Aromatase Inhibition
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Aop:7 AOP Title PPARγ activation leading to impaired fertility in adult female rodents Short name: PPAR activation leading to reproductive toxicity Authors Malgorzata Nepelska, Sharon Munn, Brigitte Landesmann Systems Toxicology Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Via E. Fermi 2749, I-21027 Ispra, Varese, Italy Corresponding author: [email protected] Status Under development: Do not distribute or cite. OECD Project 1.21: Three Adverse Outcome Pathways from Peroxisome Proliferator-Activated Receptors (PPARs) Activation Leading to Reproductive Toxicity in Rodents This AOP was last modified on 5/19/2015. Click here to show/hide revision dates for related pages Page Revision Date/Time Abstract This AOP links activation of the Peroxisome Proliferator Activated Receptorγ (PPARγ) to reproductive toxicity in adult female. The development of this AOP relies on evidence collected from rodent models and incorporates human mechanistic and epidemiological data. The PPARγ is a ligand-activated transcription factor that belongs to the nuclear receptor family, which also includes the steroid and thyroid hormone receptors. Interest in PPARγ action as a mechanistic basis for effects on the reproductive system arises from the demonstrated relationships between activation of this receptor and impairment of the steroidogenesis leading to reproductive toxicity in rodents. PPARs play important roles in the metabolic regulation of lipids, of which cholesterol, in particular being a precursor of steroid hormones, makes the link between lipid metabolism to effects on reproduction. The key events in the pathway comprise the activation of PPARγ, followed by the disruption of the hormonal balance which leads to irregularities of the ovarian cycle and further to impaired fertility. The PPARγ-initiated AOP to rodent female reproductive toxicity is a first step for structuring current knowledge about a mode of action which is neither ER-mediated nor via direct aromatase inhibition. In the current form the pathway lays a strong basis for linking an endocrine mode of action with an apical endpoint, prerequisite requirement for the identification of endocrine disrupting chemicals. This AOP is complemented with a structured data collection which will serve as the basis for further quantitative development of the pathway. Summary of the AOP Molecular Initiating Event 1 Aop:7 Molecular Initiating Event PPAR gamma, Activation PPARγ, Activation Short name: PPARγ, Activation How this Key Event works Level of Biological Organization Molecular Biological state The Peroxisome Proliferator Activated receptor γ (PPARγ) belongs to Peroxisome Proliferator Activated receptors (PPARs; NR1C) steroid/thyroid/retinoid receptor superfamily of transcription factors, which respond to specific ligands by altering gene expression in a cell-specific manner. The PPARγ gene contains three promoters that yield three isoforms, namely, PPAR-γ1, 2 and 3. PPAR-γ1 and γ3 RNA transcripts translate into the identical PPAR-γ1 protein. Biological compartments PPARγ is abundantly expressed in adipose tissue, promoting adipocyte differentiation, but is also present in various cells and tissues, for review see (Braissant et al. 1996). PPARγ expression is tissue dependent (L Fajas et al. 1997), (Lluis Fajas, Fruchart, and Auwerx 1998). PPARγ is most highly expressed in white adipose tissue and brown adipose tissue, where it is a master regulator of adipogenesis as well as a potent modulator of whole-body lipid metabolism and insulin sensitivity (Evans, Barish, and Wang 2004), (Tontonoz and Spiegelman 2008). Whereas PPARγ1 is expressed in many tissues, the expression of PPARγ2 is restricted to adipose tissue under physiological conditions but can be induced in other tissues by a high- fat diet (Saraf et al. 2012). General role in biology PPARγ is activated after the binding of natural ligands such as polyunsaturated fatty acids and prostaglandin metabolites. It can also be activated by synthetic ligands such as thiazolidinediones (TZDs) (rosiglitazone, pioglitazone or troglitazone) (Lehmann et al., 1995). PPARγ controls many vital processes such as glucose metabolism and inflammation as well as variety of developmental programs(Wahli & Desvergne, 1999), (Rotman et al., 2008), (Wahli & Michalik, 2012). This receptor itself is essential for developmental processes since targeted disruption of this gene results in embryo lethality, due in part to defective placental development, therefore modulation of PPARγ activity may impact endocrine regulated processes during development as well as later in life. How it is Measured or Detected Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? Binding of ligands to PPARγ is measured using binding assays in vitro and in silico, whereas the information about functional activation is derived from the transactivation using e.g. reporter assay with a reporter gene that demonstrates functional activation of a nuclear receptor by a specific compound. Binding of agonists within the ligand-binding site of PPARs causes a conformational change promoting binding to transcriptional 2 Aop:7 coactivators. Conversely, binding of antagonists results in a conformation that favours the binding of corepressors (Yu & Reddy, 2007) (Viswakarma et al., 2010. Transactivation assays are performed using the transient or stably transfected cells with the PPARγ expression plasmid and a reporter plasmid, correspondingly. There are also other methods that have been used to measure PPARγ activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARγ transcription factor assay kits, see Table 1. Key event PPARγ activation What is Ligand Binding Transcriptional measured? activity transactivation Method/test binding transcription reporter gene category molecular modelling assay factor assay assay PPARγ Scintillation luciferase Electrophoretic proximity (mouse/rat) Method/test molecular modelling; docking reporter gene Mobility Shift Assay Reporter name binding assay (EMSA) assay Assay Kit Test In silico In vitro In vitro In vitro, ex environment vivo PPARγ once activated by a Quantifying ligand, the changes in receptor binds luciferase to a promoter direct expression in the element in Computational simulation of binding treated reporter a candidate ligand binding to indicating the gene for Test cells provides a a receptor, Predicts the the mode target gene principle sensitive and activates strength of association or of action surrogate its binding affinity. for PPARα/ measure of the transcription. γ changes in PPAR The bound functional (activated) to activity. DNA PPAR is measured. The changes in A binding interaction Assess the activity of between a small molecule ability of reporter gene ligand and an enzyme compounds levels functionally Protein: DNA protein may result in to bind to linked to a PPAR- Test binding, DNA activation or inhibition of the PPARγ. responsive outcome binding enzyme. If the protein is a Identifies element/promoter activity receptor, ligand binding may the gives information result in agonism or modulators about the activity antagonism of PPARγ. of the PPAR activation. Gene regulation and determining protein: DNA interactions are the detected by the Transcriptional EMSA. EMSA can be activity of used qualitatively to PPARγ can be identify sequence- Predicts the preferred assessed specific DNA-binding orientation of one molecule PPARγ COS-1cell using This assay proteins (such as to a second when bound to transactivation commercially determines transcription factors) each other to form a stable assay (transient (PPRE)3- available kits whether Proprietary rodent in crude lysates and, complex. Knowledge of the transfection with luciferase like e.g. compounds cell line expressing in conjunction with Test preferred orientation in turn human or mouse reporter PPARγ interact the mouse/rat mutagenesis, to background may be used to predict the PPARγ expression construct transcription directly PPARγ identify the strength of association or plasmid and C2C12 factor assay with important binding binding affinity between two pHD(x3)-Luc kit (Abcam, PPARγ. sequences within a molecules using, for reporter plasmid Cambridge, given genes example, scoring functions. USA or upstream regulatory Cayman region. EMSA can Chemical, also be utilized USA). quantitatively to measure thermodynamic and 3 Aop:7 kinetic parameters. Assay type Quantitative Qualitative Quantitative Quantitative Quantitative Quantitative Quantitative In vitro Screening, In vitro Screening Application In vitro Functional functional studies functional activity Functional studies domain Virtual screening screening studies activity (reported (antagonist/agonist) use: agonist) Source Research/commercial Research Research Research commercial commercial Research/commercial (Lapinskas et al., (Feige et al., 2007), (Kaya, 2005), (Maloney & (Feige et Cayman, (Gijsbers Ref Mohr, Waxman, & Vajda, Abcam (Wu, Gao, Waxman, 1999) al., 2007) et al. 2013) 2006) & Wang,