Using MEAs to screen chemicals for potential … · Using MEAs to screen chemicals for potential neurotoxicity and developmental neurotoxicity ... Outline • Introduction ... Developmental

Using MEAs to screen chemicals for potential neurotoxicity and developmental neurotoxicity

Timothy J Shafer, PhDIntegrated Systems Toxicology Division

National Health and Environmental Effects Research LaboratoryOffice of Research and Development

United States Environmental Protection Agency

Disclaimer: This is a scientific presentation only. Some or all of the data presented in this presentation are preliminary and subject to change based on additional experiments or analysis. Do not cite or quote this presentation. This presentation does not represent EPA policy and mention of products or tradenames does not constitute a recommendation for use or endorsement. Some of the work presented here was done under a Cooperative Research and Development Agreement with Axion Biosystems.

Outline

• Introduction• Background on Microelectrode Array (MEA)

recording• Screening Toxcast compounds for acute effects

on neural network function• Screening DNT reference compounds to

develop an MEA-based network development assay

*This research is still in progress, and is not yet published.

The problem of too many chemicals.. not enough resources…

• Inadequate toxicity data exists for tens of thousands of chemicals

• Hazard characterization using animal based approaches is not feasible

• Faster, less expensive and predictive methods are needed for hazard characterization– Includes neurotoxicity and developmental neurotoxicity

• Drivers for higher throughput testing– REACH* in Europe– Toxicity Testing in the 21st Century report in the U.S.

*Registration, Evaluation, Authorisation and Restriction of Chemicals

Developmental Neurotoxicity < 1%


Methods for in vitro Neurotoxicity assessment

Functional Endpoints•Patch clamp electrophysiology•Ion homeostasis (e.g. Calcium imaging)•Membrane potential•Mitochondrial Function•Microelectrode array (MEA) recording

Morphological Endpoints•Neurite outgrowth•Cell type•Synapse number•Proliferation

Biochemical Endpoints(e.g. ToxCast)•ion channels•AChE•thyroid hormone metabolism•growth factor receptors•cell adhesion molecules

Structure

Individual Cell


Adverse Outcome Pathways (AOPs) for Neurotoxicity Screening

ChemicalPropertyProfile

Receptor/LigandInteraction

DNA Binding

Protein Oxidation

Gene ActivationProtein ProductionAltered SignalingProtein Depletion

Altered Physiology

Disrupted Homeostasis

Altered Tissue Developmentor Function

Lethality

Impaired Development

Impaired Reproduction

Cancer

ToxicantMolecularInitiating Event

Cellular Responses

OrganResponses

IndividualResponses

Initiating Event? (many)

Key Eventsdifferentiationneurite growthSynaptogenesis…

Nervous System↓ connectivity

Adverse Outcomecognitive deficit

Micro-Electrode Array (MEA)High-Content Screening (HCS)Biochemical assays(e.g. ToxCast)ion channelsAChEthyroid hormone metabolismgrowth factor receptorscell adhesion molecules…

Increasing Throughput

Increasing Biological Complexity*This research is still in progress, and is not

yet published.

Introduction to Microelectrode Array (MEA) Recording

• Array of extracellular electrodes• Each electrode can record event rates

and patterns of any tissue in “contact” with it.

• Spontaneous or Evoked Activity

• Cortical Neurons • Hippocampal Neurons• Spinal cord Neurons• Retinal neurons• Retina• Slices• Primary Cardiac Cells

200μm

30μm

neurons (green) astrocytes (red)

excitatory (red) and inhibitory (green) neurons



Cortical culture + MEA = “Brain-on-a-Chip”


Advantages of MEAs for Neurotoxicity Screening

• Phenotypic, cell-based screen.• Apical- integrates the responses of multiple ion channels, kinases,

receptors, etc• High Content- Rich temporal and spatial information• Amenable to pharmacological manipulations• Can be applied to neurons from multiple brain regions• Non-invasive• Complex culture system (multiple neuronal types + glia)• Repeated measurements over time

The throughput of multiwell plates is sufficient for neurotoxicity screening.


Screening the ToxCast Phase I and II Library for Acute Effects on Network Function


Multiplexed Experimental Design

Determine Effects on Spontaneous Network Activity

1. Primary cortical neurons are cultured in 48 well MEA plates and allowed to mature

for 13 days

2. Plates are placed in the Axion Maestro MEA amplifier

3. Determine firing rate in each well for 40 min prior to and after treatment with compounds

Burst of action potential spikes

Determine Effects on Cell Health

CellTiter BlueTM

5. Replace media with 200 µL of fresh media containing CellTiter BlueTM reagent, incubate for 1 h at 37 ̊ C, then read.

LDH

4. Transfer 50 µL of media from mw MEA to 96 well assay plate.


Screening ToxCast Compounds

I n c r e a s e d M F R

D e c r e a s e d M F R

T o t a l = 1 0 8 0

N o E f f e c t

D e c r e a s e d M F R + C y t o t o x i c

8

758

107

207

Single concentration (40 µM) Screen


I n s e c t i c i d e s

P h a r m a c e u t i c a l s

T o t a l = 2 0 3

H e r b i c i d e s

F u n g i c i d e s

F l a m e R e t a r d a n t s

4 9

7 9

3 3

3 7

Categories with no hits:IndustrialPesticide Breakdown Product MedicinalCosmetic

Compounds of Interest that Increased MFROrganochlorinesAldrin DDT Endrin DDEHeptachlorHeptachlor epoxideLindane

NeonicotinoidsNicotineImidaclopridThiamethoxam

Compounds of Interest that Decreased MFROrganochlorinesEndosulfanKeponeMethoxychlor

PyrethroidsAllethrinCypermethrinFenpropathrinPrallethrinTetramethrin

MectinsAbamectinEmamectin

Five Compound Categories Accounted for 2/3 of the Hits


Analysis of Hits for Chemotype (CT) Enrichment

(27/39 active)

0 10 20 30 40 50 60 70 80 90

bond:CX_halide_alkyl-X_bicyclo[2_2_1]heptene

chain:aromaticAlkane_Ph-C1-Ph

ring:hetero_[5]_O_dioxolane_(1_3-)

group:ligand_path_4_bidentate_aminoethanol

Sample Chemotype Enrichments in Hits

%total %Hits

(20/48 active)

(6/11 active)

(7/8 active)


7/8 of chemicals containing CTs #158 & 711 are “hits”

2/7 Hits decrease MEA activity

5/7 Hits increase MEA activity*This research is still in progress, and is not yet published.

Future Directions

• Concentration-Response Characterization– Ongoing for 384 compounds– Completion expected in June

• Follow-up on Chemotyping• Burst Analysis & Fingerprinting

Concentration (log M)

Mea

n Fi

ring

Rate

(% C

ontr

ol)

Concentration-Response Confirmation


HTT Task 2.2: Expanding the capability to screen and prioritize chemicals for developmental toxicity.

Faster, less expensive and predictive methods are needed for developmental neurotoxicity hazard characterization

GoalDevelop medium throughput, in vitro assays to screen and prioritize chemicals for developmental neurotoxicity hazard


Quantify key neurodevelopmental events in vitro

Phenotypic Screening for DNT Hazard


Complex network activity develops over time

Burst: A group of spikes

Burst Duration

Spike


Development of Network Activity on MEAs

Cotterill et al., J. Biomolecular Screening, 2016, in press*This research is still in progress, and is not

yet published.

Developmental Neurotoxicity Screening on MEAs

Approach (PIP year 1)Develop the Assay• SA#1: Test Assay Positive Control CompoundsEvaluate the Assay• SA#2: Test 30 compound DNT Reference chemicals (24 in vivo DNT, 6

negative controls)Screen with the Assay• SA#3: Screen 18 ToxCast Compounds for which acute MEA data existsStage 3 PIP• SA#4: Complete screening of GRADNT compounds

– Screen ~200 ToxCast Compounds • SA#5 & 6. Evaluate rat and human iPS-derived neurons• Tipping Point Analysis


Chemicals 1-6

Concentrations 1-8

con

con

con

con

con

con

con

con

Experimental Design

Record

Media change/Dose

Viability (CTB, LDH)

0 2 5 7 9 12

Dosed 2 hrs after plating. Compounds present throughout

+

^

+ + + +

^ ^

*

MEA/DNT Assay Development

SA#1: Test Assay Positive Control Compounds


General Parameters Burst Parameters

Network Connectivity Parameters Network Spike Parameters


Developmental Neurotoxicity Screening on MEAs

Progress to Date:• ~100 compounds have been screened in total (excluding

repeats)– 41 DNT reference compounds – 27 Organophosphate insecticides– 14 Negatives– 18 ToxCast– 5 Assay Controls

100 compounds X 16 endpoints X 5 timepoints X 7 concentrations =56,000 datapoints


EC50 estimates for each compound based on reduction in AUC over time

Reducing data by collapsing across time and concentration

0 5 1 0

0 . 0

0 . 1

0 . 2

0 . 3

0 . 4

0 . 5

C o r r e l a t i o n C o e f f i c i e n t

D I V

Co

rre

lati

on

Co

eff

icie

nt c o n t r o l

0 . 0 3

0 . 1

0 . 3

1

3

1 0

3 0


DNT Ref

NEG

Unknown

Assay +Control


Specificity


FDR = 1%

Statistically-significant Concentration-related changes


Tipping Point: The threshold between adaptation and adversity

Can tipping points for chemical effects on network development be defined?

Examine MFR, BR, #NS, #AE, #ABE, correlation (r)


Tipping Point Analysis- Preliminary results


Tipping PointsIn a developing network, are “adaptations” really adaptations?

0 5 1 0

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

# N e t w o r k S p i k e s

D I V

#N

etw

ork

Sp

ike

s

c o n t r o l

0 . 0 3

0 . 1

0 . 3

1

3

1 0

3 0

0 5 1 0

0

5

1 0

1 5

2 0

# B u r s t i n g E l e c t r o d e s

D I V

#B

urs

tin

g E

lec

tro

de

s

c o n t r o l

0 . 0 3

0 . 1

0 . 3

1

3

1 0

3 0

0 5 1 0

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

L e a d A c e t a t e M F R

D I V

Me

an

Fri

rin

g R

ate

(sp

ike

s/m

in)

c o n t r o l

0 . 0 3

0 . 1

0 . 3

1

3

1 0

3 0


• Preliminary Data Analysis indicates that MEA assay can separate developmentally neurotoxic from non-neurotoxic compounds.– Analysis is ongoing

• Screening compounds at a rate of ~100/year; ~$300/cmpd– Compare to ~2/yr & ~$900,000/cmpd for Guideline DNT assay– <100 environmental compounds screened in 20 years in Guideline DNT

Outcomes


• Continue Screening– Finish the ~100 DNT reference compounds– Screen ToxCast Acute MEA Hits– Goal is ~300 compounds in 2 years– Includes High Priority Compounds (e.g. Flame Retardants, OP

insecticides)

• Continue Tipping Point Analysis

• Develop “Animal Free” Complex Cell Models– Rodent Stem Cell Models – Human Stem Cell Models – Identify viable models and begin screening DNT “reference”

compounds

Future Directions


Acknowledgements:

Students/Student contractors:• Diana Hall • Jasmine Brown

EPA:• Theresa Freudenrich• Kathleen Wallace• Bill Mundy• Matt Martin• Keith Houck• Chris Frank• Imran Shah• Ann Richards

Cambridge University• Stephen Eglen

Axion Biosystems (CRADA 644-11):• Jenna Strickland• Jim Ross• Tom O’Brien


Documents

Using MEAs to screen chemicals for potential … · Using MEAs to screen chemicals for potential neurotoxicity and developmental neurotoxicity ... Outline • Introduction ... Developmental