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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.
Background image from Nature Vol.424, August 2003, p.610
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
Background image from Nature Vol.424, August 2003, p.610
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.
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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
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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
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Aroclor
AroclorAroclor
Ctrl
VinclozolinVinclozolin
Vinclozolin
Ctrl
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Ctrl
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mebiquat chloride techn.
Ctrl
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mebiquat chloride techn.
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
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
Bis-(hydroxylammonium)-sulfat
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-Methyltestosterone
17alpha-Methyltestosterone
17alpha-Methyltestosterone
17alpha-Ethynylestradiol
17alpha-Methyltestosterone
17alpha-Methyltestosterone
17alpha-Methyltestosterone
17alpha-Methyltestosterone
17alpha-Methyltestosterone
17alpha-Methyltestosterone
17alpha-Methyltestosterone
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
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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)
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
Application of Toxicogenomicsto Risk Assessment (2)
Application of Toxicogenomicsto Risk Assessment (2)
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