Effects of pharmaceuticals on gene expression and hormone production in H295R cell line using QRT-PCR/ELISA

Transcription

1 Effects of pharmaceuticals on gene expression and hormone production in H29R cell line using QRT-PCR/ELISA Tannia Gracia P. D. Jones, K. Hilscherova, M. Hecker, E. Higley, J. L. Newsted and J.P. Giesy Aquatic Toxicology Laboratory Center for Integrative Toxicology Michigan State University

2 Pharmaceuticals in the environment Around 82, drugs are registered in the U.S. for human use, which include more than 3 active ingredients; Besides the active substances, formulation adjuvants and, in some instances, pigments and dyes are also drug components. After administration in humans and animals, pharmaceuticals are excreted into wastewater and unused medications are sometimes disposed of in drains. Sewage treatment facilities, depending on their technology and the chemical s structure, are not always effective in removing the active chemical from waste-water.

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4 Previous Study A screening system to assess the potential effects of compounds on key enzymes involved in steroidogenesis. Cell line: Human H29R adrenocortical carcinoma cell line. Endpoint: Expression levels of steroidogenic enzymes mrna (1). Method: Quantitative, real time, reverse transcriptase polymerase chain reaction (Q-RT-PCR).

5 HMGR StAR Cholesterol Steroidogenesis CYP11A Pregnenolone 3β-HSD2 Progesterone CYP21 11-Deoxycorticosterone CYP11B1 Corticosterone Aldosterone Zona glomerulosa 3β-HSD 17α-OH- Pregnenolone 17α-OH- Progesterone CYP21 11-Deoxycortisol Cortisol CYP11B1 Zona fasciculata DHEA 3β-HSD Androstene -dione 17β-HSD Testosterone 17β-Estradiol Zona reticularis

6 Methodology Primer Design Gene Bank NCB (NIH) PCR Optimization Protocols and Chemistry Cell Culture Suppl.Media/C2:Air RNA Extraction/Quantitation EtOH precipitation, H2O Elution cdna Synthesis Cloned AMV-RT Exposure In.1%DMSO Q-RT-PCR SYBr Green Reporter Cytotoxicity Test Live/Dead Hormone Analysis Aldosterone, Progesterone, Testosterone and Estradiol Product Confirmation Melt.C Gel/Electr Seq. Result Analysis Ct comparative

7 Previous Results Chemical agents have the potential to alter gene expression profiles. The H29R cell line is useful for detecting up and down regulation of steroidogenic genes produced by single chemicals or mixtures. Changes in patterns of relative expression can be used to classify chemicals of unknown mechanism of action on the steroidogenic pathways. The assay responds to agonism or antagonism of the compounds when present in chemical mixtures like the real contaminants in the environment.

8 Objective Assess the potential effects of pharmaceuticals on steroidogenic genes and hormone production. Cell line: Human H29R adrenocortical carcinoma cell line. Endpoints: Expression of 4 critical steroidogenic genes. Hormone production. Methods: Q-RT-PCR and ELISA

9 Cholesterol Steroidogenesis CYP11A Pregnenolone 3β-HSD2 Progesterone CYP21 11-Deoxycorticosterone CYP11B1 Corticosterone Aldosterone Zona glomerulosa 3β-HSD 17α-OH- Pregnenolone 17α-OH- Progesterone CYP21 11-Deoxycortisol Cortisol CYP11B1 Zona fasciculata DHEA 3β-HSD Androstene -dione 17β-HSD Testosterone 17β-Estradiol Zona reticularis

10 Exposure High Concentrations Compound Therapeutic Use Conc. Acetaminophen NSAA 3μg/mL Clofibrate Lipid agent 3 μg/ml Dexamethasone Corticosteroid 2 μg/l Doxycycline Antibiotic 1 μg/ml DEET Pesticide 3 μg/ml Erythromycin Antibiotic 3 μg/ml Ibuprofen NSAA 2 μg/ml Trimethoprim Antibiotic 3 μg/ml Tylosin Antibiotic/growth 3 μg/ml Environmental Concentrations Compound Therapeutic Use Conc. Amoxicillin Antibiotic 71 μg/l Cephalexin Antibiotic 73 μg/l Cyproterone Cancer treatment 62 μg/l Oxytetracyclin Antibiotic/growth prom. 81 μ/l Salbutamol Asthma/β-agonist ng/l Trenbolone Growth prom. 2 μ/l α Zearalanol Hyperestrogen/Nat occ. 2.8 ng/l Ethynil Estradiol Oral Contraceptive 1 μg/l Fluoxetine Antidepressant 1 μg/l

11 Results Antibiotics 1 1 Amoxicillin 1 Cephalexin Doxycylin Erythromycin 1 Oxytetracyclin 1 Trimetropin Tylosin 1

12 Results Hormone Therapy 7 6 Cyproterone 7 6 Dexametasone EE2 7 6 Trenbolone

13 Results Other Contaminants Acetaminophen Clofibrate Fluoxetine DEET Ibuprofen Salbutamol α-zearalanol River Sample 1 1 1

14 Results All Contaminants Pearson correlation matrix Gene/Hormone TEST PROG ESTR TEST PROG ESTR Bonferroni probabilities matrix Gene/Hormone TEST PROG ESTR TEST PROG ESTR

15 Results Antibiotics Pearson correlation matrix Gene/Hormone TEST PROG ESTR TEST PROG ESTR Bonferroni probabilities matrix Gene/Hormone TEST PROG ESTR TEST PROG ESTR

16 What is next? Measurement of Aldosterone and Androstenedione. Exposure to mixtures of pharmaceuticals. Exposure to other environmental toxicants. Exposure to environmental samples, extracts from rivers and sea water and water treatment plants.

17 OVERALL APPROACH Model chemicals Environmental toxicants Assessment of effects of Chemicals in the Expression of ten steroidogenic Genes in H29R cell line using RT-PCR Tox.Sci. 81, (24). H29R Cell exposure Quantitative RT-PCR Methods for Evaluating Toxicant-Induced Effects on Steroidogenesis Using the H29R cell line. Environ.Sci.Tech. 39: (2). Cytotoxicity assay Q-RT-PCR (1 genes) Assessment of the Effects of Chemical Mixtures on the Expression of Ten Steroidogenic Genes in the H29R Cell Line Anal.Bio.Chem. (Submitted). Responses profiles Alteration of steroidogenesis in H29R cells by organic sediment contaminants and relationships to other endocrine disrupting effects. Environ.Tox. (Submitted). Informatic analysis

18 Conclusions Other compounds different to classified EC may produce changes in gene expression and hormone production. Not always hormone production is affected by gene expression. Compounds with the same gene expression profile may present different hormone production profile. The assay is responsive to extracts from environmental samples.

19 Conclusions This analytical procedure, which allows simultaneous analysis of the expression of key steroidogenic genes and hormones, can be used as a sensitive and integrative screen system for the many effects of chemicals or mixtures that can lead to endocrine disruption.

20 Tannia Gracia National Food Safety and Toxicology Center Michigan State University East Lansing, Michigan, 48824, USA Tel: (17) x 16 Fax: (17) gracia@msu.edu