Presentation on theme: "Evaluating Existing in vitro Endocrine Data Jeff Pregenzer, Director of Endocrine Studies, CeeTox."— Presentation transcript:
Evaluating Existing in vitro Endocrine Data Jeff Pregenzer, Director of Endocrine Studies, CeeTox
EDSP in vitro assays EDSP in vitro Tier 1 Battery ER binding ER transcriptional activation AR binding Steroidogenesis Aromatase
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
Interpretation of Binding data binder non-binder
Interpretation of Binding data
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).
Agonist ER ERE LUC +ATP = luminescence CofactorE2 Transcription apparatus luciferase Transactivation Reporter Model Agonist Induction
ER Transactivation Agonism Luciferase reporter gene results expressed as fold of vehicle control. Data calculations performed using Microsoft Excel and graphed with GraphPad Prism. Agonist: E2 Antagonist background Antagonizable induction
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
Antagonist ER ERE LUC +ATP = luminescence CofactorE2 Transcription apparatus antagonist luciferase ER Transactivation Antagonism T47D-KBluc Estrogen transactivation reporter model “spike” with agonist
Limiting False Positives Transactivation Reporter Model Non-Receptor Specific Signal Inhibition ER ERE LUC +ATP = luminescence CofactorE2 Transcription apparatus chemical luciferase
Limiting False Positives Control for Non-Estrogen Receptor related Reduction NCH appears to show antagonism with 0.01 nM E2. Test compound concentrations are co-incubated with 0.01nM E2 ICI antagonizes 0.01 nM E2 response
Antagonist – excess agonist control ER ERE LUC +ATP = luminescence CofactorE2 Transcription apparatus antagonist luciferase Limiting False Positives Transactivation Reporter ModelE2 E2 E2 E2chemical Non-Receptor Specific Signal Inhibition
Limiting False Positives: Control for Non-Estrogen Receptor related Reduction 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. Test compounds co-incubated with 0.01nM E2 and 100nM “excess agonist” controls. ICI does not affect 100 nM E2 response ICI antagonizes 0.01 nM E2 response
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.
Steroidogenesis inhibition example M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
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.
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?
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.