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Toxicity Pathways to Assessment Endpoints. P. Schmieder, S. Bradbury, G. Veith, J. McKim. Toxicity Pathway . WHAT:.

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slide1

Toxicity Pathways to Assessment Endpoints

P. Schmieder, S. Bradbury, G. Veith, J. McKim

toxicity pathway
Toxicity Pathway

WHAT:

  • A concept; a way of depicting a chain of events starting with a molecular initiating event (site of chemical –biological interaction) and ending with an adverse effect manifested in an individual, or higher level – population, community, ecosystem
  • May include a biochemical/signaling pathway, but goes beyond, to at least hypothesize how something observed at one level of biological organization is linked to response manifested at another level.
  • Chemical similarity is defined in the context of biological similarity
    • “Similar” chemicals, by definition, invoke the same toxicity pathway (within a specified biological model)
    • QSARs are developed for “similar” chemicals from a known or hypothesized “mode/mechanism” of action; hypothesis is tested to refine the models
  • QSAR requires a well-defined biological system

WHY:

slide3

Effects of toxicants occur at different levels of biological organization. Toxic effects are best known and understood at the cell and organ level, while the ecosystem and community level are least understood although most relevant. (Haux and Forlin, 1988)

Organ

Population

Individual

Ecosystem

Community

Cell

Chronic toxicity

Reproduction Growth

Productivity

Energy Flow

Contaminant dynamics in

microcosms

Acute toxicity

Lethal

Sublethal

Respiration

Osmoregulation

Structural

changes

Induction

TOXIC

CHEMICAL

Understanding

Relevance

toxicity pathway uses
Toxicity Pathway Uses
  • Assess knowledge gaps - what we know and what we don’t know about a chemical’s toxicity (toxicodynamics)
  • Assess the plausibility that a series of events are linked, i.e., degree of connectedness;
    • degree of specificity/certainty needed depends upon intended use
      • prioritization for further testing – correlation; “good” hypothesis?
      • quantitative RA - confirm cause and effect?
  • Pinpoint molecular initiating event for chemical extrapolation
    • QSAR – can be based on in vivo endpt if system is simple enough, e.g., fish acute/chronic for narcotic chemicals where applied chem conc is directly related to chemical activity in blood and further to the whole organism effect
    • Measurements closer to molecular initiating event will be more definitive for QSAR but some degree of relevance should be established (Linkage across levels of biological organization)
  • Basis for species extrapolation
  • Shifting RA paradigm - predict most likely tox pathways for a chemical to pinpoint most appropriate testing
well defined biological system know what you know and what you don t know
Well-Defined Biological System(Know what you know and what you don’t know)
  • Metabolism
    • Is the system used for collection of empirical data capable of xenobiotic metabolism?
    • Is what you’re measuring due to parent chemical or a metabolite?
  • Kinetics
    • What do you understand about the chemical kinetics within the system?
    • Is the chemical in solution
      • Bound and unavailable
      • Loss to hydrolysis

Measure chemical form and concentration in your system

slide7

Fathead Minnow Acute Toxicity Database

0

Narcosis I

-2

Narcosis III

-4

Narcosis II

Log Fathead Molar Toxicity (1LC50)

Uncoupler

-6

-8

-10

-2

0

2

4

6

8

Log P

slide8

Sorting Modes of Action

(Toxicity Pathways)

Fish Acute Toxicity Syndromes

- respiratory/cardiovascular responses (RBT)

Behavioral observations (FHM)

Mixture studies (FHM)

slide10

Assigning Chem Toxicol. Similarity for QSAR

In vivo Assays

Delineating Toxicity Pathways Across Levels of Biological Organization:

Acute Nonpolar Narcosis

Xenobiotic

MOLECULAR

TARGETS/RESPONSES

TISSUE/ORGAN SYSTEM PHYSIOLOGY

INDIVIDUAL

-Decreased Respiration

-Decreased Circulation

-Faulty Osmoregulation

Membrane

Partitioning

Ion Gradient Interruption

Failed ATP Production

Lethality

Toxicological

Understanding

Risk Assessment

Relevance

slide11

Uncoupling Toxicants

Water Solubility

LC50-96hr

MATC-30 day

LC50-96hr

MATC-30 day

slide12

Assigning Chem Toxicol. Similarity for QSAR

In vivo Assays

Delineating Toxicity Pathways Across Levels of Biological Organization:

Acute Uncoupling of Oxidative Phosphorylation

Xenobiotic

TISSUE/ORGAN SYSTEM PHYSIOLOGY

MOLECULAR

TARGETS

INDIVIDUAL

-Increased Respiration

-Increased O2 Consumption

-Decreased O2 Utilization

Lethality

Chemical Partitioning

Membrane

Proteins/

Ion Channels

Toxicological

Understanding

Risk Assessment

Relevance

slide14

Sorting Modes of Action

(Toxicity Pathways)

Fish Acute Toxicity Syndromes

- respiratory/cardiovascular responses (RBT)

Behavioral observations (FHM)

Mixture studies (FHM)

Biochemical responses – in vitro

slide15

Effects of toxicants occur at different levels of biological organization. Toxic effects are best known and understood at the cell and organ level, while the ecosystem and community level are least understood although most relevant. (Haux and Forlin, 1988)

Organ

Population

Individual

Ecosystem

Community

Cell

Chronic toxicity

Reproduction Growth

Productivity

Energy Flow

Contaminant dynamics in

microcosms

Acute toxicity

Lethal

Sublethal

Respiration

Osmoregulation

Structural

changes

Induction

TOXIC

CHEMICAL

Understanding

Relevance

slide16

Defining Toxicity Pathways Across Levels of Biological Organization:

Redox cycling_Arylation

Assigning Chem Toxicol. Similarity for QSAR

In vivo Assays

In vitro Assays

Xenobiotic

CELLULAR

GSH Oxidation

PrSH Oxidation

ROS Production

Decr. Energy Chg

Disrupt Cytoskel. (MT;IF);

Blebbing

Altered Cell Signaling

Cell Death

TISSUE/ORGAN

INDIVIDUAL

Liver Toxicity

Multiple Organ System Toxicities/Disease

MOLECULAR

Lethality

Impaired Growth

Binding to cytoskeletal components

-Redox cycling

- SH Arylation

Toxicological

Understanding

Risk Assessment

Relevance

slide18

Knoxville Workshop Framework for

Predicting Reactive Toxicity

Speciation

and

Metabolism

Molecular

Initiating

Events

Measurable

System

Effects

Adverse

Outcomes

Parent

Chemical

Rather than developing statistical models of complex endpoints, molecular initiating events are identified as well-defined QSAR endpoints…..and used to estimate the probabilities for important downstream biological effects based on transparent assumptions

slide19

Steps to the Development of QSAR for Reactive Toxicants

Speciation

and

Metabolism

Molecular

Initiating

Events

Measurable

System

Effects

Adverse

Outcomes

Parent

Chemical

Systems

Biology

QSAR

1. Establish Plausible Molecular Initiating Events

2. Design Database for Abiotic Binding Affinity/Rates

3. Explore Correlations/Pathways to Downstream Effects

4. Explore QSARs to Predict Initiating Event from Structure

slide20

Delineation of Toxicity Pathways

Linkages Across Levels of Biological Organization

In Silico Methods

In vitro Methods

In vivo Methods

Electronic

Molecular

Cellular

Organ

Individual

Chemical Reactivity

Profiles

Receptor binding

DNA alteration

Proteins adducts

Membrane effects

Gene Activation

Protein Syn/deg

Cell Signaling

GSH balance

Respiration Osmoregulation

Liver Function

Gonad Devel

Lethality

Growth

Development

Reproduction

slide21

Understanding “Specific” Toxicities

Endocrine Disruptors:

-Receptor-Mediated Toxicity Pathways

ER, AR, TR?

-Enzyme Inhibition (aromatase)

-Steroidogenesis (altered steroid metab)

slide22

QSAR

In vivo Assays

In vitro Assays

Delineating Toxicity Pathways Across Levels of Biological Organization:

Direct Chemical Binding to ER

Xenobiotic

INDIVIDUAL

POPULATION

TISSUE/ORGAN

Skewed

Sex Ratios,

Altered

Repro.

Chg 2ndry

Sex Char,

Altered

Repro.

CELLULAR

Altered Hormone Levels,

Ova-testis

MOLECULAR

Altered

Protein

Expression

ER Binding

Toxicological

Understanding

Risk Assessment

Relevance

slide23

Xenopus Metamorphosis Model for Thyroid System Disruption

Molecular

Tissue

Individual

Cellular

Gene/Protein

Expression

Thyroid Histology

Altered Morphology

Circulating TH Status

Thyroid Gland

Thyroid Hormone Synthesis

Peripheral Tissues

Deiodination

Morphology

Pituitary Gland

TSH Release

Hypothalamus

TRH (CRH) Release

slide24

Conceptual Overview of Project

Increasing Diagnostic (Screening) Utility

Increasing Ecological Relevance

Levels of

Biological

Organization

  • Molecular
  • Gene expression
  • Protein levels
  • Receptor binding
  • Enzyme activities

Cellular

Alterations in

production of

signalling molecules

  • Organ
  • Functional changes
  • Structural changes
  • (Pathology)

Individual

Altered reproduction

or development

Population

Decreased numbers

of animals

Small teleost model, well characterized genome, low ecological / regulatory relevance

Phase 2.

Zebrafish genomics proteomics

Computational modeling

HPG Systems modeling

Population modeling

Small teleost model, poorly characterized genome, high ecological / regulatory relevance

Phase 3.

Fathead minnow molecular markers metabonomics

Phase 1.

Fathead minnow 21 d reproduction test

→’s Depict the flow of information

chemical risk assessments
Chemical Risk Assessments

Linkages Across Levels of Biological Organization

Receptor-Mediated Pathways

Organ

Chemical 2-D Structure/

Properties

Individual

Molecular

Cellular

Gonad Development

(Ova-Testis)

Altered Hormone Levels

Impaired Kidney Function

Gene Activation

Protein Production

Receptor/

Ligand Interaction

Impaired

Reproduction

Chemical 3-D Structure/

Properties

Metabolism

Understanding

Relevance

slide26

In vivo

Toxicological

Understanding

Risk Assessment

Relevance

Toxicokinetics

Toxicodynamics

Molecular/

Sub-Cellular

Xenobiotic

Chemical

Cell

Organ/Tissue

Individual

Changes in Gene/Protein

Expression

Leading to Altered Cell Function

Chemical- Receptor Binding

Initiating

Altered

Gene/Protein

Expression

Impaired

Reproduction

Altered Organ Growth and Function

slide27

Chemical Kinetics

In vivo

Molecular/

Sub-Cellular

Xenobiotic

Chemical

Cell

Organ/Tissue

Individual

Gene/Protein

Cell Function

Receptor Binding

Gene/Protein

Expression

Reproduction

Growth and Function

Toxicological

Understanding

Risk Assessment

Relevance

slide28

Chemical Kinetics

In vivo

Xenobiotic

Chemical

Uptake

Molecular/

Sub-Cellular

Cell

Organ/Tissue

Individual

Trout

Toxicological

Understanding

Risk Assessment

Relevance

slide29

Chemical Kinetics

In vivo

Xenobiotic

Chemical

Uptake

Distribution/Metabolism

Molecular/

Sub-Cellular

Cell

Organ/Tissue

Individual

Trout

Toxicological

Understanding

Risk Assessment

Relevance

slide30

Chemical Kinetics

In vivo

Xenobiotic

Chemical

Uptake

Distribution/Metabolism/Excretion

Molecular/

Sub-Cellular

Cell

Organ/Tissue

Individual

Trout

Toxicological

Understanding

Risk Assessment

Relevance

slide31

In vivo

Metabolism studies across levels of biological organization

Linkages must be established

Xenobiotic

Chemical

In vitro

Uptake

Distribution/Metabolism/Excretion

Molecular/

Sub-Cellular

Cell

Organ/Tissue

Individual

Isolated

Hepatocytes

Celllines

Microsomes

S9

Purified enzymes

Trout

Isolated Perfused Liver

Tissue Slices

Toxicological

Understanding

Risk Assessment

Relevance

slide32

(E2)

(E2-gluc)

(E2)

(gluc)

slide33

Chemical Kinetics

In vivo

Xenobiotic

Chemical

Uptake

Distribution/Metabolism/Excretion

Molecular/

Sub-Cellular

Cell

Organ/Tissue

Individual

Gene/Protein

Expression

Cell Function

Receptor Binding

Gene/Protein

Expression

Reproduction

Growth and Function

Toxicological

Understanding

Risk Assessment

Relevance

slide34

Project Goal: Enhance Metabolic Simulator for EPA Regulatory Lists

Predicted inactive

parent;

“activated”

metabolites

Existing ER

Binding Model

OPP Chemicals

Existing ER

Binding Model

Expert Judgement

Existing Metabolism

Simulator

improve

ER model

enhance

simulator

Prioritized Chemicals

Verified ER

activation

Verified maps

Predicted Metabolites

Rat

liver microsomes,S9

Trout

liver slice

Analytical methods

MED; NERL-Athens; LMC

slide35

Toxicity Pathways

A useful concept for organizing toxicity data across levels of biological organization

-Linking toxicological understanding to risk assessment relevance

A conceptual framework for:

- chemical extrapolation

- molecular initiating events are the key to linking chemical reactivity continuum to biological response continuum

- species extrapolation

A useful concept in Predictive Toxicology

- Predict most likely tox pathway for a chemical to pinpoint most appropriate testing

slide36

Mapping Toxicity Pathways to Adverse Outcomes

Structure

Individual

Cellular

Molecular

Organ

Chemical 2-D Structure

Altered

Reproduction/

Development

ER

Transctivation

VTG mRNA

Vitellogenin Induction

Sex Steroids

ER Binding

Initiating Events

Impaired Reproduction/Development

Chemical 3-D

Structure/

Properties

Libraries of Toxicological Pathways

slide37

Mapping Toxicity Pathways to Adverse Outcomes

Adverse Outcomes

Initiating Events

Libraries of Toxicological Pathways