The Human Toxome Project a test case for pathway identification by multiomics. Thomas Hartung

Transcription

1 The Human Toxome Project a test case for pathway identification by multiomics integration Thomas Hartung

2 Mechanistic & evidence-based toxicology 2

3 Level of resolution Current state of the art Perturbed Molecular Network Molecular Pathway of Toxicity Toxicity Pathway / AOP Mode of Action Phenomenologic 3

4 Omics Image analysis Robotised / automated testing High content High through-put Information rich Bioinformatics & Data-mining Knowledge on pathways Systems Toxicology 4

5 Mapping the Human Toxome by Systems Toxicology Endocrine disruption Use omics to map PoT for endocrine disruption Develop software tools Identify PoT Develop a process for PoT annotation, validation Establish public database on PoT. Hewitt et al., Science, 307:

6 Use for PoT identification: Homeostasis under stress, i.e. signatures of tox Critical cell infrastructures Network knowledge Reference models Reference toxicants 6

7 Workshop on the Concept and Tools for Pathways of Toxicity October 10-12, 2012, Baltimore, MD Content side: - Mol.biol. - Biochem. - Omics SoT - Tox Mechan. ALTEX 31 (2014) Human Toxome database PoT Existing databases User side: - Regulation - Probabilistic RA - Systems Toxicology - Virtual patient 7

8 (Pre-)Validated work Robust protocols, good cell models Regulatory acceptance available or in progress Available reference substances Thresholds of adversity defined In vitro systems MCF-7 cells (pre-validated by ICCVAM) & Initial set of endocrine disrupting chemicals selected from a priority list of 53 reference compounds identified by ICCVAM Day -1 or -2 Switch to stripped serum Day -2 to -5 Plate cells Day 0 Begin treatment Cell culture SOP Gene Expression Timecourse 1nM estradiol 2, 4, 8, and 24 hours 8

9 Similar studies, dissimilar gene expression MCF-7 test failed parallel validation - Not reproducible from literature All MCF-7 cells 24h, 1nM estradiol, whole genome microarrays Meta-analysis of transcriptomics studies A test does not become better adding a sophisticated endpoint!!! Ochsner et al. Cancer Res

10 QA of cell system is of critical importance Good Cell Culture Practice (Coecke et al. 2005) Karyotyping 10

11 Extent of deviations from normal genome Classification Kilobases Percentage of genome Losses % Deletions % Amplifications % Gains % Normal % Centromeres % Total Abberations % All Entries SurePrint G3 ISCA CGH+SNP Microarray Kit, 4x180K CGH features.2440 CGH replicate probes, SNP features reference mapping: caucasian female human reference DNA 11

12 omics data generation MCF-7 cell culture and E2 exposure Sample prep and metabolite extraction Data acquisition (Q-Tof) Untargeted Targeted Peaks with mass & RT (more than 6,000 features) Peaks with metabolite names (around 200 metabolites) Peak/feature extraction with NaÏve data mining algorithm Metabolite peak extraction with database searching Workflow for Metabolomics Bio-statistical analysis (e.g. ANOVA, PCA and clustering analysis) Significant metabolite names Metabolites Identification (only for untargeted) and confirmation (for both approaches) 12 Data interpretation (e.g. Pathway analysis)

13 METABOLOMICS 3 WORKSHOPS 2 INFODAYS 13 ALTEX ,

14 Software tools LC/MS GC/MS MassHunter Qual/Quant ChemStation AMDIS Microarrays Feature Extraction GeneSpring Platform Biological Pathways NGS Alignment to Reference Genome 14 Agilent Integrated Biology Workflows 14

15 Success Consortium with intense interactions PoT concept furthered, mechanistic validation Improved cell system standardization Some problems omics overcome, QA Cloud Server, Toxome Collaboratorium, GeneSpring expansion and other tools High visibility Failure No link to high-throughput testing No annotation yet MCF-7 not really suitable Metabolite identification No real PoT identification yet No expansion to other hazards or regions Workflow established (integrated omics analysis, weighted gene connection network analysis, transcription factor analysis and systematic literature mining) PoT #1 emerging 15

16 International expansion? 16

17 Nature blog at start 2 hour event in European Parliament Science coverage Nature coverage Lay press like LeMonde, La Recherche, Sueddeutsche Zeitung, BBC, RAI-1, Laboratorio 2000 NIEHS 17

18 What this project is not. an endocrine disruptor screening project a test development project an academic publish or perish project a five year project 18

19 The difficulty lies, not in the new ideas, but in escaping from the old ones. John Maynard Keynes ( ) 19