Summary Molecular and Cellular Toxicology

Summary Molecular and Cellular
toxicoloog
and transporters
Lecture 2 :
signaaltransductie .
ubiquiti nation

Defence induced stress
against drug -




metabolisme
Drug
-




Antioxidant response
-



Apoptosis

Stress responses


-



Induction protective enzymes
protein rekking
-




protein degradatie / resynthes.is
-




ftp.optosis
'




BIOCKACK Cev
Cycle
'




'


DNA -

repair


ROS Causes and responses


chemo -




therapy SOD
NF KB
Radiation
Is
-




#
(ataiase
PPAR AD -1

Growthf
\
1- Glutathion
Yq 9 /
.
_
ROS STA-13 -
ROS Nrfz
# -1# peroxiredoxin
-
uv


Cytokines / f. ]
Thiooedo✗in reductor
kypoxiape.ro .
HIF '×
-




gp , p53
✗ idase




Major een stress
signaiing response's

oxidatie stress ER stress In Flammarion DNA damage Heat shock Heavy metal Hypoxia


.SI!
/keameffectOrS
IKBX


÷
53BPI MTF -
1 VHL

Nrf 2 ATFY / XBPI Rel A P53 HSFI / 2 MTF -
1 HIER

Srxnl (HOP / Bip ICAMI pzl USD70190 MTS HUMMR



Sensors transducers and effectors
.




Sensor :
receptor of signa/
-




Transducer of
Signals relay
-
:




Effector ion Factor :
transcript

signa/ molecules
-




-

Survival factors :
promoties Cev Survival
by Suppressing apoptosis
blocks in cel Cycle
primary by overreding
stimulate cel division
Mitogens
-
:
.




-
Growth factors : stimulatie
ceugrowth b promoting Synthesis and inhibiting
the degradaties of protein's and other macromolecules


↳
there always be Cross talk
possiblyactivatesmore
-
one molecule
win .

signa
than one
transductionpathway
ij

,Example : TNF x - -
enhanced diclofenac toxi
City
-




Hep62 Cells ( Ii ver cells . but no metabolism)
for basic toxi
City of compounds
↳ look

TNFX is a
cytokine used to Mimic an inham Motion reaction in the een
-




-

Use of a non -
toxic dose Of TNFX

diclofenac
Adding TNFX increases the
toxicity of
-




-


Common tool to Mimic certain situations in the Cev



kinase -
linked receptor
signa/
of molecule 50




GG
~
phosphorylation


§ Jr
- '

Auto -
in trans or (is

trans :
phosphoryiate eachother
-




itself
< is :
phosphoryat.es
-




ËËËË .


Dominant
negative
mutant by -
inhibitors

RTK :
no kinase activator in trans

IS this kinase important :
Inactie RTKS Cross
phosphoryiation tyrosine
-
-




activated kinase domains transfect and out
b a mutant kinase


compote the wild type -




stress -
activated protein -
kinase pathways

Stimuli Growth factors etc . oxidatie stress ,
cytokines
RTK
b. ↳ !
'

MAPYK Ras
;
t 6 b.

MAP 3K Raf ASKI MEKKI , ,
MLK}

↳ b b.

MAPZK MEK MKK3/6 MKK417

to b t t
MAPK ERK p38 JNK
6 1 t t
ceil responseS proliferatien proliferatiein
Differentialion -

differentiaties and apoptosis

ceil
Migration Inflammatoire responses




from
Dominant
negative mutant : mutant kinase over express in ce 11 preventS WT
being active
-



, ,




↳ serine to alanine Can . on ever be
phosphorylated .
blocks
pathway
Dominant active mutant :
always phosphorylated
-




-
charge surface op the protein →
introduces a
negative charge
→
mutate serine to

( as partie / acid)
a
negativey charged amino acid giutamic


Example phospho specific: -
antibodies




JNK effector : (
Jun
-




IPJNK is activated.it is
phosphorylated
-




Take cells -
seen if there is
phosphorylated JNKI
-




phospho specific antibodies recognize phosphor lated :
JNKI
-
-




phosphorylated
↳
but the
it is not the aMount of JNKI that Changes . aMount of JNKI

ij ij

, Regulation of MAPKS b GSTS


•


Certain GSTS can bind to certain MAPK
and
Regular inactivate
b binding to JNK
<




Stress leads to the disruption of these interaction5 and the activator of the
signaiing pathway
-




Kinase -

specific inhibitors


-
IS the kinase that is
phosphorylated also really important for the effect we want to
study
•


Solution : kinase -

specific inhibitor

Example Imatinib / : Gieevec
-




The BCR ABL ONCOprotein
phosphoryiates tyrosine
-
-




-
you get leukemie
-

Inhibitor birds in the pocket and preventS this



Example :
JNKI and APAP toxicity

APAP activate 5 JNK →
phosphoryiated c-
Jun
-




•

with an inhibitor -
no
phosphorylation
Serum ALT of protein from liver
Ieakage
•
:



that leads to inhibited JNK inhibitor
Downstream
signaling of JNK Cev
damage is
by a
'




Ubiquiti Nation :
proteasemal protein degradaties

El ( UB activa
ting enzyme ) activates UB
-




onjugating enzyme) cor ugates ligase)
-
Ez ( UB ( with E3 ( UB


Degraded b proteasome
-




UB Coupled via its
is
always lysine
'




-

C terminus
-
ub to
lysine residues
-


Dependant on E3
youget different Shapes

FunctionS Of ubiquitination

•



protea50 Mes : K-48 / K -
11


Endocytoseis / DNA repair 10calizatiON (Multi) mono ub
•

. : - -




signalling Ms / K -63
.

:


•


Functions unclear : K -6/-271 29 / - -
33



Example :
proteasome inhibitor

In
hibiting proteasome leads to increased proteotoxic stress
•




- can Kit1 tumour cells
ou
.


HOW can we see this →
accumulatieN Of Ub
protein 5 -




have protectOxic stress because of imbalance the
Leukemia cells
genome by
giving
-

in .




compound this cells cannot Cape with the stress and die


Study of transporters

↳
specific transporter inhibitors

ij ij
ijij

, Example : Saabte
yeast xenobiotic receptor


Drug activate 5 transcriptin factor
'




In Crease in level of ABC transporter pdrsip and others
many
-
-




Lower level of the due to export
drug in cel1
'




Importance of transporters
•




-


knock-out and look at growth
-


over expression of transporter



BSEP

-




transports bileacids
.




drugs wilI be tested For BSEP inhibitor
Al 1
↳
ifinhibitedyougetchoestas.is

Transporter vesicles
ex
pressing
-




over express transporters
•




IP make a veside
youdisrupt the membranen
you
•


.




↳
vesice can be in 2
Ways
.

Insideout vesíce : what was in síde .
is now outside
vesides that in an effect
only 90 give
-




reduced
-




efficiency is

but the rest does not
giveanynoise
-




In vivo
imaging
Build luciferase in Mouse (Nrfz)
in
gene
•




can detect nrfz stress response5 and selectie
Organ Eoxicity
-
-




( isplatin in
Kidney high amount OF OCTZ
-
-




paracetamol in li ver needs bioactiva tion
high level of
Cyp450 in river
•
-
.




Lecture 3 :(en modeIs for toxic
ity testing
Cancer (en limes


-

characterstics
abnormaal
karyotype
-




-

mutation in p53 in more than 50% of an Cancer cells
-

Immoreel . expression of telomerase or Stabilization of te10Mers
-


Loss of contact inhibítion
-

Immortalization

spontaneons
-




( papillomavirus)
virus in
during Cancer
-




-
L imitation S

over expression of MDR
-




Lock of metaboli
zing enzymes
-




Mare
oncogen es tess
suppressorgenes
-




.




-

Abnormat behavior . no controlled Cev Numbers