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1 09 May 2012
2nd McKim Workshop, Baltimore 2012
Identification of non-genotoxic carcinogens based on mechanisms
Jan van Benthem
National Institute for Public Health and the Environment
Laboratory for Health Protection Research
Bilthoven, The Netherlands
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
2
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
- no further testingnot genotoxic
In vitro genotoxicity tests
In vivo genotoxicity tests
+consider whether in vivo test is requiredcheck bioavailabilitycheck available dataconsider proper in vivo (follow up) testconsider integration into other toxicity tests
Considered genotoxic in somatic cells
+
- no further testingnot genotoxic
Check for information on a genotoxic hazard to germ cells
Carcinogenicity test
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
3
Detection of non-genotoxic carcinogens
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
• Carcinogenicity test
• (Sub)-chronic repeat dose toxicity tests 90-day toxicity test
6-month toxicity test
12-month toxicity test
• Negative in genotoxicity tests
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
4
REACHREACH
Since 1981 difference is made between new and existing chemicals (marketed before September 1981).
About 99% of the total number of compounds marketed is part of this new policy
In 1981, 100106 (existing) compounds were registered (30000 >> 1 tpa) Since 1993, 141 existing compounds have been tested
Of about 80% of these compounds toxicological data are lacking
Registration, Evaluation, Authorisation and Restriction of CHemicals
Registratie, Evaluatie en Autorisatie van CHemische stoffen
Registrement, Evaluation et Autorisation des produits CHimiques
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
5
REACH
Major aims:To ensure a high level of protection of human health and the environment• Safety assessment for new and existing chemicals
• Classification and labelling• Risk Assessment
• In less time, money and experimental animals
• Promotion of tests which do not use experimental animals.• Optimal use of in vitro tests• Stimulation of development of new in vitro tests• Minimalization of test strategies
New:• In future it is the responsibility of the industry and down stream users
to obtain toxicological knowledge bout chemicals. Every compound is notified only once (industrial collaboration)
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
6
REACH data requirements at different tonnage levels
Acute toxicity
In vitro geno-toxicity
Local toxicity
28 day toxicity
90 day toxicity
Repro-ductive toxicity
Develop-mental toxicity
Carcino-genicity
2 genera-tion study
In vivo geno-toxicity
Acute toxicity
In vitro geno-toxicity
Local toxicity
28 day toxicity
90 day toxicity
Repro-ductive toxicity
Develop-mental toxicity
2 genera-tion study
In vivo geno-toxicity
Acute toxicity
In vitro geno-toxicity
Local toxicity
28 day toxicity
90 day toxicity
Repro-ductive toxicity
Acute toxicity
In vitro geno-toxicity
Local toxicity
No studies required< 1 tpa
1 - 10 tpa
10 - 100 tpa
> 1000 tpa
100 – 1000 tpa
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
7
September 11, 2004: Testing ban on finished cosmetic products in the EU. Marketing ban on cosmetic products and ingredients tested on animals outside of the EU where validated alternative tests exist.
March 11, 2009 Testing ban cosmetic ingredients or formulations in the EU. Marketing ban cosmetic products and ingredients tested on animals with the exception of repeated-dose toxicity, reproductive toxicity and toxicokinetics.
March 11, 2013 Marketing ban cosmetic products or ingredients tested on animals, irrespective of the availability of alternative non-animal tests.
The 7th Amendment requires cosmetics manufacturers and distributors to provide certain product information for the safety of the end user.
The Cosmetics Directive and its Seventh Amendment
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
8
Risk associated with non-genotoxic carcinogensRisk associated with non-genotoxic carcinogens
Mechanisms of non-genotoxic carcinogens and importance of a weight of evidence approach
Lya G. Hernandez, Harry van Steeg, Mirjam Luijten, Jan van Benthem
Mutation Research 682, 94–109 (2009)
lya.hernandez@rivm.nl
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
9
Number non-carcinogens in IARC 1, 2A and 2B
IARC group total carc non-carc percentage
1 77 64 13 16.9
2A 57 55 2 3.5
2B 237 207 30 12.7
total 371 326 45 13.8
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
10
Margin of Exposure
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
Margin of Exposure (MoE) = Margin of Safety (MoS)
The ratio: Most relevant exposure from animal studies
(Estimated) human exposure
Potential hazard:Carcinogens MoE < 10,000 Non-genotoxic carcinogens MoE < 100 for consumersNon-genotoxic carcinogens MoE < 50 for workersCosmetics in Europe: MoS < 100
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
11
Approaches
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
Rodent dose response carcinogenicity study data of the Gold database
using LTD10 calculated from TD50
(Sub)-chronic dose response toxicity studies from literature using NO(A)EL
RfD using EPA/IRIS data
Human exposureusing data from the open literature
vs
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
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Reference dose
A dose (with uncertainty factors) of a daily exposure to
human population that is likely to be without appreciable
risk of deleterious effects during a lifetime
Potential hazard:
Average human daily exposure > RfD
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
13
Risk Assessment
MoE from LTD10 38% (7/18) with MOE < 100
MoE from NOAEL 18% (2/11) with MOE < 50
RfD 30% (3/10) RfD > average human daily exposure
Non-genotoxic carcinogens with potential human hazard: 27% (12/45) among non-genotoxic carcinogens
3.2% (12/371) among carcinogens (1, 2A, 2B)
●
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
14
MoE cut off’s of EPA
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
With an acceptable life time cancer risk of 1 in 104
MoE > 100,000 low risk for carcinogenicity
10,000 < MoE < 100,000 moderate risk for carcinogenicity
MoE < 10,000 high risk for carcinogenicity
For the present data:
1/45 (2%) in the low risk group
2/45 (4%) in the moderate risk group
22/45 (49%) in the high risk group
No data were available for 20 remaining compounds
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
15
Comparison EU vs EPA
Non-genotoxic carcinogens with potential human hazard:
- Europe, MoE < 100
- 12/45 (27%)
- 12/371 (3.2% of all carcinogens)
- U.S.A., MoE < 100,000
- 24/45 (49%) (moderate to high risk)
- 24/371 (6.5% of all carcinogens)
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
16
Putative methods to detect non-genotoxic carcinogensPutative methods to detect non-genotoxic carcinogens
• Structure-activity relationships (SAR) and quantitative (Q)SARStructure-activity relationships (SAR) and quantitative (Q)SAR
• Read acrossRead across
• Replicative DNA synthesisReplicative DNA synthesis
• In vitro In vitro cell transformation assaycell transformation assay
• ToxicogenomicsToxicogenomics
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
17
Dissecting modes of action of non-genotoxic carcinogens in primary mouse hepatocytes
Mirjam M Schaap, Edwin P Zwart, Paul FK Wackers, Ilse Hijskens, Bob van de Water, Timo M. Breit, Harry van Steeg, Martijs J. Jonker, and Mirjam Luijten.
Arch. Toxicol. submitted
For more information: mirjam.schaap@rivm.nl
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
18
Primary hepatocytes combined with toxicogenomics
Goal
Identification mechanism of action of non-genotoxic carcinogens
Advantages- relevant cell type: liver major organ for toxicity- metabolic competent cells- requires only small number of animals
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
19
Experimental design
Primary mouse hepatocytes– Two-step perfusion (based on Seglen, 1976)– Hepatocytes cultured in sandwich configuration
Compounds tested– 16 non-genotoxic carcinogens– Dose ~ 90% cell viability (based on MTT)
Microarray analysis– Cells exposed for 24 hours– Affymetrix HT MG-430 PM Array plate
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
20
Mode of Action (MoA) Pairs of substances tested (NGTXC)
Immunosuppressant and Calcineurin inhibitor
Cyclosporin A (CsA) Tacrolimus (FK506)
Organochlorine pesticide β-hexachlorocyclohexane (HCH)
Heptachlor epoxide (HCE)
Peroxisome proliferator Wyeth-14.643 (WY) Clofibrate (CF)
AHR ligand TCDD Aroclor 1254 (ARO)
CAR ligand TCPOBOP Phenobarbital (Pb)
Halogenated hydrocarbon Carbon Tetrachloride (CT) 1,1,1-trichloroethane (TCE)
Skin tumor promotor Okadaic acid (OA) Calyculin A (CA)
Metalloid Sodium Arsenite (SAR) Lead Acetate (LAc)
Non-genotoxic carcinogens
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
21
Data analyses
General– Principal component analysis– Pathway analysis per individual substance
Supervised clustering
Selection discriminative genes specific for mechanism?
Unsupervised clustering
Clustering of substances based on gene expression signature
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
22
Gene expression analysis - general
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
Differentially expressed genes:from 40 to >5,600 (FDR<0.05)
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
23
Results of the gene expression analysis
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
NGTXC mode of action DEG response PCA
TCE halogenated hydrocarbon 40 3 A
CT halogenated hydrocarbon 56 3 A TCPOBOP constitutive androstane receptor agonist 65 3 A
PB constitutive androstane receptor agonist 238 3 A
TCDD arylhydrocarbon receptor agonist 567 1 A
ARO arylhydrocarbon receptor agonist 762 1 B
CA skin tumor promotor 281 3 A
OA skin tumor promotor 282 3 A
WY peroxisome proliferator 1611 1 B
CF peroxisome proliferator 308 1 A
CSA immunosuppressant 124 1 A
FK506 immunosuppressant 2404 2 B
HCE ligand-independent estrogen receptor 2088 2 B
HCH ligand-independent estrogen receptor 115 3 A
LAC metalloid 5693 2 C
SAR metalloid 4130 2 C
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
24
Data analyses
General– Principal component analysis– Pathway analysis per individual substance
Supervised clustering
Selection discriminative genes specific for mechanism?
Unsupervised clustering
Clustering of substances based on gene expression signature
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
25
Supervised clustering
Lac
Sar
CT
TC
E
OA
CA
FK
506
CsA P
b
TC
PO
BO
P
HC
H
HC
E
TC
DD
Aro
clo
r
Clo
fib
rate
WY
Method to select genes discriminative for MoA
-2 -1 0 1 2
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
26
• Apply supervised clustering on random pairs to confirm specificity of previous results
• Level of distinction is expressed as C-value; calculated for each individual gene
• Maximum C-value obtained for random pairs ~ 40
Supervised clustering - specificity
Maximal C-value
Distribution random pairs (5000 permutations)
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
27
C-value p-value
LAC & SAR 181.0 0.0002
CT & TCE 7.1 1.0
CA & OA 15.1 0.314
CSA & FK506 14.2 0.432
PB & TCPOBOP 12.2 0.614
HCE & HCH 11.9 0.635
ARO & TCDD 73.0 0.0002
CF & WY 587.0 0.0002 Maximal C-value
Distribution random pairs (5000 permutations)
Maximal C-values ‘correct’ pairs
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
28
C
om
po
un
d 1
Co
mp
ou
nd
2
Co
mp
ou
nd
3
Co
mp
ou
nd
4
Co
mp
ou
nd
5
Co
mp
ou
nd
6
Etc.
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Etc.
Unsupervised clustering
Which substances have a similar gene expression profile and cluster together?
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
29
Unsupervised clustering – preliminary results
CsA CT
CT CA
Pb HCH
Lac Sar
WY Clofibrate
CA OA
TCPOBOP HCH
Sar Lac
Aroclor HCE
TCDD Aroclor
TCE TCPOBOP
HCE Aroclor
HCH Pb
OA CA
Clofibrate WY
FK506 Lac
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
30
Data analyses
General– Principal component analysis– Pathway analysis per individual substance
Supervised clustering
Selection discriminative genes specific for mechanism?
Unsupervised clustering
Clustering of substances based on gene expression signature
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
31
Conclusions
● Primary hepatocytes combined with toxicogenomics useful to identify mechanisms of action of some non-genotoxic carcinogens:– Peroxisome proliferators– Metalloids– Skin tumor promotors– AhR ligands
● Useful tool for risk assessment of chemicals
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
32
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
Finding maximal transcriptome differences between reprotoxic and non-reprotoxic phthalate responses in rat testis
Xiaolian Yuan, Martijs J. Jonker, Jillian de Wilde, Aart Verhoef, Floyd R.A. Wittink, Jan van Benthem, Jos G. Bessems, Betty C. Hakkert, Raoul V. Kuiper, Harry van Steeg, Timo M. Breit, and Mirjam Luijten.
J. Appl. Toxicol 31, 421 – 430 (2011)
For more information: mirjam.luijten@rivm.nl
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
3333
Category approach – hazard identification
Category: Phthalates- mainly used as plasticizers to increase their flexibility,
transparency, durability and longevity.- widely applied in children’s toys- some phthalates reprotoxic, others not
Can we separate reprotoxic and non-reprotoxic phthalates by gene expression profiling?
Experiment:- Oral exposure of rats to phthalates, reprotoxic and non-reprotoxic- Sacrifice after 24h of exposure: gene expression profiling and
histopathology of the testis
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
34
Time (days) 0 1
Treatment
Gene expression profiling
Acclimatization
-21
7-week-old male HsdCpb:WU rats; n = 5
Animals killed 24 h after treatment
Study design
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
35
Replicates Compound AbbreviationChainlength Description
5 Controle (vehicle) - -
5 2-Methoxyethanol 2-ME - Positive control
5 Dimethyl phthalate DMP C1 Non-reprotoxic
5 Diethyl phthalate DEP C2 Non-reprotoxic
4 Di-n-propyl phthalate DPrP C3 Non-reprotoxic
5 Di-n-octyl phthalate DOP C8 Non-reprotoxic
5 Di-n-butyl phthalate DBP C4 Reprotoxic
5 Di-n-pentyl phthalate DPeP C5 Reprotoxic
5 Di-n-hexyl phthalate DHP C6 Reprotoxic
5 Di(2-ethylhexyl) phthalate DEHP C6 Reprotoxic
4 Mono(2-ethylhexyl) phthalate (MEHP) MEHP - Reprotoxic, metabolite
Experimental design
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
36
Untreated control Positive control (2-ME)
DHP treatment
No clear histopathological effects 24 hours after exposure to phthalates
Histopathology
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
37
Data analysis: 3 strategies
1. Identification of differentially expressed genes between treatment with individual compounds and the untreated control
2. Identification of differentially expressed genes between compound classes:
– reprotoxic phthalates (RT)– non reprotoxic phthalates (NRT)– untreated control (NC)
3. Identification of differentially expressed individual probes between compound classes
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
38
RT NRT
Differentially expressed genes (FDR<0,1) range from 107 (DEHP) to 1,273 (DPeP)
1. Differentially expressed genes-individual compounds (I)
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
39
• Little overlap between different RT or different NRT phthalates• Only 2 genes (LCMT2 and an unknown gene) are affected by all RT
phthalates, these are however also affected by at least one NRT phthalate
1. Differentially expressed genes-individual compounds (II)
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
40
2. Differentially expressed genes-compound classes (I)
• Differentially expressed genes (FDR <0,05):
– RT vs NRT phthalates: 469 genes
– RT phthalates vs NC: 454 genes
– NRT phthalates vs NC: 764 genes
• No genes were differentially expressed in all 3 comparisons
• 70 (51 + 19) genes not only differentially expressed in RT vs NRT phthalates, but also between either class of phthalates and NC
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
41
Discrimination between RT and NRT phthalates based on 70 selected genes
2. Differentially expressed genes-compound classes (II)
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
42
3. Differentially expressed probes-compound classes (I)
• Differentially expressed probes (FDR <0,05):
– RT vs NRT phthalates: 1897 probes
– RT phthalates vs NC: 4159 probes
– NRT phthalates vs NC: 6189 probes
• No probes were differentially expressed in all 3 comparisons
• 269 (143 + 126) probes were not only differentially expressed in RT vs NRT phthalates but had also altered expression between either class of phthalates and NC
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
43
Discrimination between RT and NRT phthalates based on 269 selected probes
3. Differentially expressed probes-compound classes (II)
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
44
Discussion
• Although 24 hours after exposure to phthalates no histopathological effects were observed, gene expression signatures were clearly changed
• Comparison of the different phthalate classes rather than individual compounds results in the identification of 70 genes or 269 probes that can discriminate between RT and NRT phthalates
• We, therefore, offer a proof-of-principle for the possibility to implement toxicogenomics in hazard assessment
• A combination of toxicogenomics and a category approach would allow prioritizing chemicals for toxicity testing and will as a result:
– Be much faster than conventional toxicity testing– Reduce number of laboratory animals used for testing– Lead to a reduction of costs
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
45 21 October 2010 | Health Canada45
Phtalates:
RIVM: Mirjam Luijten, Jan van Benthem, Harry van Steeg, Jeroen Pennings, Jillian de Wilde, Joke Robinson, Raoul Kuiper, Conny van Oostrom, Jos Bessems, Cees de Heer, ZhiChao Dang, Betty Hakkert
MicroArray Department, UvA, Amsterdam: Xiaolian Yuan, Martijs Jonker, Timo Breit
Acknowledgements
Risk assessment:
Lya Hernandez, Wout Slob, Wim Mennes, André Muller, Jan van Benthem
Hepatocytes:
RIVM: Mirjam Schaap, Mirjam Luijten, Edwin Zwart, Harry van Steeg
LACDR, University Leiden: Ilse Huijskens, Bob van de Water
Merck: Jan Polman, Willem Schoonen
MicroArray Department, University Amsterdam: Paul Wackers, Martijs Jonker, Floyd Wittink, Timo Breit
Fundings:Dutch Technology Foundation, STW 06935NGI Netherlands Toxicogenomics Centre NTC 050-06-510
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
46
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
47
Number non carcinogens in IARC
IARC group total carc non-carc percentage
1 77 64 13 16.9
2A 57 55 2 3.5
2B 237 207 30 12.7
3 507 471 36 7.1
total 878 797 81 9.2
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
48
Phthalates - Toxicity based on side chain length
Reprotoxic (C4-C6) Non-Reprotoxic
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
49
Results of the gene expression analysis
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
NGTXC mode of action DEG response PCA
TCE halogenated hydrocarbon 40 3 A
TCPOBOP constitutive androstane receptor agonist 65 3 A
TCDD arylhydrocarbon receptor agonist 567 1 A
ARO arylhydrocarbon receptor agonist 762 1 B
CA skin tumor promotor 281 3 A
CT halogenated hydrocarbon 56 3 A
CF peroxisome proliferator 308 1 A
CSA immunosuppressant 124 1 A
HCE ligand-independent estrogen receptor 2088 2 B
LAC metalloid 5693 2 C
OA skin tumor promotor 282 3 A
PB constitutive androstane receptor agonist 238 3 A
SAR metalloid 4130 2 C
FK506 immunosuppressant 2404 2 B
WY peroxisome proliferator 1611 1 B
HCH ligand-independent estrogen receptor 115 3 A
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
5050
Risk estimate: many uncertainties and critical data gaps– mode of action– interspecies extrapolation– dose-response analysis
Ethics: high number of test animals involved – reduction & refinement
Time-consuming and costly– gene expression profiles representative of toxicity– prioritization new chemicals
Potential relevance -omics to risk assessment
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research
2nd McKim Workshop, Baltimore 2012 | 09 May 2012
51
Toxicogenomics
● Gene expression profiles
● Advantage• Reliable• Reduce # of animals, time and cost• Information on mechanism of action
● Disadvantage• Identify multiple pathway-associated gene expression
profiles that encompass all non-genotoxic carcinogens
National Institute for Public Health and the Environment (RIVM)
Laboratory for Health Protection Research