Dose-Response Relationships for Acute
Effects of Volatile Organic Compounds
on the Mammalian Nervous System
Philip J. Bushnell
Neurotoxicology Division
National Health and Environmental Effects Research Laboratory
Office of Research and Development, US EPA
Research Triangle Park, NC
McKim Conference on Predictive Toxicology
September 25, 2007
Rationale
• Done one chemical at a time, risk assessments can neither account for all
of the chemicals currently in commerce nor keep up with the rate of their
invention and use.
• The NAS (2007) “envisions a new toxicity-testing system that evaluates
biologically significant perturbations in key toxicity pathways by using
new methods in computational biology and a comprehensive array of in
vitro tests based on human biology.”
• Our goals:
 To facilitate the extrapolations necessary to implement risk assessments in the
current toxicity-testing system.
 To help characterize key toxicity pathways as we move into a new system
• EPA Clients
 Office of Air and Radiation (OAQPS and OTAQ)
 National Homeland Security Research Center
Approach
1. Develop Exposure-Dose-Response models to
understand and characterize the neurotoxicity of
volatile organic compounds (VOCs)
2. Use the EDR models to generate information to
replace the default uncertainty factors currently
applied in the extrapolations necessary for risk
assessments
1.
2.
3.
4.
C x t relationships for acute effects
Cross-species
High-to-low dose
Acute-to-chronic
Exposure-Dose-Response Model
Applied Dose
Concentration in air
and exposure duration
Internal Dose
Concentration in
Target Tissue
Interaction with
Target Tissue
Effect on receptor,
enzyme, etc.
Adverse
Outcome
Change in function
in vivo
Exposure-Dose-Response Model
Chemicals: Volatile organic
compounds – toluene, trichloroethylene,
Applied Dose
Concentration and
duration in air
perchloroethylene, iso-octane
Internal Dose
Concentration in
Target Tissue
Interaction with
Target Tissue
Effect on receptor,
enzyme, etc.
Adverse
Outcome
Change in function
in vivo
Exposure-Dose-Response Model
Chemicals: Volatile organic
compounds, primarily toluene
Applied Dose
Concentration and
duration in air
Internal Dose
Concentration in
Target Tissue
Interaction with
Target Tissue
Changes in ion
channel function
Effect on receptor,
enzyme, etc.
Adverse
Outcome
Change in function
in vivo
Exposure-Dose-Response Model
Chemicals: Volatile organic
compounds, primarily toluene
Applied Dose
Concentration and
duration in air
Internal Dose
Concentration in
Target Tissue
Interaction with
Target Tissue
Changes in ion
channel function
Effect on receptor,
enzyme, etc.
Adverse
Outcome
Acute, reversible effects on the
nervous system  Narcosis
Change in function
in vivo
Narcosis
• Privation of sense or consciousness, due to a narcotic
• An English heavy-metal band
• Enemy of Batman that uses gas to turn his victims
into a state of Bliss
• A reversible state of arrested physiology caused by
non-reactive toxicants
• A likely mode of action for acute effects of volatile
solvents, with a wide range of effects




Subtle CNS changes at sub-anesthetic concentrations
Clear sensory and motor effects at high concentrations
Anesthesia
Death
Narcosis as a Potential Mode of Toxic
Action of VOCs in the CNS
• Exposure scenarios
 Inhalation at various concentrations for various
durations  Internal dose via PBPK models
• Multiple endpoints
 Transform to a common scale for quantitative
comparisons
Estimating Internal Dose from
Inhalation Scenario
Inhalation
Lung
Exhalation
Brain
Physiologically-Based
Pharmacokinetic
Model
Venous
Blood
Fat
Rest of
Body
Liver
Elimination
Arterial
Blood
Toluene Concentration (mg/L)
PBPK Model Evaluation
Brain
10
Blood
Toluene concentration
in air = 585 ppm
Long-Evans rats
1
0
1
2
3
4
5
Time (hrs)
6
7
8
Kenyon et al., 2007
Effects in Mammalian Systems
• In vivo






Increased reaction time (human)
Reduced sensory function (rat)
Slowed response time (rat)
Reduced choice accuracy (rat)
Impaired shock avoidance (rat)
Lethality (rat)
• In vitro
 Inhibition of current through ion channels associated with excitatory
pathways
• NMDA (NR2A, NR2B, NR2C)
• Nicotinic ACh (nAChR)
• Voltage-Sensitive Calcium Channels (VSCC)
 Facilitation of current through ion channels associated with inhibitory
pathways
• γ-Amino Butyric Acid (GABA-A)
• Glycine
Sensory Function Method
Boyes et al., 2003
Sensory Function Method
• The rat watches TV patterns
• Brain waves are recorded
from visual cortex
• The contrast of the visual
pattern cycles on and off at
frequency F (typically 5Hz)
• The recorded and averaged
brainwave reflects visual
processing
• Spectral analysis of the
evoked potential shows
response power at the
stimulation frequency F and
higher harmonics – especially
at F2
Sensory Function Results
Visual Evoked Potential Amplitude as a
Function of Estimated Brain TCE Concentration
0 ppm
500 ppm during exposure
1000 ppm during exposure
2000 ppm during exposure
2000 ppm after exposure
3000 ppm during exposure
3000 ppm after exposure
4000 ppm during exposure
4000 ppm after exposure
5000 ppm during exposure
5000 ppm after exposure
Sigmoidal function
10
9
F2 Amp (uV)
(mean +\- SEM)
8
7
6
• F2 amplitude falls with
increasing concentration
of the VOC in the brain
of the rat.
5
• The effect is not
unambiguously related to
the concentration or
duration of exposure.
4
3
2
1
0
0
20
40
60
80
100
120
Estimated Brain [TCE] (mg/l)
140
160
Boyes et al., 2003
Effects in Mammalian Systems
• In vivo






Increased reaction time (human)
Reduced sensory function (rat)
Slowed response time (rat)
Reduced choice accuracy (rat)
Impaired shock avoidance (rat)
Lethality (rat)
• In vitro
 Inhibition of current through ion channels associated with excitatory
pathways
• NMDA (NR2A, NR2B, NR2C)
• Nicotinic ACh (nAChR)
• Voltage-Sensitive Calcium Channels (VSCC)
 Facilitation of current through ion channels associated with inhibitory
pathways
• γ-Amino Butyric Acid (GABA-A)
• Glycine
Behavioral Method
Effects of Toluene on Signal Detection
Behavior
Air Concentration and Time
Adjusted to Constant Air Session
0.8
0.8
P(Correct)
A
B
0.7
0.7
0.6
0.6
Air
1200 ppm
1600 ppm
2000 ppm
2400 ppm
0.5
0.5
22
Response Time (sec)
2.0
Air
1200 ppm
1600 ppm
2000 ppm
2400 ppm
C
34
46
58
Air
1200 ppm
1600 ppm
2000 ppm
2400 ppm
1.5
22
70
2.0
1.0
0.5
0.5
46
58
70
58
70
Air
1200 ppm
1600 ppm
2000 ppm
2400 ppm
D
1.5
1.0
34
0.0
0.0
22
34
46
58
70
Duration of Exposure (min)
22
34
46
Duration of Exposure (min)
Bushnell et al., 2007
Dose Metric: Concentration of Solvent in Brain
Exposure AUC
0.8
P(Correct)
0.7
0.6
0.6
0.5
Response Time (sec)
C
B
0.7
2.0
0.8
0.8
A
0.7
Air
1200 ppm
1600 ppm
2000 ppm
2400 ppm
0.6
0.5
0.5
0
Brain AUC
Concentration in Brain
400 800 1200 1600 2000 2400
D
0
2.0
20
E
40
60
0
80 100 120 140
2.0
Air
1200 ppm
1600 ppm
2000 ppm
2400 ppm
1.5
1.0
1.0
1.0
0.5
0.5
0.5
400 800 1200 1600 2000 2400
Cumulative Inhaled Dose (ppm-hr)
60
80
100
20
40
60
80
100
F
0.0
0.0
0
40
1.5
1.5
0.0
20
0
20
40
60
80 100 120 140
Brain [Toluene] (mg/L)
0
Cumulative Brain [Toluene] (mg-hr/L)
Bushnell et al., 2007
Effects in Mammalian Systems
• In vivo






Increased reaction time (human)
Reduced sensory function (rat)
Slowed response time (rat)
Reduced choice accuracy (rat)
Impaired shock avoidance (rat)
Lethality (rat)
• In vitro
 Inhibition of current through ion channels associated with excitatory
pathways
• NMDA (NR2A, NR2B, NR2C)
• Nicotinic ACh (nAChR)
• Voltage-Sensitive Calcium Channels (VSCC)
 Facilitation of current through ion channels associated with inhibitory
pathways
• γ-Amino Butyric Acid (GABA-A)
• Glycine
Female
Xenopus
Laevis
Frog
cDNA
Cut with Restriction Enzymes
In Vitro Transcription
Linearized
DNA
Oocyte Removal
Surgery
mRNA
Separated
Stage VI, V
Oocytes
Incubate 2-7 days at 18oC
Oocyte Injections
Voltage Current
ACh
Clamp at appropriate
test potential
Two-Electrode Voltage Clamp
EUREKA
Currents!!!
50 nA
2 sec
Toluene Reversibly Inhibits nAChRs
a7 - human
a4b2-human
1 mM TOL
1 mM TOL
ACh
ACh
ACh
ACh
ACh
40 nA
ACh
200 nA
20 sec
5 sec
a4b2-rat
a7-rat
1 mM TOL
ACh ACh
ACh
100 nA
2 sec
1 mM TOL
ACh ACh
ACh
50 nA
20 sec
Bale et al., 2005
nAChR Inhibition by Toluene:
Concentration-Dependent
and Species-Independent
75
100
a7- human
a7- rat
% Inhibition
% Inhibition
100
50
25
0
-4
10
10-3
[Toluene] M
-2
10
75
50
a4b2-human
a4b2-rat
25
0
10-4
10-3
10-2
[Toluene] M
Modified from Bale et al., 2005
Scaling Effects Across Endpoints
Generate transformations that convert measured metric to a
uniform scale from 0 (no effect) to 1 (maximum possible effect).
Visual Evoked Potentials: E(VEP)i = (VEPb - VEPi) / VEPb
Response time: Convert to speed, then
E(RS)i = (RSb - RSi) / RSb and because RT = 1/RS,
E(RT)i = 1.0 - (RTb / RTi)
Accuracy: E(Acc)i = (Accb - Acci) / Accb - 0.5)
Escape-Avoidance: E(Esc)i = (Escb - Esci) / Escb
In Vitro effects did not require scaling, because inhibition ranged
from 0 to 1.
Dose-Effect Functions – in vivo Endpoints
Human Reaction Time
Rat VEP
0.9
1.0
b.
0.9
rat VEP
0.7
0.4
0.3
0.2
0.6
0.5
0.4
0.3
0.2
0.1
0.1
0.0
0.0
Rat Response Time
0.50
0.75
1.00
1.25
1.50
0.6
rat SIGDET, Accuracy
0.5
0.4
0.3
0.2
Effect Magnitude
Effect Magnitude
Effect Magnitude
0.5
0.6
0.4
0.3
0.2
0.00
0.25
0.50
0.75
1.00
1.25
1.50
Estimated Brain Toluene Concentration (mM)
0.5
0.4
0.3
0.2
0.1
0.0
0.0
0.00
5.0
0.6
0.1
0.1
0.0
4.0
0.7
0.8
0.7
3.0
0.8
d.
rat SIGDET, RT
2.0
Comparison
0.7
c.
1.0
Estimated Brain Toluene Concentration (mM)
Rat Accuracy
1.0
0.9
0.25
Estimated Brain Toluene Concentration (mM)
0.25
0.50
0.75
1.00
1.25
1.50
Estimated Brain Toluene Concentration (mM)
0.0
y
-0.1
0.00
0.30
ra
c
0.25
-A
cc
u
0.20
T
0.15
-R
T
0.10
SI
GD
E
0.05
Estimated Brain Toluene Concentration (mM)
Ra
t
0.00
-0.1
0.0
IG
DE
T
0.0
EP
0.1
0.5
Ra
tS
0.2
rat ES-AV
0.7
0.6
Effect Magnitude
Effect Magnitude
0.3
e.
0.8
Ra
tV
0.8
human CRT
CR
T
a.
Hu
ma
n
0.4
Effect Magnitude
Rat Shock Avoidance
-AV
Rat ES
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Estimated Brain Toluene Concentration (mM)
Taken from Benignus et al., 2007
Effects of Inhaled Toluene In Vivo
Effect Magnitude
1.00
0.75
Human Reaction Time
Rat Visual Evoked Potential
Rat Signal Detection - Response Time
Rat Signal Detection - Accuracy
Rat Shock Avoidance
0.50
0.25
Rat
Lethality
0.00
0.01
0.1
1
Estimated Brain Toluene Concentration (mM)
10
Effects of Inhaled Toluene In Vivo
Effect Magnitude
1.00
0.75
Human Reaction Time
Rat Visual Evoked Potential
Rat Signal Detection - Response Time
Rat Signal Detection - Accuracy
Rat Shock Avoidance
0.50
Increasing
Motivation
0.25
Rat
Lethality
0.00
0.01
0.1
1
Estimated Brain Toluene Concentration (mM)
10
Effects of Inhaled Toluene In Vitro
Effect Magnitude
1.00
0.75
nAChR
VSCC
NMDA NR1/NR2A
NMDA NR1/NR2B
NMDA NR1/NR2C
0.50
0.25
Rat Lethality
0.00
0.01
0.1
1
Toluene Concentration in Culture Medium (mM)
10
Conclusions
• Volatile solvents exert reversible effects on the nervous system
• Effects are
• Graded in severity and quality
• Directly related to the concentration in the brain
• Quantitatively comparable via scaling to a common metric
• Consistent with a mode of action based on narcosis
• Modeling based on brain concentration
• Accounts for differences in exposure scenario
• Permits extrapolation across dose, duration, species
• Mode of action for acute effects
• Classic view: Membrane fluidity
• Current thinking: Alteration of membrane-resident proteins,
especially certain voltage- and ligand-gated ion channels
• Interactions with membrane proteins may provide a basis for
predicting toxicity based on physico-chemical properties of the
compounds…
Invaluable Collaborators
Vernon Benignus – Dose-Response Modeling
Will Boyes – Visual Neurophysiology in vivo
Tim Shafer – In vitro Neurophysiology
Elaina Kenyon – PBPK Modeling
Ambuja Bale – In vitro Neurophysiology
Wendy Oshiro – Inhalation exposures, behavioral testing
Tracey Samsam – Inhalation exposures, behavioral testing
STOP HERE
Stages of Anesthesia
Stage 1: Induction
The period between the initial administration of the agent and loss of
consciousness. Progression from analgesia without amnesia to analgesia
with amnesia.
Stage 2: Excitement
The period following loss of consciousness and marked by excited and
delirious activity.
Stage 3: Surgical Anesthesia
Relaxation of skeletal muscles, slowed respiration and eye movements. Loss
of pain sensation.
Stage 4: Overdose
Severe depression of medullary activity, cessation of respiration, potential
cardiovascular collapse. Lethal without cardiovascular and respiratory
support.
Signal Detection Task
Trial Type
Signal Period
Choice
"Signal"
0.3” - 24”
2- 4”
Outcome
Hit
Signal
"Blank"
Miss
Extend Levers
"Signal"
False
Alarm
"Blank"
Correct
Rejection
Blank
Sensitivity of Ion Channel Function to Selected Solvents and Pharmacological Agents
Compound
Ion Channel
Solvents
NMDA Receptor
nAChR
GABA Receptor
Glycine Receptor
VSCC*
Toluene
Trichloroethylene
Perchloroethylene
1,1,1-Trichloroethane
Ethanol
Halothane
Inhibits
ND
ND
Inhibits
Inhibits
Inhibits
Inhibits
ND
Inhibits
ND
Inhibits/Potentiates
Inhibits
Potentiates
Potentiates
ND
Potentiates
Potentiates
Potentiates
Potentiates
Potentiates
ND
Potentiates
Potentiates
Potentiates
Inhibits/Potentiates
Inhibits/Potentiates
Inhibits/Potentiates
Inhibits
Inhibits
Inhibits
nAChR
GABA Receptor
Glycine Receptor
VSCC*
Antagonist
Antagonist
-Antagonist
Agonist
Antagonist
---Antagonist
-----Antagonist
Antagonist
Agonist
-Antagonist
----Agonist
-Antagonist
-Agonist
Antagonist
-----------
Pharmacological
Agents
NMDA Receptor
MK-801/Dizocipline
Memantine
NMDA
Mecamylamine
Nicotine
Bicuculline
Picrotoxin
GABA
Glycine
Strychnine
Antagonist
Antagonist
Agonist
-Potentiates
---Agonist
--
-- No Effect
ND - Not Determined
*VSCC - Voltage Sensitive Calcium Channel
Bushnell et al., 2005