Review of the in vitro Assays Validation Results and Methods for Improving the Tier 1 Endocrine Disruption Screening Assays

Transcription

1 Review of the in vitro Assays Validation Results and Methods for Improving the Tier 1 Endocrine Disruption Screening Assays Presented by: Colleen Toole, Ph.D. Director of Project Management for CeeTox, Inc.

2 CeeTox Experience In Vitro Laboratory Lots of experience with the Assays > 90 in vitro studies following EDSP guidelines ~ 30 of the Tier 1 test articles

3 Tier 1 In Vitro Assays Five assays that assess potential interaction with: Estrogen Receptor ER Transcriptional Activation Assay (OPPTS ) ER Competitive Binding Assay (OPPTS ) Androgen Receptor AR Competitive Binding Assay (OPPTS ) Steroidogenesis Steroidogenesis assay (OPPTS ) Aromatase Assay (OPPTS ) General study design Lessons learned; improvements to the assay and efficiency Challenges running the assay and reporting 3

4 ER Transcriptional Activation Assay (ERTA) Purpose of the assay is to assess the ability of a substance to bind the Estrogen Receptor (ER) and subsequently transactivate an ER responsive element (ERE) driven reporter gene The ERα transactivation assay (ERTA) utilizes herα-hela immortal cell line hera expression vector 5X ERE-luciferase reporter vector Cells are treated with a test substance and the induction of the reporter gene products is utilized to measure the response. 4

5 Transactivation Assay Model Estrogens and Xenoestrogens Estrogen Receptor hsp90 EREs Estrogen Responsive Gene mrna EREs Luciferase Gene Increased Estrogen- Responsive Gene Product New Polypeptides Translation Luciferase Activity Provided by Mike Denison UC-Davis 5

6 ERTA Lessons Learned, Challenges Proficiency Compounds (7 pos/3 neg) Good number of test materials, clear results Solubility Assessment Added nephelometry (light scattering) coupled with visual detection Cytotoxicity Ability to use alternative assays (live-dead) 4 Reference Controls/compound 17b Estradiol (E2) 17a Estradiol 17a Methyltestosterone (17MT) Corticosterone 6

7 % of Maximal Induction Control % of Maximal induction Control ERTA Control Data - Challenges Name logpc 50 logpc 10 logec 50 Hill slope Test range 17β-Estradiol (E2) ~ < ~ ~ ~ 10-8 M 17α-Estradiol -9.6 ~ ~ ~ ~ ~ 10-6 M Corticosterone ~ 10-4 M 17α-MT -6.0 ~ ~ ~ 10-5 M 17a-MT E Log Concentration (M) Log Concentration (M)

8 ERTA Stability of 1 nm E2 (PC)

9 ERTA Lessons Learned, Challenges % of Maximal Induction Control Edge Effects and Non-Specific ER Responses Changes in Plate format Edge effect- How do we deal with outliers (increasing n=3 to n=6)? Addition of ER antagonist, ICI 182,780 at each concentration of Test Article (n=2) allows for identification of non-specific (i.e. non heramediated) induction of luciferase gene. Identification of potential false positives Identification of high background issues Concentration [LogM] Test Article X Test Article X + ICI 182,780 9

10 ERTA Lessons Learned, Challenges Antagonism (Appendix 2 of the guideline) More robust description of antagonism assay (details) Use of 4 OH-Tamoxifen (Selective Estrogen Response Modulator, SERM) 10

11 ERTA Future Testing Flexibility for time of incubation following plating of cells Change 3 hour to > 3 hours and < 24 hours Enhance recovery time after plating Difficult to stagger plating and exposures List of alternative solvents (DMSO and EtOH) How to run controls? Criteria for controls (historical data), 95% confidence limits Appendix 2 Antagonism Change 4OH tamoxifen to ICI 182,780 Outlier Analysis (edge effect) BG-1 cell line (expensive)

12 Estrogen Receptor Competitive Binding Assay: Lessons Learned, Challenges Competitive radioligand binding ER assay have been used for decades and is considered the gold standard for identifying substances that bind to ER The ER competitive binding assay measures the ability of radiolabeled 17β-estradiol (E2) to competitively interact with the ER, using rat uterine cytosol, in the presence of increasing concentrations of a substance If the substance interacts with the ER binding domain, less radioligand can bind, so an active competitor produces a descending concentration-response plot. 12

13 ER Preparation of Cytosol Experimental design Collection of Uteri (Sprague-Dawley female rats) Preparation of Uterine Cytosol Protein determination Saturation Binding Assay (Scatchard plot) K d - information of ER binding affinity for the radioligand B max - number of receptors for a batch of uterine cytosol Competitive Binding Assay 13

14 Mean Specific Binding (%) Mean Specific Binding (%) ER Binding Performance Standards Parameter Unit Estradiol Lower limit Upper limit Lower limit Norethynodrel Upper limit Loge(Syx) (i.e., Loge(Residual Std.Dev) -- NA 2.35 NA 2.60 Bottom plateau level % binding Top plateau level* % binding (Hill) Slope log10(m) Estradiol Norethindrone Log Concentration (M) Log Concentration (M) 14

15 Determination of False Positives by Determining the K i K i is the equilibrium dissociation constant (the concentration of the competing ligand/compound that will bind to half the binding sites at equilibrium) Performed when non-sigmoidal binding curves or partial curves are observed. Susan Laws, Toxicological Sciences

16 Determination of False Positives by Determining the K i Increasing concentrations of 3 H-E2 are incubated with ERs in the presence of test material (~0, 0.5, 1 and 2 X IC50) Lineweaver-Burke plots are constructed The slopes obtained from the double reciprocal plots are replotted vs. concentration of the test material. The negative intercept of the slope replot is the K i 16

17 17β-Estradiol True Binding Event All the trendlines for 17β- Estradiol intersect at the same point Results in a linear slope replot nm 1 nm 0.5 nm 0 nm

18 Test Material A False Positive The trendlines for Test Material A do not intersect at the same point Results in a non-linear slope mm 0.6 mm 0 mm

19 AR Competitive Binding Assay Competitive radioligand binding AR assay like the ER binding assay has been used for decades and is the gold standard for identifying substances that bind to AR. The AR binding assay measures the ability of radiolabeled R1881 to competitively interact with the AR using rat prostate cytosol, in the presence of increasing concentrations of a substance. If the substance interacts with the AR binding domain, less radioligand can bind, so an active competitor produces a descending concentration-response plot. 19

20 AR Preparation of Cytosol Experimental design Collection of Prostate (Sprague-Dawley male rats 60 to 90 days old) Preparation of Prostate Cytosol Protein Determination Saturation Binding Assay (Scatchard plot) K d - information of AR binding affinity for the radioligand B max - number of receptors present batch of prostate cytosol Competitive Binding Assay 20

21 AR Performance Standards Chemical Parameter Lower limit Upper Limit Standard Curve Slope Top (%) Bottom (%) Weak Positive Slope Top (%) Bottom (%) R1881 Dexamethasone 21

22 Comparison of AR and ER ER Test tube based assay Uses 100 ml of cytosol Less waste and prep time for assay Storage at -80ºC up to 90 days Decrease runs for requalifying prep Solubility constraints Reporting Categories for binding Binder Equivocal Non-binder AR Test tube based assay Uses 300 ml of cytosol and discards 200 ml 2Xs the amount of test tubes increases prep time No Negative Control Storage discard after 6 months Decrease runs for requalifying prep Solubility constraints Reporting Categories for binding Interactive Non-interactive

23 Steroidogenesis Purpose of the assay is to identify xenobiotics that affect the steroidogenesis pathway after receptor binding (FSHR and LHR) through the production of estradiol/estrone and testosterone The steroidogenesis assay utilizes H295R cells (human adrenocortical carcinoma cell line). Cells are used between passages 5 and 10 Cells are treated with a test substance and estradiol and testosterone are measured by ELISA or HPLC/MS/MS along with viability measurements and visual solubility assessment 23

24 Steroidogenesis Criteria Testosterone Estradiol Minimal Basal Production 500 pg/ml 25 pg/ml (or 2.5 X detection limit) Basal Production > 5 X the MDL > 2.5 X MDL Induction (10 mm Forskolin) > 2 X SC > 7.5 X SC Inhibition (1 mm Prochloraz) < 0.5 X SC < 0.5 X SC

25 Viability Steroidogenesis, Challenges 22RHC can be used to increase basal production of Estradiol and Testosterone, however cytotoxicity and solubility issues were identified at concentrations between mm as outlined in guideline RHC Viability Assessment: MTT in H295R Concentration 22HC (mm) Delicate balance between Estradiol and Testosterone

26 Steroidogenesis, Lessons Learned 22R-hydroxycholesterol (22RHC) can be used to increase basal production of estradiol (E2) and testosterone (T) Testosterone Blank (pg/ml) Media + 22RHC Testosterone Background (pg/ml) No 22RHC at Exposure Estradiol Blank (pg/ml) Media + 22RHC Estradiol Background (pg/ml) No 22RHC at Exposure Changing plate format from 24 to 48 well (increase from n=3 to n=6) Increase number of runs No decrease in E2 or T levels with increasing passage number NuSerum (potential variability) o Stripped versus non-stripped

27 Steroidogenesis, Challenges Criteria QC Results Test Article Results Average Estradiol (pg/ml) Estradiol Fold Change over SC Concentration Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Background Blank DMSO µm Forskolin µm Forskolin µm Prochloraz µm Prochloraz Concentration (µm) Fold Change over SC Run 2 Testosterone Estradiol Minimal Basal Production 500 pg/ml 25 pg/ml (or 2.5 X detection limit) Basal Production > 5 X the MDL > 2.5 X MDL Induction (10 mm Forskolin) > 2 X SC > 7.5 X SC Inhibition (1 mm Prochloraz) < 0.5 X SC < 0.5 X SC Fold Change over SC Run 3 Fold Change over SC Run 4 Mean SD Mean SD Mean SD ** ** N/A N/A 1.53** ** N/A N/A N/A N/A N/A N/A

28 Steroidogenesis: Challenges Guidance for interpretation of the data OECD versus EPA guidelines OECD TG 456: H295R Steroidogenesis Assay: Decision matrix for possible outcome scenarios Run 1 Run 2 Run 3 Decision Scenario Decision Scenario Decision Scenario Positive Negative Negative Confirma Negative Stop X Negative Confirma Positive Refineb Negative X Equivocalc Refineb Negative Confirma Negative X Equivocalc Refineb Negative Confirma Positive X Equivocalc Refineb Positive X Positive Refineb Negative Confirma Positive X Negative Confirma Positive Refineb Positive X Positive Refineb Positive Stop X a Confirm previous run using the same experimental design. b Re-run assay at ½-log concentration spacing (bracketing the concentration that tested significantly different in the preceding experiment). c Fold-change at one concentration is statistically significantly different from the SC

29 Aromatase (CYP19), Lessons Learned and Challenges Purpose of the assay is to identify chemicals that may inhibit the catalytic activity of aromatase Assay measures the conversion of androgen to estrogen in microsomes containing aromatase H 3 -androstenedione and NADPH are incubated with microsomes 3 H20 is released during conversion of androstenedione to estrone and quantified as direct measurement of aromatase activity

30 Aromatase Activity (%) Aromatase (CYP19); Lessons Learned and Assay Performance Challenges Parameter Lower limit Upper Limit Positive Control Slope Top (%) Bottom (%) Log IC OH-ASDN Log Concentration (M)

31 Aromatase (CYP19), Lessons Learned and Challenges 4 proficiency compounds- 3 inhibitors/1 non-inhibitor Perform well in assay Test tube based assay Every sample is divided into 2- increased read times (200 tubes becomes 400 tubes), labor intensive Microsome addition to the assay is based upon protein versus specific activity With purchased supersomes have to rerun protein concentration versus using CofA Miniaturization of assay (decrease waste)

32 In Vitro Assays: Remaining Challenges Technical Support for individual questions (contact at EPA) Guidance for: Alternative vehicles Calculations Outlier analysis Miniaturization of the assays General Questions Reporting SEPs differ from guidelines Data Templates

33 QUESTIONS?

34 Criteria for the QC plate Testosterone Estradiol Minimal Basal Production 500 pg/ml 25 pg/ml (or 2.5 X detection limit) Basal Production > 5 X the MDL > 2.5 X MDL Induction (10 mm Forskolin) > 2 X SC > 7.5 X SC Inhibition (1 mm Prochloraz) < 0.5 X SC < 0.5 X SC Testosterone CeeTox Values (MS/MS) Run 1 Run 2 Run 3 Minimal Basal Production (Bk wells) pg/ml Basal Production (SC wells) pg/ml Induction (10 mm Forskolin) Inhibition (1 mm Prochloraz)

35 ERTA Lessons Learned, Challenges Typical Response for fold induction Between ~10 and ~80-fold (1 nm E2 (PC)) Proficiency Compounds Use high purity, potential false positive 0.1% DMSO Solubility issues at high concentration Other vehicles Increase to 0.5%? 0.1% DMSO Solubility issues at high concentration Other vehicles

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