair)
5. Transfer of multiple ADP-ribose moieties from NAD+ to repair
DNA single strand breaks
⮚ poly (ADP-ribose) polymerase enzyme (PARP), (Molecular repair) 36
Q17. TGF- β (Transforming growth factor β)
a) It triggers the release of secondary signaling molecules to
initiate cell division
b) It mimics Ca2+, a physiological protein kinase activator to
promote cell division
c) It triggers the activation of stellate cells during tissue
repair
d) It binds to nuclear receptors to initiate cell proliferation

Q18. Apoptosis is most advantageous in ………..

a) Neoplastic prostate cells
b) Female germ cells
c) Cardiac myocytes
d) CNS neurons 37
Step 4: Repair or Dys-repair:

DysRepair
Mechanism

Necrosis Fibrosis Carcinogenesis

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Q19. Fibrosis is caused by……….

a) Abnormal ECM deposition

b) TGF-β overproduction

c) TGF-a overproduction

d) A & b

e) All of the above

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Q20. The main driving force which controls the
transformation of a normal cell to a malignant cell is
regulated by the balance between

a) Tumor suppressor gene- apoptosis

b) Oncogene- proto-oncogene

c) Proto-oncogene- Tumor suppressor gene

d) Oncogene- necrosis

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Good Luck
☺
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