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Evaluating Existing in vitro Endocrine Data Jeff Pregenzer,

Evaluating Existing in vitro Endocrine Data Jeff Pregenzer, Director of Endocrine Studies, CeeTox. EDSP in vitro assays. Receptor Binding Assays. Potential false positives receptor denaturation due to test chemical non-specific displacement at high concentrations

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Evaluating Existing in vitro Endocrine Data Jeff Pregenzer,

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  1. Evaluating Existing in vitro Endocrine Data Jeff Pregenzer, Director of Endocrine Studies, CeeTox

  2. EDSP in vitro assays

  3. Receptor Binding Assays • Potential false positives • receptor denaturation due to test chemical • non-specific displacement at high concentrations • Examine curve fit parameters • Potential false negatives • Solubility issues • Measure precipitation in buffer • Detection method interference (assay specific) • Test for detection interference

  4. Interpretation of Binding data non-binder binder

  5. Interpretation of Binding data

  6. Transactivation Reporter Assays Cell-based - reporter human cell lines Provide functional biological response data (agonist vs antagonist) Highly sensitive, High throughput Validated for ER agonism as of 9-2009, (antagonism and AR to follow).

  7. Transactivation Reporter Model Agonist Induction E2 Transcription apparatus Agonist ER Cofactor +ATP = luminescence luciferase ERE LUC

  8. Agonist: E2 Antagonist ER Transactivation Agonism Antagonizable induction background Luciferase reporter gene results expressed as fold of vehicle control.  Data calculations performed using Microsoft Excel and graphed with GraphPad Prism.

  9. Controls in Transactivation Assays Blank, positive, and negative controls in all plates use of dextran-charcoal stripped serum Solubility check (i.e. via nephelometry) Cytotoxicity Assay “Agonist” plates - Specific antagonist for receptor specificity “Antagonist” plates - Excess agonist for non receptor related signal interference

  10. ER Transactivation AntagonismT47D-KBluc Estrogen transactivation reporter model E2 Transcription apparatus Antagonist “spike” with agonist antagonist ER +ATP = luminescence Cofactor luciferase ERE LUC

  11. Limiting False Positives Transactivation Reporter Model Non-Receptor Specific Signal Inhibition E2 Transcription apparatus chemical ER +ATP = luminescence Cofactor luciferase ERE LUC

  12. Limiting False PositivesControl for Non-Estrogen Receptor related Reduction Test compound concentrations are co-incubated with 0.01nM E2 ICI antagonizes 0.01 nM E2 response NCH appears to show antagonism with 0.01 nM E2.

  13. Limiting False Positives Transactivation Reporter Model E2 E2 E2 E2 E2 Transcription apparatus Antagonist – excess agonist control • Non-Receptor Specific Signal Inhibition chemical antagonist ER +ATP = luminescence Cofactor luciferase ERE LUC

  14. Limiting False Positives:Control for Non-Estrogen Receptor related Reduction Test compounds co-incubated with 0.01nM E2 and 100nM “excess agonist” controls. ICI does not affect 100 nM E2 response NCH appears to show antagonism with 0.01 and 100 nM E2. Suggests apparent antagonism may really be result of non binding site related signal inhibition. ICI antagonizes 0.01 nM E2 response

  15. Transactivation Assay Plate Layout (CeeTox)

  16. in vitro Metabolism check (possible future assay) Metabolism testing -/+ S9 microsomes. Phase I and Phase II enzymes both in the liver and in hormonally active tissues could lead to: false-positive data (due to lack of detoxification) or false-negative data (lack of activation) in vitro metabolism testing could test potential for metabolism.

  17. End

  18. Steroidogenesis inhibition example M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124

  19. Steroidogenesis • 5.2.9 Known False Negatives and False Positives • The assay will almost certainly produce false negative results. As for false negatives, this is most likely to occur for those test substances that require metabolic activation, since the testes do not include pathways for metabolism. Other examples of false negatives involve those instances when a substance evokes an indirect effect on steroidogenesis, e.g., site of action is at the hypothalamus or pituitary gland. • Finally, if the effect of the toxicant is delayed for a time greater than the duration of the incubation period, then a false negative result will occur. An example of a delayed effect was observed when lead was tested for its effect on steroidogenesis, which inhibited steroid hormone production 4 hours after initiation of the incubation (Thoreux-Manlay et al., 1995). • There are no known false positive instances to report at this time.

  20. Transactivation • Potential reasons for false positives • non-specific interaction (agonism) • Solution – specific inhibitor control • Assay signal inhibition (antagonism) • Solution – controls: constitutive luciferase or excess agonist to out compete specific antagonism • Potential reasons for false negatives • Solubility issues in assay medium • Edge effect • Plate layout • outlier rejection Binding data and Transactivation data corroborate?

  21. AR Transactivation Antgonism In the example graph above luciferase reporter gene results are expressed as fold of vehicle control.  Data calculations are performed using Microsoft Excel and graphed with GraphPad Prism.

  22. Limiting False Positives – Assay inhibition

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