The document discusses the challenges of predictive toxicology due to biological complexity and the potential role of cell-based assays. It describes the BioMAP platform which uses primary human cell systems to model disease biology and responses to drugs and chemicals. BioMAP profiles compound effects on multiple biomarkers and can identify toxicity mechanisms and pathways. However, data analysis is complicated by concentration effects, cytotoxicity, and heterogeneous assay readouts. Specialized approaches are needed to analyze the complex, multiparameter data generated by these human cell-based systems.

1. Defining Chemical Target and Pathway Toxicity Mechanisms with Primary Human Cell Systems Ellen L. Berg, PhD BioSeek, Inc. eChemInfo, Philadelphia PA October 2009 Bio Seek

3. Biological Systems Are Complex Scale (meters) (Time) 10 -9 M 10 -8 M 10 -7 M 10 -6 M 10 -5 M 10 -4 M 10 -3 M 10 -2 M 10 -1 M 1 M Human exposure Molecular targets 10 -6 sec 10 2 sec 10 4 sec 10 5 sec 10 8 sec Modular design / Networked architecture molecules pathways cells tissues humans

8. BioMAP ® Technology Bridging the Gap Scale (meters) (Time) 10 -9 M 10 -8 M 10 -7 M 10 -6 M 10 -5 M 10 -4 M 10 -3 M 10 -2 M 10 -1 M 1 M Human exposure Molecular targets 10 -6 sec 10 2 sec 10 4 sec 10 5 sec 10 8 sec BioMAP Technology molecules pathways cells tissues humans

9. BioMAP ® Technology Platform Assays Human primary cells Disease-like culture conditions LPS BF4T SM3C Profile Database Informatics Biological responses to drugs and stored in the database Specialized informatics tools are used to mine and analyze biological data

13. BioMAP Systems are Validated Corticosteroids (Prednisolone) Are Active in Inflammation Systems Profiles retain shape over multiple concentrations BioMAP Systems Readout Parameters (Biomarkers) Cytotoxicity Readouts Log expression ratio (Drug/DMSO control) Control (no drug) 99% significance envelope Dose Response

14. E-selectin TNF-  IL-8 BioMAP Systems are Validated Activities of Corticosteroids Match Clinical Effects PGE 2 IL-8 MCP-1 Readouts in BioMAP show the same pattern as has been reported for patients receiving steroid therapy Log expression ratio (Drug/DMSO control) MCP-1, IL-8, E-sel. decrease Leukocyte recruitment Many, e.g. Jilma et al., 2000 PGE 2 decrease Pain, swelling Sebaldt et al., 1990 Collagen I & III Collagen I, III decrease Skin atrophy Autio et al., 1994 MMP-1 PAI-1 SAA PAI-1, SAA increase CV complications Sartori et al., 1999 Fyfe et al., 1997 PAI-1

16. Microtubule Stabilizers Mitochondrial ET chain Retinoids Hsp90 CDK NF  B MEK DNA synthesis JNK Protein synthesis Microtubule Destabilizers Estrogen R PI-3K Ca ++ Mobilization Classification of Drugs By Mechanism Pairwise Correlation of BioMAP Reveals Functional Similarities mTOR PKC Activation p38 MAPK HMG-CoA reductase Calcineurin Transcription Mechanism of Action (On-Target) Pathway Relationships

17. Defining Toxicity Mechanisms Sodium Azide Chlorambucil Cycloheximide A23187 Thapsigargin Oxidative /Nitrosative Stress ER Stress Unfolded Protein Response Rotenone

18. ToxCast Chemicals Diversity of Mechanisms Houck, 2009

22. ToxCast Chemicals Diversity of Mechanisms cAMP Elevation DNA Alkylation NF  B Tubulin Inhibition Mitochondrial Dysfunction