Anne Krug - Lush Prize Conference 2014
Transcript of Anne Krug - Lush Prize Conference 2014
04/15/2023 1
Developmental neurotoxicity assessment in vitroNeurite growth as DNT endpoint and the underlying mechanisms of toxicity
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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Developmental neurotoxicity (DNT)
• DNT: Any adverse effect on the chemistry, structure or function of the nervous system during development, induced by chemical or physical influences
• Exposure to toxicants can lead to cognitive deficits, as shown for arsenic, methyl mercury, lead, PCBs and toluene
• Prenatal exposure to some compounds, e.g. methyl mercury, are suspected to trigger psychiatric diseases in later life
• Over 1000 animals needed to test one single chemical• DNT tests in animals do not guarantee human safety
Alternative test systems are urgently needed for DNT testing
04/15/2023Anne Krug
Doerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
How to assess DNT in vitro
Sensitive processes during neurodevelopmentBrain development
neural plate
NC NCED ED
rd
ctx
MBHB
c
v
FB
SC
v
dKadereit et al. Front Biosci (Landmark Ed) 17: 2442-60
Brain development can be split into biological processes
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How to assess DNT in vitro
Sensitive processes during neurodevelopment Neurite growth
Modified according to Radio et al. NeuroTox: 29, 361–376
Advantages of neurite outgrowth testing within a DNT integrated test battery:
• Correct wiring of the brain ( neuronal connectivity) absolutely depends on neurite outgrowth
• Compounds disturbing neurite growth are very likely to be developmental toxicants
• This ‚functional assay‘ integrates many different toxicity pathways
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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LUHMES System
DNAb-III Tubulin
Scanning electron microscopy
Fluorescence microscopy
Thanks to Diana Scholz
Neurite growth in vitro
Human neuronal cells as biological system to assess DNT
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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Nuclei
Neuron-specific Toxicity LUHMES Neurite Growth Assayhealthy
sick
Neurite growth assay
Neurite growth assay in vitro
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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Neuron-specific Toxicity LUHMES Neurite Growth Assayhealthy
sick
Neurite growth assay in vitro
Neurite growth assay
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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Subtract somata from neurite network
Aut
omat
ical
ly
iden
tify
nucl
ei
CalceinH-33342
ii
iii
iv
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Automated high content image analysis
U0126 ROCK InhControl
Neurite growth assay in vitro
Automated analysis provides basis for high-throughput capable screening
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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Automated high content image analysis
Cells grown on 96 well or 384 well plates allow testing of many substances in one run
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
Neurite growth assay in vitro
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METH
Puromycin
Okadaic acid
HonokiolCisplatin
ChlorpyrifosChlorpyrifos Oxon
Oligomycin
SP600125 PTP IV
ODQ
PiericidinAntimycin A
Menadione
Acrylamide
Cytochalasin
Fipronil
IPA3
-2 -1 0 1 2 3-2
-1
0
1
2
3
U0126
Bis I
Na3VO4
MeHg
Brefeldin A
Flavopiridol
Cycloheximide
Diquat
Colchicine
Rotenone
Vincristine
Nocodazole
Paraquat
Narciclasine
H1152
HA-1077Thiazovivin
Blebbistatine
SDSCd2Cl
K2CrO4
H-33352
Tween-20
CCCPBSO
Etoposide
2,4-DNP
tBuOOH
log EC 50 neurite area
log
EC
50 v
iab
ility
Identification of putative developmental neurotoxicants possible
No effect
Altered neurite growth
Search for pathways of toxicity
Screening chemicals with the neurite growth assay
Krug et al. (2014) Arch ToxicolAnne Krug
Doerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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EPA - http://www.epa.gov/ncer/rfa/2010/2010_star_comptox.html
Systems toxicology approach
Search for pathways of toxicity
(Neurite growth)
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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EPA - http://www.epa.gov/ncer/rfa/2010/2010_star_comptox.html
Systems toxicology approach
Search for pathways of toxicity
(Next step: omics)
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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Metabolomics - 2005, Volume 1, Issue 1, pp 1-2
Systems toxicology approach
Omics technologies
Combine for pathway identification
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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Epidemiologic data
Transcriptomics / Metabolomics
Identification of perturbed molecules
Comparison
Pathways of toxicity (PoT)
Proof-of-principle Study – MPP+
MPP+
MPP+
Omics technologies applied on LUHMES
MPP+ = 1-Methyl-4-phenylpyridinium
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Upregulated genes/metabolites
Downregulated genes/metabolites
ATF4 target genes
Underlying biological processes
0 6 12 24 36 48
ATF 4
GAPDH
GADD 34
eIF2a[pS52]
5 µM MPP+
50 kDa
37 kDa
100 kDa
Time [h]:
GAPDH
GAPDH
ConfirmationIdentified pathways
Proof-of-principle Study – MPP+
Krug et al. (2014) Cell Death DiseaseAnne Krug
Doerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine
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• Screen big library of chemicals with neurite growth assay
• Apply omics technologies to reveal pathways of toxicity (PoT) of neurite growth disturbing chemicals
• Classify chemicals on the basis of PoT
Outlook
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Acknowledgement
Doerenkamp-Zbinden-Chair, Universität Konstanz: Simon Gutbier, Cornelius Kullmann, Dominik Pöltl, Sunniva Förster, Martina Adam, Stefan Schildknecht, Marcel Leist
Anne KrugDoerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine