1. Modeling of Early Key Events Based on Genomics and potential applications for nuclear-receptor-mediated toxicity Harvey Clewell Director, Center for Human Health Assessment The Hamner Institutes for Health Sciences Research Triangle Park, NC

2. Mode of Action from a Systems Biology Perspective: Chemical Perturbation of Biological Processes Systems Inputs Biological Function Impaired Function AdaptationDisease Morbidity & Mortality Exposure Tissue Dose Biological Interaction Perturbation Molecular Target(s) “Chemical Mode of Action Link”

3. Uses of Genomic Data (1): Hazard Identification – Identify pattern of gene changes associated with a particular effect - can provide insights into key elements in mode of action - essentially qualitative - typically, little concern for tissue dosimetry (Liu et al. 2005)

4. Uses of Genomic Data (2): Functional Genomics – Characterize interactions of compound with gene regulatory network using temporal analysis and iterative gene over-expression / inhibition - can elucidate key elements of cellular dose-response (e.g., switch-like behaviors) - time-consuming, requires sophisticated analyses - modeling of gene regulation is in its infancy Increasing Stimulus Input Pulse (Conolly 2004)

5. Uses of Genomic Data (3): Dose-Response – Collection of data on genomic responses to a compound over a range of cell/tissue exposure concentrations to identify dose-response for key genomic bio-indicators of response - provides evidence for dose-dependent mode of action - requires tissue dosimetry or phenotypic anchoring Activity / Amount (% control) µM As III (Snow et al. 2002) 0 100 200 300 400 0 510152025 Trx mRNA APE/Ref-1 mRNA Pol  Ligase I

6. Analysis of Mutation Frequency in p53 and K-ras Oncogenes in Nasal Tissues of Rats Exposed to Inhaled Formaldehyde NCTR analysis Found no increase in p53 or K-ras mutations after 90 days of exposure to formaldehyde at up to 15 ppm Demonstrates lack of mutagenic activity in vivo at carcinogenic concentrations

7. High Content Imaging Assays (HCA) in vitro, cell-based imaging assays in multi-well plates Primary cells or cell lines similar to target tissue (human and rodent) Capable of performing large numbers of replicates, doses, and time points High statistical power to detect departure from linearity or threshold Can apply to provide oxidative and DNA stress measures 7

8. Example 1: Formaldehyde causes nasal cancer in rats… 0 10 20 30 40 50 60 Tumor Response (%) 00.7261015 Exposure Concentration (ppm ) Kerns et al., 1983 Monticello et al., 1990

9. …but it’s a normal constituent of cells Glutathione Conjugation Oxidation Formaldehyde Dehydrogenase (FDH) Labile methyl groups and one-carbon metabolism HCOOH Formic acid GSH Endogenous Production NAD + NADH + H + + DIsplacement Formaldehyde Hydrate DNA & Protein DNA - Protein Crosslinks Addition Products

10. Genomic Dose-Response Analysis Results No evidence of genomic changes at 0.7 ppm exposure Transient response (at 5 days) in animals exposed to 2 ppm –different from response at 6 ppm –suggestive of cellular adaptation Inflammatory and oxidative stress responses at 6 ppm –maintained throughout entire exposure period Evidence of change in mode of action between 2 and 6 ppm –Consistent with transition from adaptive to toxic state –Indications of cell-surface targets at lower concentrations vs. internal targets at higher concentrations

11. Genomic Data Benchmark Analysis Genomic Data Benchmark Analysis Gene Expression Dose Response Data One-Way Analysis of Variance to Identify Genes Changing with Dose Power Model Linear Model Polynomial Model (2°) Polynomial Model (3°) Select Best Model Remove Genes with BMD > Highest Dose Group Genes by Gene Ontology Category Estimate BMD and BMDL for each Gene Ontology Category Nested test to Select Best Polynomial Model

12. Comparison Between Transcriptomic Dose Response and Tumor Response Time Point 6 hr 5 day 19 day Mean (ppm)SD Min (ppm)Count Mean (ppm)SD Min (ppm)Count Mean (ppm)SD Min (ppm)Count Lowest Response Categories Protein Import into NucleusGO:00000592.020.341.534 Complement Activation, AlternativeGO:00069571.580.281.373 Positive T-cell SelectionGO:00433681.180.171.023 Selected Categories Related to Carcinogenicity Positive Regulation of Cell ProliferationGO:00082847.824.260.641007.523.780.631146.853.900.57137 Response to DNA Damage StimulusGO:00069746.824.070.641037.123.790.841056.573.730.70124 Inflammatory ResponseGO:00069547.614.640.861008.163.780.791327.154.030.49140 Formaldehyde Time Course BMD for tumors: 6.4 ppm (Schlosser, Risk Anal., 2003) BMD for cell labeling index: 4.9 ppm

13. Comparison Between Transcriptomic Dose Response and Tumor Response 0 10 20 30 40 50 60 Tumor Response (%) 00.7261015 Exposure Concentration (ppm) Kerns et al., 1983 Monticello et al., 1990 Critical Gene Changes

14. Conclusions: Formaldehyde Genomic analysis demonstrates the need to differentiate regions of adaptive response from those with overt tissue damage (> 6 ppm) Mutation analysis demonstrates no evidence of mutagenic activity at clearly toxic and tumorigenic concentrations Results provide mechanistic support for U-shaped cell proliferation dose-response curves seen in cancer bioassay and the modeling results of Conolly et al. 2004

15. Example 2: Genomic Analysis to Identify the Dose-Response for Early Cellular Responses to Inorganic Arsenic Normal Epithelial Cell Adaptive State Stressed State Necrosis Apoptosis Tumors Biochemical effects GSH/GSSG ratio Interactions with proteins Arsenite, trivalent MMA HSP proteins Oxidative stress Inflammation Cytotoxicity DNA damage Proliferation Goal of Genomic Dose-Response Studies: To identify key elements of each state and the points of transition

16. Dose-Response for the In Vitro Effects of Arsenite in Primary Cells (Gentry et al. 2009)

17. Inorganic arsenic concentrations in mouse bladder after 12 weeks of exposure to arsenic in drinking water

18. Gene expression changes (up/down) at weeks 1 and 12 (1.5-fold or greater, p<0.05) 17/7 0 0/70/7 490/16 19/259 831/84 25/294 1677/125 Down-regulation at week 1 changes to up-regulation at week 12

20. Summary of BMD Analysis Conducted for GO categories with genes showing D/R at p<0.05 1 week: –Majority of GO category median BMDs: 9-15 ppm –Lowest BMDs for categories (N>6): 1.5 ppm –Lowest BMDs for single genes: 0.7 ppm 12 week: –Majority of GO category median BMDs: 6-11 ppm –Lowest BMDs for categories (N>6): 1.7 ppm –Lowest BMDs for single genes: 0.7 ppm

21. GO categories (N>6) with lowest BMDs at week 1 DNA packaging chromatin assembly or disassembly chromatin assembly nucleosome organization protein-DNA complex assembly nucleosome assembly

22. GO categories (N>6) with lowest BMDs at week 12 apoptotic mitochondrial changes double-strand break repair cellular response to oxidative stress regulation of DNA recombination negative regulation of gene expression, epigenetic epidermal growth factor receptor signaling pathway oxygen and reactive oxygen species metabolic process response to gamma radiation nucleotide-excision repair positive regulation of cell division activation of protein kinase activity histone deacetylation regulation of protein stability response to ionizing radiation response to UV positive regulation of cell cycle

23. Exposure: As(5); As(3); mixed forms Active chemical in tissue: As(III), MMA(III) Protein binding: As(III) + RSH RS - As Oxidative stress DNA repair inhibition Inflammation Cell proliferation Proposed “Sequence of Events” for Inorganic Arsenic Carcinogenicity DNA mutation Tumors DNA damage

24. Conclusions Dose and duration of arsenic exposure interact to produce an cellular response that evolves over time Median BMDs for gene categories cluster around the 10 ppm dose, with a small number of BMDs around the 2 ppm dose. The lowest BMDs for single genes are above the lowest dose of 0.5 ppm. The genomic responses are largely consistent with an adaptive response at lower concentrations / shorter durations, but with increasing toxicity as concentration and duration increase

25. In Vitro Exposure of Human Urothelial Cells to Mixtures of Trivalent Arsenic Compounds Relative Concentrations Based on Measurements of Arsenic Compounds in Urine of Humans Exposed to Arsenic in Drinking Water Treatment GroupArsenite (µM)MMA (µM) (trivalent) DMA (µM) (trivalent) Control000 Exposure 10.01 0.04 Exposure 20.03 0.12 Exposure 30.1 0.4 Exposure 40.3 1.2 Exposure 5114

26. Dose LevelTotal Arsenic (uM)Number of Significant Genes 1 0.06 0 2 0.18 0 3 0.6 0 4 1.8 1 5 6.0 36 Results: In Vitro Exposure of Human Urothelial Cells to Mixtures of Trivalent Arsenic Compounds Number of Genes with Significant Change in Expression Genomic alterations restricted to total arsenic concentrations above 1 micromolar The most significant genes at Dose 5 are consistent with oxidative stress response

27. The inter-individual variability is much larger than the change in expression elicited by arsenic treatment.

28. Conclusions: Arsenic Genomic responses to arsenic in vitro exposures were observed in human uroepithelial cells at total arsenic concentrations greater than 1 micromolar These results are consistent with the in vivo genomic responses observed in bladders of mice exposed to inorganic arsenic in drinking water, where significant genomic changes were observed at bladder concentrations above 1 micromolar Human interindividual variability in gene expression is large in comparison with the effect of environmental arsenic exposures, complicating the use of genomic changes as biomarkers

29. Potential Impact of Population Variability on Cancer Dose-Response for Arsenic in Drinking Water 0.10.010.0011.0 Concentration in Drinking Water (mg/L) Risk 0.01 0.001 0.0001 0.00001 Susceptibility Factors: - Dietary intake - Nutritional status - Other exposures - selenium - mutagens - Genetic factors - metabolism (GST) - cell control (P53) Linear Extrapolation Average Individual Dose-Response Population Dose-Response Sensitive / Resistant Individual Dose-Response (Clewell, 2001)

30. Application to Nuclear-Receptor-Mediated Toxicity Nuclear receptor binding is just one of many possible early events linking an active compound or metabolite to a cellular response Downstream events from receptor activation can include multiple effects -- induction of metabolism -- mitogenic signaling -- DNA damage The dose-responses for these pleiotropic effects can differ significantly, resulting in dose-dependent transitions and changes in mode of action Genomic, proteomic, and mutational dose-response analysis can be used to identify key early events, dose-dependent transitions, and the mode-of-action element that drives carcinogenicity (toxicity, proliferation, or mutation)