How can Toxicogenomics inform Risk Assessment? · Toxicokinetics external dose/ internal dose/...

How can Toxicogenomics inform Risk

Assessment?

Ursula Gundert-Remy

Background image from Nature Vol.424, August 2003, p.610

Hazard identificationRegulatory requirements1 */ testing endpointsHazard identificationRegulatory requirements1 */ testing endpoints

* human health- relatedchemicals/ biocides

1SIDS (Screening Information Data Set)

endpoint-basedhazard identificationendpoint-basedhazard identification

Repeated DoseToxicity

oraldermalinhalation

Acute Toxicityoraldermalinhalation

ReprotoxicityFertilityDevelopmental

Toxicity

Genotoxicity2 endpoints, e.g.:

point mutationchromosomal

aberration

requires toperform multipleanimal studies

Toxicokinetics

external dose/ internal dose/ tissue dose/ cellular/exposure exposure exposure subcellular

dose

Toxicokinetics & Toxicodynamics- from exposure to effect -

Toxicokinetics & Toxicodynamics- from exposure to effect -

irreversible late early cellular/pathology response response subcellular

interaction

Toxicodynamics

Combines Combines geneticsgenetics, genomic, genomic--scale mRNA scale mRNA expression (expression (transcriptomicstranscriptomics), cell and tissue), cell and tissue--wide wide protein expression (protein expression (proteomicsproteomics), metabolite profiling ), metabolite profiling ((metabonomicsmetabonomics), and ), and bioinformaticsbioinformatics with with conventional conventional toxicologytoxicology in an effort to understand the role of genein an effort to understand the role of gene--environment interactions in effect/ disease. environment interactions in effect/ disease.

Toxicogenomics (TXG)

Toxicogenomics (TXG)

Study of the response of a genometo environmental stressors and toxicants.


III. Metabonomics

changedcell metabolism

exposurein-vivo/ in-vitro

concentration/dose at effect site

metabolism +distribution

ToxicogenomicsToxicogenomics

changed gene expressiontranscription/ RNA

I. Genomics/Transcriptomics

changed protein expressiontranslationPTM*

II. Proteomics

*PTM = Post-Translational Modifications

interactionsDNA/ proteins/metabolites etc. signal transduction

TXG: Basic Tools and StrategyTXG: Basic Tools and Strategy

detectiondetection of of biochemicalbiochemical endpointsendpoints in in tissuestissues, , cellcell culturescultures etc.etc.

transcriptional level : DNA microarrays (toxicogenomics)

translational level: protein expression (toxicoproteomics)

metabolite level: metabolite profiling (metabonomics)

toxicogenomicstoxicogenomics


Mostly unknown

Mostly known

DNA40,000 genes

RNA150,000 transcripts

Proteins1,000,000 proteins

Metabolites2,500 metabolites

Genomics

Transcriptomics

Proteomics

Metabolomics

variability in susceptibilityvariability in exposureinternal

Doseearly

biologicaleffect

exposurealtered

function/structure

disease/pathology

targetorgandose

biologicallyeffective

dose

Toxicokinetics (TK) Toxicodynamics (TD)

The Steps from Exposureto Effect and Disease

The Steps from Exposureto Effect and Disease

marks steps where toxicogenomic markersmay provide additional information

Copyright restrictions may apply.

Ellinger-Ziegelbauer, H. et al. Toxicol. Sci. 2004 77:19-34; doi:10.1093/toxsci/kfh016

Characteristic gene expression profiles induced by the genotoxic carcinogens 2-NF (2-nitrofluorene), DMN (dimethylnitrosamine), NNK (4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone), and AB1 (aflatoxin B1) in rat liver after 1, 3, 7, or 14 days of treatment

MOAMode of action information from gene profiling

Copyright restrictions may apply.

Ellinger-Ziegelbauer, H. et al. Toxicol. Sci. 2004 77:19-34; doi:10.1093/toxsci/kfh016

Genotoxicant-induced DNA damage response

3.

1.

4.

2.

5.

22--DD--GelGel--Electrophoresis Electrophoresis of of mousemouse liverliver extractextracttreatedtreated withwith TCCDTCCD

Ras

RTKs Integrins Ion channels

MEK 1/2

Erk 1/2

c-myc (mRNA)

Annexin A2

Ca2+

c-Myc (protein)

PUR-a

Caldesmon-1

Cdc2-kinase

Actin

Caldesmon-1

Coronin-1A

F-Actin capping protein

Macrophage capping protein

Actin

Cell cycle progession

M-phase progression

initiates transcription

stabilization

Crosslink of actin

Regucalcin

TCTP

microtubules

P

cell membrane

nucleus

DNA

Tropomyosin 1ainteract

A B

ATPADP

ATP

ADP

Cap (+) ends of actinActin

polymerization

Malignanttransformation

Rho-GDP

Rho-GTP

Rho-GDIGTP

GDP

Regulated Pathways in GS(-) tumors

glutathione (red)glutathione (ox)glycinealaninecysteine

NADPH NADP+

EC 2.5.1.18

2-oxoglutarate

fumarate

oxalacetate

EC 1.1.1.41

glutamate

histidine

EC 4.2.1.49

glutamine

NH4

EC 6.3.1.2

arginino-succinate

ornithine

citrullinearginine

urea

EC 3.5.3.1

EC 4.3.2.1

acetyl-CoAglucoseEC 3.1.3.11

ribose-5-P + erythrose-4-P

phenylalaninetyrosinetryptophanhistidine

phenylalanine tyrosineEC 1.14.16.1

acetoacetate

EC 1.13.11.5

NADP+

NADPH

EC 1.1.1.44

L-amino acids

EC 2.6.1.13

flavine (ox)

flavine (red)

EC 1.5.1.30

N-formimino-L-glutamate

EC 2.1.2.5

Regulated metabolic pathways in GS(+) tumors

Clofibratenetwork 1

Clofibratenetwork 2

Phenobarbital

Mechanistic information as an excellent predictor fordistinction between genotoxic, non-genotoxic and non-hepatocarcinogens

Mechanistic information as an excellent predictor fordistinction between genotoxic, non-genotoxic and non-hepatocarcinogens

Nephrotoxicity: sensitivity of genomicbiomarkers vs conventional markersNephrotoxicity: sensitivity of genomicbiomarkers vs conventional markers

Genomics

Transcriptomics

Proteomics

Metabolomics

DNA40,000 genes

RNA150,000 transcripts

Proteins1,000,000 proteins

Metabolites2,500 metabolites

Metabolomics

Closer to daily toxicology

Mostly unknown

Mostly knownUsing blood

Why Metabolomics ?

Three Profiling Data Sets from “One Analysis”

Metabolite Structure Identification

Known-Unknown Metabolites• Additional 400 – 900 compounds• Metabolite ID not final but indexed• Relative concentrations reliably measured

SAMPLEca. 100 ul plasma

Target Metabolites• 200 – 500 per sample matrix• Structure ID established• Sensitive detection at actual levels• Absolute quantitation possible

Total Metabolome Signature• Up to 9.000 analyte signals per sample• Directly used in Data Mining• Based on original 3D MS data sets

The Data Mining Process

PRINCIPLE COMPONENT ANALYSISMETABOLITE PROFILES

SINGLE VARIABLESTATISTICS

MULTIPLE VARIABLESTATISTICS

PLS-DA

Each SAMPLE is represented by a single „bubble“The positioning of each SAMPLE is determined by the whole METABOLITE PROFILESAMPLES which group together share similar metabolic characteristics or „patterns“

The Data Mining Process

METABOLITE PROFILES

SINGLE VARIABLESTATISTICS

MULTIPLE VARIABLESTATISTICS

INTERPRETATION OFMETABOLISM

METABOLIC PATHWAYS to challenge metabolic and statistical results in biological contextPathway diagrams are customised to match project objectives.EXAMPLE: PathwayExplorerTM a metanomics proprietary software tool.

PROJECT SPECIFICANALYSIS

The Basis is high Reproducibility & Reliability

Shown are plasma profiles from metanomics Toxicology & Pharmacology:

Subset of about 700 control male & female ratsControls of different experiments spanning about 5 monthsData represent biological, handling and profiling variability

Colors indicate different experiments over 5 months

Comp.1

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-3 -2 -1 0 1 2 3

-1.5

-1

-0.5

0

0.5

1

1.5

2

femalesmales

main differentiator in control animalsand experiments is gender only

Results Toxicology: dose response

dose 3

control

dg2dg3

dg1

Metabolite profiling can differentiate between dose groupsMapping of metabolite changes onto pathways usingmetanomics‘ proprietary software tools supportsmechanistic understanding

Ptu

PbFlu

control

Phenobarbital: liver enzyme inducer Propylthiouracil: thyroid hormone formation blockerFlutamide: anti-androgenic

☺ MoA Identification

☺ MoA Classification

☺ Dose-response relationship

Vinclozolin

controls

Flu

Results Toxicology: MoA Identification

Double Blind StudyNovember 2005

„identify correctly the MoA of 10 unknown compounds usingthe profile of 85 investigated compounds (representingtypical MoAs) based on met abolome profiling“

Goal:

5 compounds correct on spot 12 compounds correct on spot 21 compound correct on spot 3

all 10 compounds within top 6 Statistical significance: p = 10 –11

Result:

Comp.1

Ctrl

CtrlAroclor

Vinclozolin

Vinclozolin

Vinclozolin

VinclozolinVinclozolin

Vinclozolin

VinclozolinVinclozolinVinclozolin

Pentobarbital

Vinclozolin

ErythrosinErythrosinErythrosin

Erythrosin

ErythrosinPentobarbital

PentobarbitalPentobarbital

Pentobarbital

EthylenthioureaEthylenthiourea

Ethylenthiourea

Ctrl

Ethylenthiourea

Aroclor

AroclorAroclor

Ctrl

VinclozolinVinclozolin

Vinclozolin

Ctrl

Erythrosin

Ctrl

Ctrl

Ctrl

Ctrl

Aroclor

Aroclor

Ctrl

DMPP

mebiquat chloride techn.

Ctrl



mebiquat chloride techn.MCPA-acid

MCPA-acidMCPA-acid

MCPA-acid

MCPA-acid

PB

MCPA-acidMCPA-acidMCPA-acid

MCPA-acid

Flt

Anilin

Anilin

Anilin

Anilin

Flt

Bis-(hydroxylammonium)-sulfat


Dicloprop-p

Trilon A 92 R

Dicloprop-p

Trilon A 92 R

BAS 670 H

Dicloprop-p

Flt

Dicloprop-p

BAS 505 F

BAS 505 F

BAS 505 F

BAS 505 F

Ctrl

Dicloprop-p

BAS 505 F Anilin

MCPP-acid


MCPP-acid

Trilon A 92 R

Trilon A 92 R

Trilon A 92 R

BAS 670 H BAS 670 H

MCPP-acid

4-N-Nonylphenol

MCPP-acid

MCPP-acid

MCPP-acid

MCPP-acidcorn oil

4-N-Nonylphenol

DMPP

4-N-NonylphenolVinylglycoldiacetat

Flt

DMPP

Terbufos (BAS 316 I)

BAS 520 F

BAS 520 F

Dimethoate

Genistein

Genistein

Genistein

Genistein

Beta-Ionon RBeta-Ionon R

Beta-Ionon R

Beta-Ionon R

Beta-Ionon R

Trimethylphenol

Trimethylphenol

Mesotrione

Genistein

Beta-Ionon R

Beta-Ionon R

Beta-Ionon R

Trimethylphenol

PTU

TBSA

TBSA

PTU

Product 1999

diet restriction -20%

Dicloprop-p

Dicloprop-p

Dicloprop-p

Dicloprop-p Dicloprop-p

MCPP-acid

MCPP-acid

Ctrl

MCPP-acidMCPP-acid

MCPP-acid

MCPP-acid

MCPP-acid

PB

DMPP

DMPP

BAS 480 FBAS 480 F

Din-butylphtalate

Methoxychlor

DES

DESDES

DIPP

DIPP

DIPP

DIPPDIPP

BAS 520 F

BAS 520 F

DIPP

L-thyroxine

17alpha-Methyltestosterone




17alpha-Ethynylestradiol








Nitrofen

MGDA

Dicamba

Ephedrine Sulfate

Ephedrine Sulfate

Caffeine

Caffeine

Caffeine

Caffeine

Caffeine

Caffeine

Caffeine

Caffeine

Caffeine

BAS 625 H

BAS 510 F

BAS 625 H

BAS 625 H

Pentachlorobenzene

Pentachlorobenzene

Pentachlorobenzene

N-Methylpyrrolidon

N-Methylpyrrolidon

N-Methylpyrrolidon

N-Methylpyrrolidon

Lysmeral

Lysmeral

Lysmeral

Lysmeral

Lysmeral

Lysmeral

Linuron

Linuron

Linuron

Linuron

Linuron

Lysmeral

Lysmeral

Linuron

Linuron

LinuronLinuron

Linuron

Linuron

Linuron

Acetone

Acetone

Emulgator 2000

Acetone

Acrylamide

BAS 570 F

BAS 455 H (Pendimethalin)BAS 455 H (Pendimethalin)

Metazachlor

MetazachlorMetazachlor

BAS 455 H (Pendimethalin)

Metazachlor

Metazachlor

MetazachlorMetazachlor

Metazachlor

Metazachlor

Compound 3

-4 -2 0 2 4 6

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0

1

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4

C 1-3 -2 -1 0 1 2 3 4

-4

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-2

-1

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C 1

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metanomics & GV/TMoA Assignment for unknown compounds

HPPD inhibitors: metabolites connection

Metabolite flT1 flT2 flT3 fhT1 fhT2 fhT3 mlT1 mlT2 mlT3 mhT1 mhT2 mhT3 flT1 flT2Threonine 1.25 1.24 1.18 1.16 1.29 1.32 1.26 1.13 1.11 1.24 1.19 1.5 1.41 1.51Citrulline 0.77 0.91 0.83 0.86 0.92 0.9 0.76 0.69 0.78 0.77 0.7 0.83 0.82 0.81Tyrosine 10.9 9.39 10.4 11.3 10.8 9.24 20.21 12.23 10.69 17.09 11.08 12.13 19.14Glycine 1.31 1.33 1.27 1.42 1.27 1.29 1.11 1.06 1.08 1.14 1.13 1.27 1.4Threonine 1.23 1.34 1.13 1.17 1.38 1.3 1.37 1.18 1.02 1.24 1.16 1.38 1.33 1.445-Oxoproline 0.56 0.57 0.62 0.57 0.68 0.59 0.48 0.55 0.59 0.49 0.57 0.53 0.66 0.61Lysine 1.3 1.67 1.17 1.2 1.43 1.08 1.31 1.39 1.22 1.39 1.44 1.31 1.27 1.28Glutamine 0.56 0.55 0.54 0.5 0.54 0.52 0.42 0.42 0.4 0.42 0.43 0.4 0.52 0.58Serine 1.28 1.49 1.19 1.21 1.26 1.16 1.29 1.17 1.07 1.17 1.04 1.17 1.16 1.38

BAS 660HUrea cycle Ammonia formation

Lysine

Serine

Glycine

Threonine

Proline

Arginine

Ornithine Citrulline Aspartate

5-oxoprolineGlutamate

Glutamine GLUL

2-oxoglu-tarate

TyrosineGOT14-hydroxyphenylpyruvateHPPDHomogen-

tisate

Impact of ToxicogenomicsImpact of Toxicogenomics

characterize sequence of genes that could make subpopulationsor individuals more or less sensitive to chemical exposure(genetic polymorphisms)

Improved understanding of chemical exposureon human health and the environment

categorize chemicals according to modes or mechanisms of toxicity

relationship between alterations of gene expression and toxicity:identify genes that are mechanistically linked to toxicity

patterns of gene expression as a biomarker of exposure and effect(mode of toxicity)

more advanced approach: identify and understand mechanisms of toxicity

detect mode of action/ mechanism of action

improvement of species extrapolation

toxicological fingerprint (pattern recognition)

new biomarkers of effect

detect organ-specific effects

use in short-term bioassays (in vivo + in vitro)to obtain basic toxicity information of chemicals more quicklycompared to established toxicity testing(animal studies, long-term animal studies)

hazard identification

Application of Toxicogenomicsto Risk Assessment (1)


will enable development of enriched testing methods

provides enhanced understanding of mechanisms of actionin biological systems

TXG:

rapid detection of exposure levels

evaluate toxicity of mixtures

identify sensitive life stages and subpopulations

new specific and sensitive biomarkers of exposure

exposure assessment

obtain dose-response relations at low doses

miscellaneous

international organizations, in particular IPCS, have a role to address technicalchallenges, interpretation of results and their implementation into risk assessment



reduction and refinement of animal studies

Toxicogenomics in Risk Assessmentin conclusion- my personal view

Toxicogenomics in Risk Assessmentin conclusion- my personal view

opens multiple chances to developing enriched testing protocols

enables measurement of exposure and effect inhuman epidemiological studies

in future, with the development of improved techniques,it will be used like measurements of biochemical parameters

provides a wealth of additional mechanistic information

TXG:

at present, it is a cost-intensive tool with limited accessibility for the entire scientific community

How can Toxicogenomics inform Risk Assessment? · Toxicokinetics external dose/ internal dose/...

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