A Cell Culture Based Assay for the Measurement of Xenobiotic Effects on Steroid Production

Transcription

1 A Cell Culture Based Assay for the Measurement of Xenobiotic Effects on Steroid Production P.D. Jones, M. Hecker, T. Gracia, X. Zhang, J. T. Sanderson*, J. P. Giesy, J. L. Newsted Aquatic Toxicology Laboratory, Center for Integrative Toxicology, National Food Safety and Toxicology Center, Michigan State University, USA *RITOX, Utrecht University, Utrecht, The Netherlands

2 EDSTAC Endocrine Screening and Testing Advisory Committee -Independent federal advisory committee -Stakeholders: academia, government, industry, advocacy Recommendations - Assess effects in both humans and wildlife - Tiered strategy including in vitro and in vivo assays - Focus on estrogenic, androgenic, and thyroid-like activity - Include assessment of complex mixtures - Validate all assays FPQA and SDWA mandate testing of EDCs

3 U.S. Environmental Protection Agency Endocrine Disruptor Screening Program Initial Sorting Priority Setting Tier 1 Screening Tier 2 Testing Zoology Dept., National Food Safety and Toxicology Center and

4 Proposed Tier 1 Screening Battery In Vitro Screens ER Binding / Reporter Gene Assay* AR Binding / Reporter Gene Assay* Steroidogenesis Assay with minced testis In Vivo Screens Rodent 3-day Uterotrophic Assay (sc) Rodent 20-day Pubertal Female Assay with Thyroid Rodent 5-7 day Hershberger Assay Frog Metamorphosis Assay Fish Reproduction Screening Assay Alternate assays have also been proposed * These assays are in the HTPS Zoology Dept., National Food Safety and Toxicology Center and

5 Uncertainties and Concerns 1. Exposure - outcome linkages - are effects occurring in humans? 2. Comparative toxicology - extrapolations between species? 3. Multiple mechanisms of action 4. Cumulative exposure/latency between exposure and outcome 5. Cost - $200,000 per substance for Tier 1 6. Animal welfare - Estimate million animals per 1,000 substances 7. Do EDCs require special consideration in risk assessment 8. Assay and test validation (Interagency Coordinating Committee for the Validation of Alternative Methods - ICCVAM)

6 EDCs mechanisms of action: Receptor-mediated processes that control sexual development and homeostasis involving estrogen (ER), androgen (AR) and thyroid (ThR) hormone receptors. Non-receptor-mediated mechanisms. Modulation of steroid hormone production or breakdown that causes ED without acting as hormone mimics.

7 ENDOCRINE DISRUPTOR Receptor-mediated processes (ER), androgen (AR) and thyroid (ThR) hormone receptors. Non-receptor-mediated mechanisms. Compounds that can alter steroidogenic enzyme activities may lead to disrupted steroidogenesis (synthesis and catabolism). Alterations in the concentrations of circulating steroid hormones have the potential to adversely affect homeostasis and reproduction.

8 Cholesterol Pregnenolone 3β-HSD Progesterone CYP11B1 Corticosterone Aldosterone 3β-HSD 11-Deoxycorticosterone 17α-OH- Pregnenolone 17α-OH- Progesterone 11-Deoxycortisol Cortisol CYP11B1 Zona fasciculata DHEA Androstene -dione Testosterone Testosterone Zona reticularis Zona glomerulosa Zoology Dept., National Food Safety and Toxicology Center and 3β-HSD 17β-HSD 17β-estradiol 17β-Estradiol

9 8BrcAMP 8BrcAMP PMA PMA forskolin forskolin lovastatin lovastatin Inducers androstenedione androstenedione spironolactone spironolactone Inhibitors

10 Inducer Time Response Forskolin 10 um Zoology Dept., National Food Safety and Toxicology Center and HOURS

11 Objective A screening system to assess the potential effects of compounds on key enzymes involved in steroidogenesis Endpoint: Expression levels of steroidogenic enzymes mrna (10). Production of Steroid Hormones. Method: Quantitative, real time, reverse transcriptase polymerase chain reaction (Q-RT-PCR). Steroid ELISA and GC/MS techniques.

12 Primer Design Gene Bank NCBI (NIH) PCR Optimization Protocols and Chemistry Cell Culture Suppl.Media/C02:Air RNA Extraction/Quantitation ETOH precipitation, H2O Elution Exposure In 0.1%DMSO Cytotoxicity Test cdna Synthesis Cloned AMV-RT Q-RT-PCR SYBr Green Reporter Medium Hormone Measurement Product Confirmation Melt.C Gel/Electr Seq. Result Analysis Ct comparative

13 Objectives Develop and optimize a rapid screening test to determine effects of chemicals on sex steroid synthesis: Progesterone Testosterone 17β-estradiol Demonstrate the performance of the assay with known inhibitors and inducers of steroidogenic pathways Assess and quantify sources of variability in the assay to: Performance criteria for large scale screening of chemicals Ensure Transferability of the protocol to other laboratories prior to conducting ring tests

14 Portability Commercially available cell line (ATCC) Normal cell culture procedures/equipment Commercially available hormone test kits

15 H295R Cell Basal Hormone Production Time Series (6 well plate) pg/ml (P&E2) pg/ml (T) E2 P T h Zoology Dept., National Food Safety and Toxicology Center and

16 Comparison of hormone production and gene expression Progesterone/3β-HSD II (48 hours) 200 3β HSD II pg/ml/medium Fold Change blank solvent control 1uM 3uM Forskolin 10uM

17 Comparison of hormone production and gene expression Testosterone/17β-HSD pg/ml/medium Fold Change 17β HSD blank solvent control 1uM 3uM Forskolin 10uM

18 Comparison of hormone production and gene expression Estradiol/ pg/ml/medium Fold Change blank solvent control 1uM 3uM Forskolin 10uM 1

19 Conclusion I Zoology Dept., National Food Safety and Toxicology Center and H295R cells can be used to measure the effects of chemicals on hormone production. Rapid, easy, reproducible. Can determine changes in hormone production with high precision and accuracy. Flexible System - Gene expression, enzyme activities, Hormone production.

20 Conclusions II Has the potential to identify multiple mechanisms of action based on gene expression profiles. Alterations in gene expression are accompanied by alterations in hormone production.

21 Future Directions Establish exposure/expression profiles (dose response/time response) for additional compounds. Relate expression profiles to chemical modes of action. Relate expression profiles to effects on hormone production.

22 Acknowledgement Funding for this project was provided by the US-EPA Computational Toxicology Program, through a contract from ENTRIX Inc. to Michigan State University

23 Thank You Paul D Jones National Food Safety and Toxicology Center Michigan State University East Lansing, Michigan, 48824, USA Tel: (517) Fax: (517) jonespa7@msu.edu