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contact ORD_Webmaster@epa.gov.

US EPA CSS-HERA
Board of
Scientific
Counselors
Chemical Safety
Subcommittee
Meeting

US EPA CSS-HERA BOSC Meeting - February 2-5, 2021

SW(

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The work presented within represents US EPA Office of Research and Development research
activities. Material includes both peer reviewed, published results and work-in-progress
research. Please do not cite or quote slides.


-------
Table of Contents

Development and Harmonization of Organotypic/Co-Culture Models and Assays to Improve Throughput
and In Vivo Relevance in Inhaled Chemical Testing (Shaun McCullough)	3

An Approach Using NAMs for the Evaluation of Inhalation Toxicity in OCSPP Chemical Registrations
(Mark Higuchi)	21

Neurovascular Unit Modeling and Blood Brain Barrier Function (Thomas Knudsen)	39

The work presented within represents US EPA Office of Research and Development research activities. Material
includes both peer reviewed, published results and work-in-progress research. Please do not cite or quote slides.


-------
oEPA

Development and Harmonization of Organotypic/Co-
Culture Models and Assays to Improve Throughput and In

Vivo Relevance In Inhaled Chemical Testing

2021 CSS BoSC
2/3/2021

Shaun D. McCullough, PhD
Principal Investigator
ORD/CPHEA/PHITD/CRB
mccullough.shaun@epa.gov

Progress for o Stronger Future


-------
oEPA

Conflict of Interest and Disclaimer

•	No conflicts of interest

•	The information presented here does not necessarily reflect the views or
policies of the U.S. Environmental Protection Agency. Mention of trade
names or commercial products does not constitute endorsement or
recommendation for use.


-------
oEPA

In Vitro Approaches for Inhaled Materials Testing and
Research

•	In vitro models are required for testing the thousands of inhalable materials

•	The lung is a complex organ with over 40 cell types, but nearly all in vitro models
include only a single cell type

•	Individual inhaled materials impact distinct cell types and physiological functions
differently

• These effects cannot be represented by current systems

•	There is a lack of consensus on methods, parameters, thresholds, and reporting
standards for models, assays, and exposures

•	Fit-for-purpose models and assays are needed for accurate, reliable, and
defensible inhalation toxicity testing

•	In vivo human data from environmental inhaled materials {e.g., ozone, acrolein,
particulate matter) are invaluable in guiding the development of lung models
and allowing validation for acceptance


-------
Case Study Using A Data-Rich Inhaled Material

•	In vitro assessment at 24hr

•	Not cytotoxic

•	No change in bronchial epithelial barrier
permeability

•	Marginal changes in stress-responsive
gene expression (RNA) at 24 hours

•	In vivo human exposures

•	Acute and chronic lung disease

•	Airway inflammation

•	Susceptibility to infection

•	Asthma

•	COPD

•	Cardiovascular morbidity and mortality

•	Ml/stroke/arrythmia

•	Attributed to 4-10 million deaths per year
worldwide

Fine Particulate Matter (PM2 5)

www.nasa.gov


-------
oEPA

So, Why Didn t It Work?

•	Traditional in vitro inhalation models do
not represent in vivo biology and
dosimetry

•	Models/endpoints are often not "fit-for-
purpose"

•	e.g., bronchial epithelial cells are not
representative of lung microvascular
endothelial cells, et cetera

•	Lack of time course data and limited
scope of endpoints resulted in failure to
identify adverse effects

•	Looking in the wrong place at the wrong
time

•	Few in vitro inhalation models are
representative of individuals most likely
to experience adverse effects (i.e.,
susceptible populations)

THIS IS WHERE YOU
LOST YOUR WALLET?

\

NO, I LOST tT IN THE PARK.
IT THIS IS WHERE THE LI6HT IS.


-------
oEPA

Lung Structure and Function

Trachea
Bronchi
Bronchioles

Terminal
bronchioles

Respiratory
bronchioles

Alveoli

Large conducting

airways

"large airways"


-------
oEPA

Bronchial Airway Tissue and Dosimetry

Epithelial

barrier

Stroma

Cilia 	

Lymphocyte
Goblet cells

Basement
membrane

Connective tissue
Fibroblasts —=
Collagen fibers

Mucous acinus —

Serous acinus

Basal cells

Basement membrane

Blood vessels

Serous acini

Adapted from Lab Chip, 2014, 14, 3349-3358

Cilia

Basal bodies
Lymphocyte
Goblet cells


-------
xv EPA

Trans-Epithelial Exposure Model

Cilia
Lymphocyte
Goblet cells

Basement
membrane

Connective tissue
Fibroblasts •
Collagen fibers

Mucous acinus —
Serous acinus —

• win 1

LLT"

- -	M

Ci

¦ :i X

' O .

I

Bronchial
epithelium

Basal bodies
Lymphocyte
Goblet cells

Basal cells

Basement membrane Bronchial

stroma

Blood vessels
Serous acini

I O* I OIOIOIOIO)

]
]

Direct
Exposure

Trans-Epithelial

Exposure

Faberef. al., (2020) Toxicological Sciences 177(1):140-155


-------
oEPA

Trans-Epithelial Exposure Model TEEM)

TEEM-Mark 2









o

1 J

o

>
f

o

0

r jw*
°°

i



o

S,

l> 1 •1 • 1 • 1 • I • )

•	Research/testing use

•	Effects on bronchial epithelium

•	Effects on bronchial microenvironment

•	Parallel analysis

•	Suitable for many endpoints

•	Hybrid primary/cell line-based model

•	Mark 1 (cell lines)

•	Very low cost (~$4 per well)

•	Set-up to assay time = 2 days

•	Mark 2 (primary cells)

•	Low cost (<$10 per well)

•	Set-up to assay time = 24+ days

•	Key findings:

•	Trans-epithelial exposure effects on fibroblasts are similar
between Mark 1 and Mark 2 models

•	Bronchial epithelial cells are minimally responsive to
exposures and may not be the primary targets/mediators of
exposure effects

•	Mark 1 model is representative of bronchial epithelial barrier
function


-------
VOLUME |

Toxicological Sciences

The Official Journal of the Society of Toxicology







Direct
Exposure

Trans-Epithelial
Exposure

EP

us pension
ular Matrix

ToxSci

••

•• • • »*• * |
0 *1*1*1*1*1*1*1*

t Epithelial . ... C
Call S

«¦ Fibroblast 	 Extracsll

Reproducible

Transparent

Impactful

Rigorous

Timely

OXPORD

UNIVERSITY PRESS

SEPTEMBER 2020 |

C/^' I ' I Society of
OvJ L | Toxicology-
academic.oup.com/toxsci

TOXICOLOGICAL SCIENCES, 177(1), 2020,140-155
doL- 10.1093/toxsci/kfaa085

Dryad Digital Repository Dd: http://doi:10.5061/dryad.8931zcm3
Advance Access Publication Date: 11 June 2020
Research Article

Exposure Effects Beyond the Epithelial Barrier:
Transepithelial Induction of Oxidative Stress by Diesel
Exhaust Particulates in Lung Fibroblasts in an
Organotypic Human Airway Model

Samantha C. Faber,* Nicole A. McNabb,t Pablo Ariel,* Emily R. AungstJ and
Shaun D. McCullough ©* 11

'Curriculum in Toxicology and Environmental Medicine, UNC Chapel Hill, Chapel Hill, North Carolina 27599
TPublic Health and Integrated Toxicology Division, Center for Public Health and Environmental Assessment,
US Environmental Protection Agency, Chapel Hill, North Carolina 27599 and J Micros copy Services Laboratory,
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599

Disclaimer: The contents of this article have been reviewed by the US. Environmental Protection Agency and approved for publication and do not neces-
sarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use
'To whom correspondence should be addressed at Public Health and Integrated Toxicology Division, US Environmental Protection Agency, EPA Human
Studies Facility, 104 Mason Farm Road, CB #7315, Chapel Hill, NC 27599. Fax: 919-966-6271. E-mail: mccullough.shaunOepa.gov.

ABSTRACT

In vitro bronchial epithelial monoculture models have been pivotal in defining the adverse effects of inhaled toxicant
exposures; however, they are only representative of one cellular compartment and may not accurately reflect the effects of
exposures on other cell types. Lung fibroblasts exist immediately beneath the bronchial epithelial barrier and play a central
role in lung structure and function, as well as disease development and progression. We tested the hypothesis that in uitro
exposure of a human bronchial epithelial cell barrier to the model oxidant diesel exhaust particulates caused
transepithelial oxidative stress in the underlying lung fibroblasts using a human bronchial epithelial cell and lung fibroblast
coculture model. We observed that diesel exhaust particulates caused transepithelial oxidative stress in underlying lung
fibroblasts as indicated by intracellular accumulation of the reactive oxygen species hydrogen peroxide, oxidation of the
cellular antioxidant glutathione, activation ofNRF2, and induction of oxidative stress-responsive genes. Further, targeted
antioxidant treatment of lung fibroblasts partially mitigated the oxidative stress response gene expression in adjacent
human bronchial epithelial cells during diesel exhaust particulate exposure. This indicates that exposure-induced oxidative
stress in the airway extends beyond the bronchial epithelial barrier and that lung fibroblasts are both a target and a
mediator of the adverse effects of inhaled chemical exposures despite being separated from the inhaled material by an
epithelial barrier. These findings illustrate the value of coculture models and suggest that transepithelial exposure effects
should be considered in inhalation toxicology research and testing.

Key words: in uitro; coculture; oxidative stress; lung; fibroblast; transepithelial; epithelial.

Published by Oxford University Press on behalf of the Society of Toxicology 2020.

This work is written by US Government employees and is in the public domain in the US.

140


-------
oEPA Moving Forward

•	TEEM Mark 2 model

•	Assess culture longevity, key parameter
values, and expected variability over time
(differentiated + up to 90 days) for
subsequent sub-cnronic exposures

•	Barrier permeability

•	Histology/immunofluorescent staining

•	Viability

•	Redox potential

•	Ciliary beat frequency

•	Marker gene expression

•	Mucus production

•	Metabolism

•	Inter-donor and inter-experimental variability
using a group of donors

•	Power calculations to strengthen future studies


-------
oEPA

Lung Structure and Function in In Vitro Testing

Trachea
Bronchi
Bronchioles

Terminal
bronchioles

Respiratory
bronchioles

Pulmonary airways

Alveoli


-------
oEPA

Alveolar Region

Alveolar
epithelium

Alveolar
/ interstitium \

Microvascular
endothelium

\

\





( ^









Direct
Exposure

Trans-Alveolar
Exposure


-------
xv EPA

Alveolar Capillary Region Exposure (ACRE) Model
Mark 1



Indirect co-culture model

•	Submerged - completed

•	Air-liquid interface - in progress

•	Incorporation of immune cells - under
development

Research/testing use

•	Effects on alveolar compartment

•	Effects on lung microvasculature

Cell line or hybrid model

•	NCI-H441

•	IMR90/pHlF

•	HULEC/pLMVEC

Cost per sample: ~$4 (cell lines)

Set-up to assay = 5 days
Key findings to date:

•	Trans-alveolar exposures to diesel exhaust
particulates cause effects that reflect in vivo
human outcomes


-------
EPA X Alveolar Region

Alveolar
epithelium \

Alveolar
interstitium \

Microvascular
endothelium

Circulation

\

\

\


-------
xv EPA

Alveolar Capillary Region Exposure (ACRE) Model
Mark 2

o

0—J-^Q/—L~vQ/—L~\Qr-HQr-L-vQ/—\Q^

Currently under development

•	Submerged arid air-liquid interface

•	Planned incorporation of immune cells

Research/testing use

•	Effects exposure on pulmonary barrier

•	Release of mediators/metabolites into the
circulation

•	Bioavailability of inhaled materials

Acute or repeated/sub-chronic/chronic
exposure scenarios

Cell line or hybrid model

•	NCI-H441

•	IMR90/pHLF

•	HULEC/pLMVEC

Cost per sample: ~$5

Set-up to assay = 4-5 days (expected)


-------
£EPA V Summary

•	Bronchial epithelial cell models provide valuable information but are not representative
of other aspects of the structure and function of a complex tissue

•	Fit-for-purpose multi-cellular models are necessary for accurate, reliable, and
defensible inhalation toxicity testing and computational model development

•	Human data from environmental inhaled materials is invaluable in lung model
development and validation

•	Bronchial epithelial/stromal co-culture model indicates that trans-epithelial exposure
effects on the stroma may exceed direct effects in the epithelium

•	Outcomes in ACRE-Mark 1 model align with	human data

•	ACRE-Mark 2 model for alveolar-vascular permeability and trans-alveolar bioavailability
is in progress

•	Incorporation of immune cells into all models is in development

•	Protocols/methods are designed to be accessible, cost-effective, and compatible with
high throughput assays, ancfwill be made publicly available


-------
Questions:

Shaun D. McCullough, PhD
mccullough.shaun@epa.gov


-------
An Approach Using NAMs for the
Evaluation of Inhalation Toxicity
in OCSPP Chemical Registrations

Mark Higuchi, PhD
ORD/CPHEA/PHITD/ITFB

2/2/2021


-------
oEPA Challenges to traditional in vitro exposure methodology

Problem: VOCs and Aerosols are
incompatible with traditional
testing methods.

•	Over 30% of the TSCA chemical
inventory are volatile or insoluble

•	Require biologically relevant air-liquid
interface (ALI) exposures to mimic
human exposures

•	Currently	lock informat
compounds to support


-------
SEPA In vivo vs. in vitro Inhalation Studies



In vivo Inhalation
Studies

Traditional in vitro
studies

NAMs for in vitro
Inhalation Studies

Eligible Compounds

Aerosols and VOCs

Soluble compounds only

Aerosols and VOCs

Dosing Methods

Known concentration
over a certain time (C*t)

Direct dosing

Known concentration
over a certain time (C*t)

Realistic Exposure?

Yes, whole animal with
intact airway

No, submerged culture

Yes, airway cells cultured
at ALI

Dosimetry

Lacks analytical animal
dosimetry

Known concentration
applied to media

Lacks analytical cell
dosimetry

Conditions

Control temp and RH for
animal

Submerged culture n
incubator

Control temp and RH for
ALI cultures

Eligible Endpoints

Porta l-of-entry and
systemic effects

Only assesses cellular
effects

Porta l-of-entry and
cellular effects

Repeated Dosing
Possible?

Yes

Limited

Limited


-------
oEPA

EPA Cell Culture Exposure System (CCES)

To address this need, we developed a novel system: the EPA's Cell Culture

Exposure Systems (CCES) permits the exposure of mammalian lung cells at air-

liquid interface (ALI).

1

j	• Delivers VOCs to advanced ALI

j	airway models to produce a

j	biologically realistic inhalation

j	exposure

I	• Medium-throughput testing strategy

¦	• Unlike other ALI exposure

!	technologies, the CCES maintains

!	ideal temperature and humidity

!	conditions for cells at ALI
I

Humidified
Dilution
Air

Heated	

Enclosure
37°C	I


-------
oEPA

CCES Provides Superior Exposure Conditions

Post-Exposure TEER comparison

CM

E

o

«
E

1500 —i

1000-

OC 500-

UJ

Li!

«

i—r

T	T

^	^ xO^ ^ xO^









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ALI

Submerged

D 16HBE
HPBE

Oil Submerged 16HBE
C3 Submerged HPBE

Cell Viability (ATP Generation)

4000000-I

3000000-

o
o
c
o
o

«5

-------
v>EPA Pilot and Proof-of-Concept Studies ^

Pilot Study Overview

Proof-of-Concept Study Overview

Cell Types
at ALI

Primary Human Bronchial Epithelial Cells*
BEAS-2B cells

Cell Types
at ALI

Primary Human Bronchial Epithelial Cells
16HBE cells

Mattek Epi-Airway cells

Chemicals
Tested

1,3-Butadiene Acetaldehyde Carbon Tetrachloride*
Acrolein Trichloroethylene* Dichloromethane*
Formaldehyde 1-Bromopropane*

*Tested in both B2B and HBECs

Chemicals
Tested

Naphthalene 1,3-Dichloropropene Chloropicrin
Methylisothiocyanate Zinc pyrithione* Metribuzin*
Didecyldimethyl ammonium chloride* Tetramethrin*
2-phenylphenol* Indoxacarb* Naled*
Azoxystrobin* Oxamyl*

*aerosol exposure necessary

Exposure
Regimen

•	2h exposure, endpoints collected 4h later

•	6 concentrations, sham + incubator controls

•	Temp and RH monitored

Exposure
Regimen

•	2h exposure, endpoints collected 4h later

•	6 concentrations, sham + incubator controls

•	Temp and RH monitored

Assay
Formats

•	TempO-Seq

•	Cytotoxicity [LDH Release, Cell Titer Glo]

Assay
Formats

•	TempO-Seq

•	Cytotoxicity [LDH Release, Cell Titer Glo]

•	Trans Epithelial Resistance (TEER)

•	Inflammatory response [ELISA for IL-6 and IL-8]


-------
oEPA

Proof-of-Concept: 1,3-Dichloropropene

140n

£	E

o	w

'§	°

2	0

o	>

>	ro

O	a)

Concentration (ppm)

| I I I I -r I I I I |

20	30

Concentration (ppm)

16-HBE

Human Primaries
Mattek

Technical Replicates • Viability, n=2; Cytotoxicity, n=4

Biological Replicates

• Conducted over three days, n=3

Exposure Regimen

• 6 concentrations, Sham exposure control, incubator control



• Several sub-cytotoxic doses are ncluded


-------
VOC Benchmark Dose Modeling

•••••0
• ••••
• •••«
• ••••

Express2

Build 3

S^NTP &EPA

¦	Health	r

¦	Canada JciQvn*

7.0
6.5
6.0
5.5
.2 5,0

¦4.5

L4.0

' 3.5
3.0
2.5
2.0
1.5

a"

1

Hill

Dichlorometharie [BEAS-2B]
CYP24A1 11891

.7

Data generated by High-Throughput
Transcriptomics (HTTr) shows promise for
quantitative human health risk assessment

BMD analysis is the current standard for
submerged high-throughput chemical screening
• We are uniquely suited to provide missing
BMD values for VOCs

Exp 2

Poly 2

0 Exploratory analysis - modeling criteria not finalizea

8.5
8.0
7.5
7.0
r 6,5

1

16.0
I* 5.5

n

I 5.0

I

4.5
4.0
3.5
3.0

1-Bromopropane [BEAS-2B]
TNFAIP2 17927

4.0
3.5

£ 2.0
a

LU

^1-5

o a

Dichloromethane [BEAS-2B]
SPRR2A 33635

1E0	1E1

Dose

Data6 — Model

Dose
h Data6 — Model

Dose
h Data6 — Model


-------
Comparison of in to exposure studies

in vitro	in vivo

Chemical Name

BEAS-2B BMD
(ppm)

HPBE BMD

(ppm)

Representative
LOAEL (ppm)

Representative
NOAEL (ppm)

TLV (ppm)

Acrolein

0.586

—

0.4

NR

O.lppm

1-Bromopropane

2.246

N/A

125

250

O.lppm

Formaldehyde

N/A

—

404

152

O.Bppm

1,3-Butadiene

13.979

—

625

8000, 200

lOppm

Carbon Tetrachloride

9.563

N/A

10

5,1

lOppm

Acetaldehyde

N/A

—

2

1

25ppm

Trichloroethylene

44.842

28.148

25, 2.6

50, 5.2

50ppm

Dichloromethane

142.127

226.73

500-1000

200

lOOppm


-------
xv EPA

. ~.

~-

• ~

• ~
• •

Next Steps: New Priority Compounds Must Be Generated
as Aerosols for ALI Exposures

* • . ~

-~ Inhalable aerosols



Nasopharynx

5-10 pm

Trachea

3-5pm

a Pulmonary aerosols



Bronchi

2-3 pm

Bronchioles

l-2pm

• • •

Ultrafine particles 0.1 -1 pm

Alveoli and bloodstream

-> Must be generated and delivered as
1-2 pm particles; possess different
transport mechanisms than VOCs.

Particle Size

100

Mobility Diameter (nm)

mr

dv~p
dt

aerosols
Langevin Equation for Transport

•	Particle Acceleration (>0.5 |im)

•	Gravitation forces (>0.5 \ivn)

•	Diffusion (<0.5 [im)

I

VOCs


-------
a cda	CAD and CFD Modeling Guide Development of

Aerosol-Specific CCES

Computer Aided Design + Computational Fluid Dynamics allow virtual testing of CCES

Generation 4: Aerosol Dilution Manifold

ANSYS

2020 R2


-------
oEPA

CAD/CFD Increase Testing Efficiency

•	Time and cost-effective method to develop aerosol-specific dilution and delivery systems

• 70+ geometry and flow combinations tested in 3 months

•	Each aerosol may require CFD testing to optimize flow parameters for each chemical exposure
(account for range of particle diameters and densities)

iii VYTiil Tiiiii ¦¦¦¦¦¦

123456 123456 123456 123456

Nozzle Position/Dose

123456 123456

Generation 1 - Original Flows
Generation 1 - New Flows
Generation 2
Generation 3
Generation 4

Goal: Half-Log Serial Dilution

Generation 4-v2 Achieves Half-Log Dilution

1000000-1

<0
&

(0

a
*

Gen4-v2
Half-Log


-------
Next Steps: Quantitative Dosimetry

•	Inhalation studies currently rely on [C]*t
and lack analytical dosimetry

•	ITFB received CE-funding for High-
Resolution Hybrid Orbitrap Mass
Spectrometer

• Fg-level sensitivity with sub-ppm specificity

•	High sensitivity/specificity vital to quantify
toxicant deposition, cellular uptake, and
biomarkers of internal exposure

rt •« .«¦} m


-------
oEPA

Alignment with Program Office Needs

Submerged monoculture

7)o |o|o|o|o

High Throughput
Lower Complexity

ALI human airway models with appropriate
inhalation exposures aims to reduce our
reliance on animal testing
Strategy will provide missing risk
assessment data for VOCs/aerosols that
cannot be tested with submerged methods

^|oTo'o[o

2D Monoculture 3D Organotypic

In vivo Inhalation

High Cost
Human Relevance
Low Throughput


-------
oEPA

Conclusions and Future Research

•	Novel exposure approach transects traditional	submerged dosing
and in vivo inhalation exposures

•	Support Program Office(s) risk assessors by providing NAMs to directly test
chemicals of interest in similar fashion to	inhalation exposures

•	Provide data from HTTr analysis to be used by ToxCast for SAR, IVIVE, etc

•	Aim to develop NAMs for analytical dosimetry in cell cultures to translate
to in vivo inhalation studies


-------
SEPA Acknowledgements

Thank you to ORD, CPHEA, and CCTE collaborators:

Engineers, Toxicologists, and Data Scientists

•	Adam Speen

•	Jessica Murray

•	Jose Zavala

•	Elise Carlsten

•	Wyatt Zander

•	Todd Krantz

•	Paul Evansky

•	Dave Davies

•	Jason Weinstein

•	George Hudson

•	Lisa Dailey

Josh Harrill
Logan Everett
Joseph Bundy
Rusty Thomas
Maureen Gwinn

Adam Speen, PhD
• HTTr, BMD Analysis

Jessica R. Murray, PhD

• CAD/CFD Modeling,
Analytical Dosimetry


-------
oEPA

References

•	Zavala J, Ledbetter AD, Morgan DS, Dailey LA, Puckett E, McCullough SD, Higuchi M. A new cell culture exposure system for studying the toxicity of volatile chemicals at the air-liquid
interface. Inhal Toxicol. 2018 Mar-Apr;30(4-5):169-177. doi: 10.1080/08958378.2018.1483983. Epub 2018 Aug 8. PMID; 30086657; PMCID; PMC6516487.

•	Zavala J, Greenan R, Krantz QT, DeMarini DM, Higuchi M, Gilmour Ml, White PA. Regulating temperature and relative humidity in air-liquid interface in vitro systems eliminates cytotoxicity
resulting from control air exposures. Toxicol Res (Camb). 2017 May 23;6(4):448-459. doi: 10.1039/c7tx00109f. PMID: 30090513; PMCID: PMC6062410.

References for LOAEL/NOAEL values:

•	Elf Atochem S.A. (Elf Atochem Societe Anonyme). (1997). Study of Acute Toxicity of N-Propyl Bromide Administered to Rats by Vapour Inhalation. Determination of the 50% Lethal
Concentration (Lc50/4 Hours). Ineris-L.ET.E. Study No. 95122. Study Performed by Laboratoire Detudes De Toxicologie Experimentale.

•	Kim, HY; Chung, YH; Jeong, JH; Lee, YM; Sur, GS; Kang, JK. (1999). Acute and repeated inhalation toxicity of 1-bromopropane in SD rats. Journal of Occupational Health, 41, 121-128. DOI
10.1539/Joh.41.121.

•	National Toxicology Program. (2011). NTP technical report on the toxicology and carcinogenesis studies of 1-bromopropane (CAS no. 106-94-5) in F344/N rats and B6C3F mice (inhalation
studies). Research Triangle Park, NC: National Toxicology Program, U.S. Dept. of Health and Human Services, National Institutes of Health.

•	U.S. Department of Health and Human Services. (1993). Registry of Toxic Effects of Chemical Substances (RTECS, online database). National Toxicology Information Program, National
Library of Medicine, Bethesda, MD.

•	Prendergast, JA; Jones, RA; Jenkins, LJ, Jr; et al. (1967) Effects on experimental animals of long-term inhalation of trichloroethylene, carbon tetrachloride, 1,1,1-trichloroethane
dichlorodifluoromethane, and 1,1-dichloroethylene. Toxicol Appl Pharmacol 10:270-289.

•	Agency for Toxic Substances and Disease Registry (ATSDR). (1997). Toxicological Profile for Trichloroethylene (Update). U.S. Public Health Service, U.S. Department of Health and Human
Services, Atlanta, GA.

•	Selgrade, MK; Gilmour, Ml. (2010) Suppression of pulmonary host defenses and enhanced susceptibility to respiratory bacterial infection in mice following inhalation exposure to
trichloroethylene and chloroform. Journal of Immunotoxicology, 7, 350-356. DOI 10.3109/1547691X.2010.520139.


-------
References cont.

Aranyi, C; O'Shea, WJ; Graham, JA; Miller, FJ. (1986) The effects of inhalation of organic chemical air contaminants on murine lung host defenses. Fundamental and Applied Toxicology, 6, 713-720. DOI 10.1016/0272-
0590(86)90184-3.

Burek, JD; Nitschke, KD; Bell, TJ; Wackerle, DL; Childs, RC; Beyer, JE; Dittenber, DA; Rampy, LW; McKenna, MJ (1984). Methylene chloride: A two-year inhalation toxicity and oncogenicity study in rats and hamsters.
Fundamental and Applied Toxicology, 4, 30-47. DOI 10.1093/toxsci/4.1.30.

National Toxicology Program. (1986). NTP technical report on the toxicology and carcinogenesis studies of dichloromethane (methylene chloride) (CAS No. 75-09-2) in F344/N rats and B6C3F mice (inhalation studies).
Research Triangle Park, NC: National Toxicology Program, U.S. Dept. of Health and Human Services, National Institutes of Health.

Nitschke, KD; Burek, JD; Bell TJ; Kociba, RJ; Rampy, LW; McKenna, MJ. (1988). Methylene chloride: A 2-year inhalation toxicity and oncogenicity study in rats. Fundamental and Applied Toxicology, 11, 48-59. DOI
10.1016/02.72-0590(88)90269-2.

Agency for Toxic Substances and Disease Registry (ATSDR). (2007). Toxicological Profile for Acrolein. Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA.

U.S. Environmental Protection Agency. (1988). Health and Environmental Effects Profile for Formaldehyde. EPA/600/x-85/362. Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment,
Office of Research and Development, Cincinnati, OH.

Monteiro-Riviere, NA; Popp, JA. (1986) Ultrastructural evaluation of acute nasal toxicity in the rat respiratory epithelium in response to formaldehyde gas. Fundam Appl Toxicol 6:251-262.

Javdan, M; Taher, TE. (2000) The short and long term effects of formaldehyde vapour (2 ppm and 5 ppm) on rat nasal epithelium. Pathol Int 5Q:A77.

Woutersen, RA; Appelman, LM; Wilmer, JW; et al, (1987) Subchronic (13-week) inhalation toxicity study of formaldehyde in rats. J Appl Toxicol 7:43-49.

Feron, VJ; Bruyntjes, JP; Woutersen, RA; et al. (1988) Nasal tumours in rats after short-term exposure to a cytotoxic concentration of formaldehyde. Cancer Lett 39:101-111.

Zwart, A; Woutersen, RA; Wilmer, JW; et al. (1988) Cytotoxic and adaptive effects in rat nasal epithelium after 3-day and 13-week exposure to low concentrations of formaldehyde vapour. Toxicology 51:87-99.

Shugaev BB. 1969. Concentrations of hydrocarbons in tissues as a measure of toxicity. Arch Environ Health 18:878-882.

NTP. 1993. NTP technical report on the toxicology and carcinogenesis studies of 1,3-butadiene (CAS No. 106-99-0) in B6C3F1 mice (inhalation studies). Research Triangle Park, NC: National Toxicology Program. NTP TR 434.
Crouch CN, Pullinger DH, Gaunt IF. 1979. Inhalation toxicity studies with 1,3-butadiene 2. 3 month toxicity study in rats. Am Ind Hyg Assoc J 40:796-802,

U.S. Department of Health and Human Services. (1993). Registry of Toxic Effects of Chemical Substances (RTECS, online database). National Toxicology information Program, National Library of Medicine, Bethesda, MD.
U.S. Environmental Protection Agency. (1999) Integrated Risk Information System (IRIS) on Acetaldehyde. National Center for Environmental Assessment, Office of Research and Development, Washington, D.C.


-------
„•	%	U.S. EPA ORD, CSS Research Program

I vwy *

\	^ NAMS Research and Development, Session D: System-specific Models and Approaches

§	|	Board of Scientific Counselors Subcommittee

saz.

pro^	February 2, 2021

Neurovascular Unit Modeling and
Blood Brain Barrier Function

Thomas B. Knudsen, PhD

Developmental Systems Biologist
Center for Computational Toxicology and Exposure
Research Triangle Park NC 27711

knudsen.thomas@epa.gov
ORCID 0000-0002-5036-596x

DISCLAIMER: The views expressed are those of the presenters and do not reflect Agency policy.

1


-------
Fetal Brain Barriers

The Neurovascular Unit (NVU) is a relatively recent concept describing the relationship
between neuronal and vascular compartments, particularly for two key processes:

X
Oi
O

0
u

1

o

v.
QJ


-------
BBB pathophysiology

•	Evidence linking BBB dysfunction with prenatal/antenatal pathophysiological states:

defective brain transport of leptin (obesity)
reduced CNS insulin (baroreceptor deficiency in pregnancy)
microglial activation and neuroinflammation (Zika-microcephaly, FIRS)

GLUT1 deficiency syndrome (epilepsy, learning disabilities)

SL75A (LAT1) dysfunction (autism)

SL16A2 (MCT8) deficiency (altered thyroid delivery and neurological impairment)
DNT- hypoxia, metal toxicity, pesticide toxicity,...

•	OECD Test No. 424: Neurotoxicity Study in Rodents - does not directly evaluate BBB
function but can be influenced by a breakdown in the function in the various cell types.

•	We know that chemicals interact with the BBB, but to what extent do chemicals of interest
disrupt its development and function?

3


-------
BBB microvasculature: late fetal to adult lifestages

Endothelial cells: continuous tight junctions, no
fenestrations, limited transcytosis.

Pericytes: produce a basement membrane continuous with
that produced by the endothelial microvasculature.

Astrocytes: processes (end-feet) interact directly with the
basement membrane; appear after formation of the BBB.

Microglia: resident macrophages of the brain, are of
hematopoietic origin in the early embryonic yolk sac.

Reseorchgote.net

• Microglia orchestrate neurovascular patterning through local signaling; however,
when activated they can invoke a local neuroinflammatory response.

astrocyte

Capillary
Endothelial Cell

"Orhrocy*6

endothelium

Basal Lamina

4


-------
BBB phylogeny

Key BBB transporters are conserved

Species

GLUT1

P-gly

SLC7A5

Human

100

100

100

Chimpanzee

99.7

99.5

99.6

House mouse

97.3

87.1

81

Zebrafish

81.3

64.8

77.7

Australian ghostshark

82.7

65.7

72.7

Amphioxus

38

54.5

61.6

Waterflea

46.1

48.5

45.4

House fly

50

41.5

48.5

Arctic lamprey

64.4

—

—

Giant Pacific octopus

—

—

—









DOt 10.1(K)2/bdr2.1180

R E V I E W A R 1 I C L E

Blood-brain barrier development: Systems modeling and
predictive toxicology

Ratcrinc S. Saili1 © I Tocltl J. Zurlindcn1 O I Andrew J. Schwab2 G I

Aymeric Silvin3 I Nancy C. Baker1 © I E. Sidney Hunter III2 I Florent Ginhoux3 © I

Thomas B. Knudsen1

Saili et al. 201 7, B

r"\

(Human)

\	Homo

sapiens

A"



Pan
troglodytes

(Chimpanzee)

Mus
musculus
(House mouse)

Endothelial



Danio
rerio

(Zebrafish)	fV

Callorhinchus 1
milii

(Australian
ghostshark)

Endothelial

Glial


-------
BBB ontogeny

E8.25-E8.5

•	Different components emerge and mature at different
stages of prenatal development.

•	Commences with angiogenic sprouting from the
perineural vascular plexus (PNVP).

•	ECs + PCs invade the embryonic neural epithelium on
E9-10 (mouse) and GD 26 (human).

•	Circulating microglia from the yolk sac colonize the
neuroepithelium resident macrophages of the brain.

•	BBB properties (tight junctions, GLUT1) and barrier
function (TEER) evident by Ell and increases to birth.

6


-------
Microglia are required to establish BBB/microvasculature

• promote vascular patterning and BBB barrier development in the embryonic forebrain.

SlgN

Vasculature (CD31) Y[

Macrophage (Ibal)

Dextran 3KDa	Microglia depleted (anti-CSFRl)

EPA-A*STAR collaboration with A Silvin, F Ginhoux - A*STAR/SlgN

1


-------
Hypothesis: 'microglial sensing' is a key event in BBB developmental toxicity

BBB Knowledgebase sifter

513 records main subject (MeSH) on BBB in an article

> nt-itnrl f o r rli-iiinlnnmnrit infl -i rlin nr> i i- -i I nffn r-t	

115 DNT chemicals -> main subject (MeSH) in an article
annotated for development/embryology AND neurological effects

82 chemicals5 with DNT effects on BBB
BPA, 5FU, Pb, paraquat, retinoic acid

Irt J Dev Neurosci. 2Q04 Feb;22(1):31-7

Mosquito repellent (pyrethroid-based) induced dysfunction of blood-brain barrier permeability in
developing brain.

Siriha C1, Aarawal AK. Islam F Seth K Chaturvedi RK Shukla S Seth PK
© Author information

Abstract

Pyrethroid-based mosquito repellents (MR) are commonly used to protect humans against mosquito vector New born babies and children are
often exposed to pyrethroids for long periods by the use of liquid vaporizers. Occupational and experimental studies indicate that pyrethroids
can cause clinical, biochemical and neurological changes, and that exposure to pyrethroids during organogenesis and early developmental
period is especially harmful. The neurotoxicity caused by MR has aroused concern among public regarding their use. In the present study, the
effect of exposure of rat pups during early developmental stages to a pyrethroid-based MR (allethrin, 3.6% w/v, 8h per day through inhalation)
on blood-brain barrier (BBB) permeability was investigated. Sodium fluororescein (SF) and Evan's blue (EB) were used as micromolecular
and macromolecular tracers, respectively. Exposure during prenatal (gestation days 1-20), postnatal (PND1-30) and perinatal (gestation days
1-20 + PND1-30) periods showed significant increase in the brain uptake index (BUI) of SF by 54% (P < 0.01), 70% (P < 0.01), 79% (P <
0.01), respectively. This increase persisted (68%, P < 0.01) even 1 week after withdrawal of exposure (as assessed on PND37). EB did not
exhibit significant change in BBB permeability in any of the group. The results suggest that MR inhalation during early
prenatal/postnatal/perinatal life may have adverse effects on infants leading to central nervous system (CNS) abnormalities, if a mechanism
operates in humans similar to that in rat pups.

development

N Baker, NCCT/Leidos

8


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HTS profiling of angiogenic-neurogenic chemical bioactivity

Reproductive Toxicology 96 (2020) 300-315



ELSEVIER

Contents lists available at ScienceOirect

Reproductive Toxicology

journal homepage: www.elsevier.com/locate/reprotox

Hi'lHuduclivc
Toxicology

A cross-platform approach to characterize and screen potential
neurovascular unit toxicants

Todd J. Zurlinderr\ Katerine S. Saili ', Nancy C. Bakerb, Tarja Toiniela' , Tuula Heinonenc,
Thomas B. Knudsen ' *

• lIS. Envronmental Proiecaeri Agency, Office of Research and DevekipmenL Center fa- Computational Toxicalcgy and Exposure. United States
b Leidos, linned States

'fJCAM. Faculty of Medicine and Heath Technology, Tampere University, Finland

Phase A

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3
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o

2

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cr

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5'

CL
CD
<
CD
O
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3

CD
D

EC
migration

EC tubule
formation

hNP migration/
proliferation

hNC migration/
proliferation

hNN network
formation

Phase B

Angiogenic Neurogenic

Cluster activity
*

NVU hazard
prioritization

Li>

<7,

Literal

re

ii) Lit. profile

Zurlinden et a I. 2020, Reprod Toxicol

•	HTS data generated on up to 58 reference chemicals across 18
diverse cell-based angiogenic and neurogenic features.

•	ToxPi bioactivity signatures used to train a logistic regression
literature model to annotate clusters with PubMed MeSH.

•	Chemical-specific pairwise mutual information score predicts
NVU developmental hazard potential for advanced modeling.

inflammatory

T i , ~'"r "f:

- -> / A

^ neurogenic

angiogenic

* * Vrf


-------
BBB systems model for predictive toxicology

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0l>

C

55

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S

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3

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24 molecular targets
(99 ToxCast assays)

Saili et at. 2017, Birth Defects Res

10


-------
Advanced Modeling:	neovascu

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CM

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ro

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E9.75

E10.0

E10.5

Neuroepithelium

NICD > Threhoid





Vessel migration

Stalk Cell



SVP formation

C^vegfX^)

Reduced VEGF-A/Cresponse

Ventricle/NPC population

VEGF-A gradient: NPCs in subventricular zone

endothelial tip cell
endothelial stalk ce
microglial cell

CCTE, work in progress

11


-------
Executing a simulated concentration-response

Summary plots - Representative samples only

2 8

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o

c 1
o

Q.

OT

a)

a:

a)

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c

 deficiency of SVZ).

Quantitative microvascular 'cybermorphs' predicts an
AC50 for Mancozeb disruption at 0.5 [iM.

12


-------
Checking the prediction: microglial integration in a synthetic microsystem

THE UNIVERSITY

Wisconsin

MADISON

FULL PAPER



ADVANCED
HEALTHCARJ
_/MATERIAL! Check for

www.advhealthmat.cL

Engineered Perineural Vascular Plexus for Modeling
Developmental Toxicity

Gaurav Kaushik, Kartik Gupta, Victoria Harms, Elizabeth Torr, Jonathan Evans,
HunterJ.Johnson, Cheryl Soref, Suehelay Acevedo-Acevedo, Jessica Antosiewicz-Bourget,
Daniel Mamott, Peyton Uhl, Brian P.Johnson, Sean P. Palecek, David J. Beebe,

James A. Thomson, William T. Daly* and William L. Murphy*

Microglia Migration into Neural Layer

Microglia migration

&0

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T

U

! -





1

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Critical concentration (PoD) for Mancozeb
on neural tube vascularization:

-	predicted by in silico cNVU = 0.5 fxM

-	observed in organotypic culture = 0.3 \xM.

20 Device Microfluidics
Plate (96 well format)

Neuronal layer

Neuronal chamber

Vascular
chamber

Side channels
Cross-Section of 1/20 Device

Hydrogel barrier
(Glial limiting
membrane)

Vascular
Plexus

EPA STAR Center Co-operative grant #835737, University of Wisconsin (W Murphy)

13


-------
Incorporating the neurogenic domain (preliminary)

500

300

000

200

Embryonic Neuroeoithelium

(	\

Endothelial Stalk Cell

Endothelial Tip Cell

Reduced
VEGF-A/C
Response

Neuroproaenitor Cell





Migration of
Blood Vessels

Ventricle

^VEGF-yO-

V



O Nophode, work in progress

14


-------
Neurovascular Unit-on-a-Chip Module

Human endothelial cells, astrocytes,
pericytes, and neurons.

Microanalyzer for real time data
(glucose, lactate, oxygen, pH, and 4
neurotransmitters).

Testing neuroinflammation (LPS) and
neurotoxicity (CPF) pathways.

Rotary Planar Peristaltic
Micropumps

(RPPM) for Perfusion

Power
Controller
Connection

(Endothelial Cell Channels)

Brain Compartment

(Neurons, Astrocytes, Pericytes)
Additional Brain Side Perfusion
Membrane Opaque

Perfusion Vials

with Septa

Microvasculature,
Brain Side

Microvasculature
Input Tube

Waste
Collection

Output Ports

NEMA17
Pump Motor

Brass Pump Driveheads

Brain Side
Input Tube

EPA STAR grant (new), Vanderbilt UniversityD Cliffel and J Wikswo

15


-------
Embryonic Human Neurovascular Unit (hNVU):

impact of chemical-induced disruption of neural morphogenesis and function.

A. Transwell

Matrigel Matrix
0.4 uM membrane

B. Ibidi Microfluidic

Matrigel Matrix
0.5 uM membrane

Lower Channel

C. Experimental Timeline

_u,	

Flow-

Plate Endothelial
Cells

Plate Plate Neural Progenitors
Pericytes	(EZ Spheres)

S Hunter; work in progress

m Endothelial Cells
^ Pericytes

Neurons and Astrocytes
(EZ Spheres)















Jv



1.5 glass coverslip

: 1.5 glass coverslip

FIX, ICC

A

Barrier Formation - Day 7

Barrier Formation - Day 14

150 n

X
3

150n

x

=3

Control 10kDa 4kDa Control 10kDa 4kDa

Control 10kDa 4kDa Control 10kDa 4kDa

Transwell Ibidi Microfluidic

Transwell Ibidi Microfluidic

Impact of the endothelial-pericyte barrier on
developmental neurotoxicity,

Assess chemical effects on barrier function in a

human cell-based in vitro system(s).

16


-------
Qualification of barrier function in the hNVU

96-well	Transwell	Ibidi Microfluidic 	

DMSO

Y-27632
(10 (J.M)

BCH

(10 mM)

Transwell
4 kDa FITC-Dextran Flux

150-

100

E

i-

o
Z

Empty TW
DMSO
Y-27632
BCH

Day 7

Day 14

Neuron Count

_n o
> >

tS 20

iiili

96-well Transwell Microfluidic

DMSO

Y-27632

BCH

Ibidi Microfluidic
4 kDa FITC-Dextran Flux

150-1

100-

Day 7

Day 14

Neurite Length

500-,

o 400-

Empty Device
DMSO
Y-27632
BCH

ill ill ill

96-well Transwell Microfluidic

DMSO

Y-27632

BCH

•	Y-27632 is a cell permeable rock inhibitor that cross the NVU barrier - increases proliferation and differentiation of
human 'EZ neurosphere' cells to neural and astrocytic phenotypes (green fluorescence) in all models.

•	BCH is a LAT-1 transporter inhibitor that does not cross the barrier; with BCH there are few NPCs cells, little
differentiation (bright green neural structure) and almost no red astrocytes. In the MPS devices, proliferation and
differentiation are similar to control cultures.


-------
Embryo-fetal NVU Barrier: application to developmental neurotoxicity

•	Microelectrode array (MEA) assay
platform developed in Tim Shafer's lab.

•	Monitors rat cortical neuronal network
formation and electrochemical activity.

•	Used to profile ToxCast chemicals for
direct effects on neuronal networks.

•	Rat cortical MEA system has been
integrated with the transwell hNVU.

T Shafer, S Hunter - work in progress

18


-------
Summary

•	NVU composed of multiple cells types and >400 genes, at least 86 of which play
important roles in BBB development and function.

•	BBB becomes functional soon after it forms during organogenesis (6-14 weeks in
human gestation).

•	Development and function is perturbed by multiple pathophysiological conditions
and may underlie neurodevelopmental disorders linked to chemical exposure.

•	Dynamics of the system modeled in silico and in vivo focusing on microglial sensing
as potential roles in neurodevelopmental toxicity linked to their activation.

19


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Selected References

•	Knudsen TB, Klieforth, B and Slikker W Jr. (2017) Programming microphysiological systems for children's health
protection. Exp Biol Med. 242: 1586-1592.

•	Baker NC, Knudsen T and Williams A (2017) Abstract Sifter: a comprehensive front-end system to PubMed.
FlOOOResearch 2017, 6(Chem Inf Sci):2164 https://fl000research.com/articles/6-2164/vl

•	Saili KS, Zurlinden TJ and Knudsen TB (2017) Modeling the neurovascular unit in vitro and in silico. Chapter for
'Handbook of Developmental Neurotoxicology', 2nd edition W Slikker, Jr (ed), Published by Elsevier, Inc. pp 127-142.

•	Saili KS, Zurlinden TJ, Schwab A, Ginhoux F, Silvin A, Baker NC, Hunter ES III, Ginhoux F and Knudsen TB (2017) Blood-
Brain Barrier Development: Systems Modeling and Predictive Toxicology. Birth Defects Res. 109: 1680-1710.

•	Saili KS, Franzosa JA, Baker NC, Ellis-Hutchings RG, Settivari RS, Carney EW, Spencer R, Zurlinden TJ, Kleinstreuer NC, Li
SZ, Xia M and Knudsen TB (2019) Systems modeling of developmental vascular toxicity. Curr Opin Toxicol 15: 55-63.

•	Zurlinden TJ, Saili KS, Baker NC, Toimela T, Heinonen T and Knudsen TB (2020) A cross-platform approach to
characterize and screen potential neurovascular unit toxicants. Reprod Toxl 96: 300-315.

•	Schwab AJ, Jeffay SC, Nichols HP and Hunter ES III (2021) Development of complementary 3-dimensional human
neurovascular unit models using static transwells and dual-compartment microfluidic devices. (In Revision).


-------
Acknowledgements

v-Emttpyo Ja
•8?

Nancy Baker (Leidos)

Jessica Conley (CCTE)

Florent Ginhoux (A*STAR Singapore)

James Glazer (Indiana University)

Sid Hunter (CCTE)

Tom Knudsen (CCTE)

Om Naphade (Brown University)

Jocylin Pierro (CCTE)

Kate Saili (now OAQP)

Andrew Schwab (now Metabolon, Inc.)

Aymeric Silvin (A*STAR Singapore)

Richard Spencer (General Dynamics)

Douglas Young (VTM Matrix Interface)

Todd Zurlinden (now CPHEA)



www.epa.go

3

Research

science in ACTION

INNOVATIVE RESEARCH FOR A SUSTAINABLE FUTURE



Virtual Tissue Models: Predicting How Chemicals Impact Human Development

EPA contract EP-D-13-056 Aruna Biomedical
Tracey St ice
Steven Stice
Forrest Goodfellow

EPA contract "P-D-1.3-053 VALA Sciences
Lily Feng

EPA STAR Center Grant 835737 Univ Wisconsin
William Murphy, PI
Bill Daly

Eric Nguyen (now NIH/NEi)

Gaurav Kaushik

EPA STAR Center Grant R839504 Vanderbilt Univ
David Cliffel, PI
John Wikswo

21


-------