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

04/15/2023Anne Krug

Doerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine 3

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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


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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


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Nuclei

Neuron-specific Toxicity LUHMES Neurite Growth Assayhealthy

sick

Neurite growth assay

Neurite growth assay in vitro


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Neuron-specific Toxicity LUHMES Neurite Growth Assayhealthy

sick


Neurite growth assay


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Subtract somata from neurite network

Aut

omat

ical

ly

iden

tify

nucl

ei

CalceinH-33342

ii

iii

iv

i

Automated high content image analysis

U0126 ROCK InhControl


Automated analysis provides basis for high-throughput capable screening


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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



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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


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EPA - http://www.epa.gov/ncer/rfa/2010/2010_star_comptox.html

Systems toxicology approach


(Neurite growth)


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EPA - http://www.epa.gov/ncer/rfa/2010/2010_star_comptox.html



(Next step: omics)


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Metabolomics - 2005, Volume 1, Issue 1, pp 1-2


Omics technologies

Combine for pathway identification


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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


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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 krug - lush prize conference 2014

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