Philip J. Bushnell Neurotoxicology Division National Health and Environmental Effects Research...

Philip J. Bushnell Neurotoxicology Division

National Health and Environmental Effects Research LaboratoryOffice of Research and Development, US EPA

Research Triangle Park, NC

McKim Conference on Predictive ToxicologySeptember 18, 2008

Toxicity Pathways Mediated by Ion Channels

• Model the acute toxicity of organic solvents for purposes of extrapolation from experimental observations to exposures relevant to public health

• Explore potential toxicity pathways for the acute effects of these chemicals

• Use the large existing database of acute effects of anesthetic agents as source of mechanistic information

Goals and Approach

Effects of Four Solvents on Behavior

Benignus et al., in preparation

0 500 1000 1500

0.0

0.1

0.2

0.3

0.4

0.5

0.6

TOLTCAPERCTCE

RAT, VEPHUMANRAT, REWRAT ES/AV

ESTIMATED BRAIN SOLVENT (M)

BE

HA

VIO

RA

L E

FF

EC

T

0 500 1000 1500

0.0

0.1

0.2

0.3

0.4

0.5

0.6

TOLTCAPERCTCE

RAT, VEPHUMANRAT, REWRAT ES/AV

ESTIMATED BRAIN SOLVENT (M)

BE

HA

VIO

RA

L E

FF

EC

T

• Ion channels are a prominent target of “nonpolar narcotics”• Organic solvents• Anesthetic agents• Inert gases

• Ion channels are pervasive in the nervous system and other excitable tissues

• Ion channels carry out essential homeostatic and signaling functions in the nervous system

Why Ion Channels?

• Gated “pores” in membranes• Control electrical potentials across membranes• Allow ions (typically Na+, K+, Ca++, Cl-) to flow between intra- and

extra-cellular fluids• Activated by voltage changes and/or ligand binding

• Functions of ion channels in nerves• Modulate electrical excitibility via membrane potential• Propagate signals along axons and dendrites• Release neurotransmitters at synapses

• Neuron – neuron• Neuron – effector (e.g., muscle)

What are Ion Channels?

Voltage-gated: Sensitive to changes in electrical potential across membrane

Types of Ion Channels

Feldman et al., 1997

• Ligand-gated channels • Sensitive to neurotransmitters, drugs, and solvents• Endogenous ligands – neurotransmitters• Excitatory (depolarizing) transmitters

• Glutamate• NMDA (N-methyl-D-aspartate)• Kainate• AMPA (α-amino-3-methyl-4-isoxazole propionic acid)

• nACh (Nicotinic acetylcholine)• 5-HT3 (5-Hydroxytryptamine type 3, or Serotonin)

• Inhibitory (hyperpolarizing) transmitters• GABA-A (γ-Amino butyric acid type A)• Glycine

Selected Types of Ion Channels

Representative Ligand-Gated Ion Channels

Campagna et al., 2003

Effects of Anesthetics on Selected Ion Channels

Modified from Dilger, 2002

NMDA nAchR a4b2 nAChR a7 GABA-A Glycine V-G Na V-G CaExcite Excite Excite Inhibit Inhibit Propagate Release NT

Toluene ↓↓ ↓↓ ↓↓ ↑↑↑ ↓↓Trichloroethylene ↑↑ ↓Perchloroethylene ↓↓↓ ↓↓↓ ↓↓↓1,1,1-Trichloroethane ↑↑

Isoflurane ↓ ↓↓↓ 0 ↑↑ ↑↑ ↓ ↓Halothane ↓ ↓↓↓ 0 ↑↑ ↑↑ ↓ ↓Ether ↑ ↓ ↓Cyclopropane ↓↓ 0 0 ↓ ↓Butane 0 0 ↓ ↓Urethane ↓↓ ↑↑ ↑↑ ↑↑ ↓ ↓Short Alcs ≤5 ↓↓ ↑↑ ↓↓ ↑↑ ↑↑Long Alcs >5 ↓↓ ↑↑Nitrous Oxide ↓↓↓ ↓↓ ↑ ↑Xenon ↓↓↓ ↓ ↑ ↑ Facilitation ↑↑↑Barbiturates ↓↓ ↓↓ ↓↓ ↑↑ ↑ ↑↑Ketamine ↓↓ ↓↓ ↓↓ ↑↑ 0 ↑Propofol 0 ↓ 0 ↑↑ ↑↑ No effect 0Etomidate 0 0 ↑↑ 0 ↓Steroids 0 ↓↓ ↑↑ ↓↓Non-immobilizers 0 ↓↓ 0 Suppression ↓↓↓

Glutamate and GABA Pathways in the CNS

Feldman et al., 1997

Glutamate (NMDA etc) Pathways GABA Pathways

Cortex

Thalamus

Hippo-campus

Brain Stem

Spinal Cord

Light SedationAmnesia Anxiolysis

Moderate Sedation

Slowed responses

Slurred speech

UnconsciousnessLoss of awareness

No response to verbal commands

Immobility

Loss of response to pain

Characteristics of Anesthesia

Rudolf & Antkowiak, 2004


Moderate Sedation

Slowed responses

Slurred speech

UnconsciousnessLoss of awareness

No response to verbal commands

Immobility

Loss of response to pain

Dose-Effect Relationships

Campagna et al., 2003

“Narcosis” Pathways for Volatile Anesthetics

nACh

Primary Brain

Region

Behavioral Effects

Hippocampus

Light SedationAmnesia

Anxiolysis

Ion Channel Receptor

Agent

CellularResponse

GABAA

Glycine

NMDA

Decreased channel-open time

Reduced membrane current

Increased channel-open time

Increased duration of mIPSCs

TissueResponse

Reduced excitatory transmission


Facilitated inhibitory transmission


Cortex - Thalamus

Brain Stem

Spinal Cord

Unconsciousness Loss of perceptual

awareness

Heavy Sedation Slow responses

Immobility Loss of pain

response

Increasin

g Depth

of An

esthesia

Kinetics Dynamics

nACh

Immobility Pathway for IsofluranePrimary

Brain Region

Behavioral Effects

Hippocampus

Light SedationAmnesia

Anxiolysis


Agent

CellularResponse

GABAA

Glycine

NMDA





TissueResponse





Cortex - Thalamus

Brain Stem

Spinal Cord


awareness



response

Increasin

g Depth

of An

esthesia

Kinetics Dynamics

nACh

Amnesia Pathway for Isoflurane Primary

Brain Region

Behavioral Effects

Hippocampus



Agent

CellularResponse

GABAA

Glycine

NMDA





TissueResponse





Cortex - Thalamus

Brain Stem

Spinal Cord


awareness



response

Kinetics Dynamics

Increasin

g Depth

of An

esthesia

Can these qualitative pathways be quantified?

1. Do dose-effect relationships in vitro support the pathway scheme for anesthetic vapors?• Depth of anesthesia depends on dose, but• Receptor sensitivity does not predict the anesthetic

depth:

IsofluraneEC10 EC50 (uM)

GABA 120 300nACh 80 870NMDA 300 2400Glycine 75 270

Light sedationHeavy sedationUnconsiousnessImmobility

• Pharmacological analysis of drug interactions• Injected anesthetics (e.g., benzodiazepines) acting at

specific receptors (e.g. GABA) interact synergistically with agents acting at other receptors.• Implication: Different modes / sites of action

• Inhaled anesthetics interact dose-additively • Implication: inhalants act at a single site or by a

single mode of action

2. Can specific sites or modes of action be identified for each pathway?


Interactions between

Anesthetics

Hendrickx et al., 2008

• Review of anesthesia literature• Two well-defined endpoints

• Hypnosis (Unconsciousness)• Immobility

• Drugs grouped by receptor• Inhalants listed separately• Synergy?

• Yes between iv drug pairs• Yes between iv drugs and inhalants• No between inhalant pairs

• Implication: Inhalants act at a common site / common MOA

Interactions Among Inhaled

Anesthetics

Hendrickx et al., 2008

• Experimental studies• Immobility to shock• Additivity most frequent • No case of synergy• One case of infra-additivity• Implication: Inhalants act at a common site / common MOA

3. However, analysis of dose-effect functions does not support a single MOA for volatile anesthetics• Dose-effect curves for single agents have a very steep

‘slope’ (Hill coefficient γ = 6 – 20) in vivo• Effects of individual agents in vitro have shallow slopes

(γ ~ 1)• In linear circuits with multiple receptors, γ 1.5 at limit

• Thus a single site model with simple linear circuits cannot account for dose-effect relationships


4. Comparative Molecular Field Analysis (Sewell et al., in press)


Correlate IC50 at NMDA receptor and MAC-I for 16 anesthetics, find poor relationship:

Models predict observed IC50 and MAC-I data very well:


Find partial overlap between the high-potency isocontour maps for IC50 and MAC-I derived by CoMFA:

Implications: • Potency at NMDA receptor contributes to anesthetic potency, but does not account for it• Could similar models for other receptors reveal contributions of each receptor to each effect?

IC50

MAC-I

Electrostatic Molecular bulk


The inevitable, likely possibility:• Anesthesia involves amplification of effects on specific channels via dynamic interactions among inter-related CNS pathways

• Understanding those interactions is necessary for a complete mechanistic understanding of anesthesia• Understading those interactions may not be necessary to make quantitative predictions of the potency and efficacy of untested compounds

• For example• Could CoMFA be used to estimate the contributions of the major ion channels to various endpoints?

Conclusions

• Volatile solvents and anesthetics reversibly affect the nervous system

• Effects are • Graded in severity and quality (anesthetic ‘depth’)• Mediated in large part by interactions with ion channels

• Qualitative pathways can be drawn that are consistent with known modes of action in relevant CNS structures• Quantifying these pathways will require a great deal more knowledge about dynamic interactions among CNS structures and interconnections• Structure-activity relationships can be developed at both the receptor and functional levels (e.g., CoMFA)

ReferencesEger, E. I., 2nd, D. M. Fisher, et al. (2001). "Relevant concentrations of inhaled anesthetics for in

vitro studies of anesthetic mechanisms." Anesthesiology 94(5): 915-21. Eger, E. I., 2nd, M. Tang, et al. (2008). "Inhaled anesthetics do not combine to produce

synergistic effects regarding minimum alveolar anesthetic concentration in rats." Anesth Analg 107(2): 479-85.

Grasshoff, C., B. Drexler, et al. (2006). "Anaesthetic drugs: linking molecular actions to clinical effects." Curr Pharm Des 12(28): 3665-79.

Hendrickx, J. F., E. I. Eger, 2nd, et al. (2008). "Is synergy the rule? A review of anesthetic interactions producing hypnosis and immobility." Anesth Analg 107(2): 494-506.

Sewell, J. C. and J. W. Sear (2004). "Derivation of preliminary three-dimensional pharmacophores for nonhalogenated volatile anesthetics." Anesth Analg 99(3): 744-51, table of contents.

Sewell, J. C. and J. W. Sear (2004). "Derivation of preliminary three-dimensional pharmacophoric maps for chemically diverse intravenous general anaesthetics." Br J Anaesth 92(1): 45-53.

Sewell, J. C. and J. W. Sear (2006). "Determinants of volatile general anesthetic potency: a preliminary three-dimensional pharmacophore for halogenated anesthetics." Anesth Analg 102(3): 764-71.

Sewell, J.C., Raines, D.E., Eger II, E.I., Laster, M.J., Sear, J.W. "Comparison of the molecular bases for NMDA-receptor inhibition versus immobilizing activities of volatile aromatic anesthetics." Anesth. Analg., in press .

Shafer, S. L., J. F. Hendrickx, et al. (2008). "Additivity versus synergy: a theoretical analysis of implications for anesthetic mechanisms." Anesth Analg 107(2): 507-24.

Sonner, J. M., J. F. Antognini, et al. (2003). "Inhaled anesthetics and immobility: mechanisms, mysteries, and minimum alveolar anesthetic concentration." Anesth Analg 97(3): 718-40.

Philip J. Bushnell Neurotoxicology Division National Health and Environmental Effects Research...

Documents

Transcript of Philip J. Bushnell Neurotoxicology Division National Health and Environmental Effects Research...