Using MEAs to screen chemicals for potential … · Using MEAs to screen chemicals for potential...
Transcript of Using MEAs to screen chemicals for potential … · Using MEAs to screen chemicals for potential...
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 Do not cite or quote this presentation.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%
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
Cortical culture + MEA = “Brain-on-a-Chip”
*This research is still in progress, and is not yet published.
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.
*This research is still in progress, and is not yet published.
Screening the ToxCast Phase I and II Library for Acute Effects on Network Function
*This research is still in progress, and is not yet published.
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.
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
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)
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
Quantify key neurodevelopmental events in vitro
Phenotypic Screening for DNT Hazard
*This research is still in progress, and is not yet published.
Complex network activity develops over time
Burst: A group of spikes
Burst Duration
Spike
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
General Parameters Burst Parameters
Network Connectivity Parameters Network Spike Parameters
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
FDR = 1%
Statistically-significant Concentration-related changes
*This research is still in progress, and is not yet published.
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)
*This research is still in progress, and is not yet published.
Tipping Point Analysis- Preliminary results
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.
• 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
*This research is still in progress, and is not yet published.
• 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
*This research is still in progress, and is not yet published.
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
*This research is still in progress, and is not yet published.