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Event: 228

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

peroxisome proliferator activated receptor promoter demethylation

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
demethylation, PPARg promoter

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
hepatocyte

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
peroxisome proliferator activated receptor signaling pathway peroxisome proliferator-activated receptor gamma increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
LXR Activation to Liver Steatosis MolecularInitiatingEvent Marina Goumenou (send email) Not under active development
NR1I3 suppression to steatosis MolecularInitiatingEvent Michelle Angrish (send email) Under Development: Contributions and Comments Welcome
PPARG mod to adipogenesis MolecularInitiatingEvent Michelle Angrish (send email) Under Development: Contributions and Comments Welcome

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
mice Mus sp. High NCBI
human Homo sapiens Moderate NCBI
rat Rattus norvegicus Moderate NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

No help message More help

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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 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. The transactivation (stable transfection) assay provides the most applicable OECD Level 2 assay aimed at identifying the initiating event leading to adverse outcome (LeBlanc, Norris, & Kloas, 2011). Currently no internationally validated assays are available.

Key event PPARγ activation
  What is measured? Ligand Binding   Transcriptional activity        
Method/test category molecular modelling binding assay transactivation reporter gene assay     transcription factor assay  
Method/test name molecular modelling; docking Scintillation proximity binding assay luciferase reporter gene assay     PPARγ (mouse/rat) Reporter Assay Kit Electrophoretic Mobility Shift Assay (EMSA)
Test environment In silico In vitro In vitro     In vitro, ex vivo  
Test principle Computational simulation of a candidate ligand binding to a receptor, Predicts the strength of association or binding affinity. direct binding indicating the mode of action for PPARα/γ Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in PPAR functional activity.     PPARγ once activated by a ligand, the receptor binds to a promoter element in the gene for target gene and activates its transcription. The bound (activated) to DNA PPAR is measured.  
Test outcome A binding interaction between a small molecule ligand and an enzyme protein may result in activation or inhibition of the enzyme. If the protein is a receptor, ligand binding may result in agonism or antagonism Assess the ability of compounds to bind to PPARγ. Identifies the modulators of PPARγ. The changes in activity of reporter gene levels functionally linked to a PPAR-responsive element/promoter gives information about the activity of the PPAR activation.     Protein: DNA binding, DNA binding activity  
Test background Predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using, for example, scoring functions. This assay determines whether compounds interact directly with PPARγ. PPARγ COS-1cell transactivation assay (transient transfection with human or mouse PPARγ expression plasmid and pHD(x3)-Luc reporter plasmid (PPRE)3- luciferase reporter construct C2C12 Proprietary rodent cell line expressing the mouse/rat PPARγ Transcriptional activity of PPARγ can be assessed using commercially available kits like e.g. PPARγ transcription factor assay kit (Abcam, Cambridge, USA or Cayman Chemical, USA). Gene regulation and determining protein: DNA interactions are the detected by the EMSA. EMSA can be used qualitatively to identify sequence-specific DNA-binding proteins (such as transcription factors) in crude lysates and, in conjunction with mutagenesis, to identify the important binding sequences within a given genes upstream regulatory region. EMSA can also be utilized quantitatively to measure thermodynamic and kinetic parameters.
Assay type Quantitative Qualitative Quantitative Quantitative Quantitative Quantitative Quantitative
Application domain Virtual screening In vitro screening In vitro Screening, functional studies activity (reported use: agonist)   In vitro Screening functional activity (antagonist/agonist) Functional studies Functional studies
Source Research/commercial Research Research Research commercial commercial Research/commercial
Ref (Feige et al., 2007), (Kaya, Mohr, Waxman, & Vajda, 2006) (Lapinskas et al., 2005), (Wu, Gao, & Wang, 2005) (Maloney & Waxman, 1999) (Feige et al., 2007) Cayman, (Gijsbers et al. 2013) Abcam[1]  

Table 1 Summary of the chosen methods to measure the PPARγ activation.

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

PPARγ have been identified in frog (Xenopus laevis), mouse, human, rat, fish, hamster and chicken (Wahli & Desvergne, 1999).

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

1.1 Binding and activation of receptor

PPARγ ligands thiazolidinediones (Rosiglitazone, Pioglitazone, Troglitazone) (Lehmann et al. 1995), (Forman et al. 1995), (Willson et al. 2000).

Phthalates

MEHP (CAS 4376-20-9) directly binds to PPARγ (Lapinskas et al. 2005), (ToxCastTM Data)in vitro and in silico (Feige et al. 2007), (Rotman et al. 2008), (Kaya et al. 2006) and activates this receptor in transactivation assays (Maloney & Waxman 1999), (Hurst & Waxman 2003b), (Venkata et al. 2006), (ToxCastTM Data). In summary, there is experimental in vitro evidence for binding and transcriptional activation of PPARγ. DEHP (CAS 117-81-7) was not found to bind and activate PPARγ (Lapinskas et al. 2005), (Maloney & Waxman 1999). However recent studies show activation of PPARγ by DEHP(ToxCastTM Data), (Pereira-Fernandes et al. 2013). DEHP was also found to increase the levels of PPARγ in vitro (Lin et al. 2011). Notably, PPARγ is responsive to DEHP in vitro and is translocated to the nucleus (in primary Sertoli cells) (Dufour et al. 2003), (Bhattacharya et al. 2005).

Parabens

Butylparaben was not found to bind to the PPARγ (ToxCastTM Data), but activated the human PPARγ (ToxCastTM Data), (Pereira-Fernandes et al. 2013) and mPPARγ in reporter gene assay (Taxvig et al., 2012).

Phenols

Bisphenol A was not found to bind to the PPARγ (ToxCastTM Data), but activated the human PPARγ (ToxCastTM Data), (Pereira-Fernandes et al. 2013) but not mouse PPARγ (Taxvig et al., 2012) in reporter gene assay. BPA was also reported to increase PPARγ (mRNA) in ovarian granulosa cell line and human luteinized granulosa cells (Kwintkiewicz, Nishi, Yanase, & Giudice, 2010).

Organotin

Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009).

1.2 Activation of target genes

MEHP activation of endogenous PPARγ target genes was evidenced by the stimulation of PPARγ-dependent adipogenesis in the 3T3-L1 cell differentiation model (Hurst & Waxman, 2003).

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

Barak, Y., Nelson, M. C., Ong, E. S., Jones, Y. Z., Ruiz-Lozano, P., Chien, K. R., … Evans, R. M. (1999). PPAR gamma is required for placental, cardiac, and adipose tissue development. Molecular Cell, 4(4), 585–95.

Braissant, O., Foufelle, F., Scotto, C., Dauça, M., & Wahli, W. (1996). Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinology, 137(1), 354–66.

Burns, K. A., & Vanden Heuvel, J. P. (2007). Modulation of PPAR activity via phosphorylation. Biochimica et Biophysica Acta, 1771(8), 952–60. doi:10.1016/j.bbalip.2007.04.018

Fajas, L., Auboeuf, D., Raspé, E., Schoonjans, K., Lefebvre, A. M., Saladin, R., … Auwerx, J. (1997). The organization, promoter analysis, and expression of the human PPARgamma gene. The Journal of Biological Chemistry, 272(30), 18779–89.

Fajas, L., Fruchart, J.-C., & Auwerx, J. (1998). PPARγ3 mRNA: a distinct PPARγ mRNA subtype transcribed from an independent promoter. FEBS Letters, 438(1-2), 55–60. doi:10.1016/S0014-5793(98)01273-3

Feige, J. N., Gelman, L., Michalik, L., Desvergne, B., & Wahli, W. (2006). From molecular action to physiological outputs: peroxisome proliferator-activated receptors are nuclear receptors at the crossroads of key cellular functions. Progress in Lipid Research, 45(2), 120–59. doi:10.1016/j.plipres.2005.12.002

Feige, J. N., Gelman, L., Rossi, D., Zoete, V., Métivier, R., Tudor, C., … Desvergne, B. (2007). The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis. The Journal of Biological Chemistry, 282(26), 19152–66. doi:10.1074/jbc.M702724200

Gijsbers, Linda, Henriëtte D L M van Eekelen, Laura H J de Haan, Jorik M Swier, Nienke L Heijink, Samantha K Kloet, Hai-Yen Man, et al. 2013. “Induction of Peroxisome Proliferator-Activated Receptor Γ (PPARγ)-Mediated Gene Expression by Tomato (Solanum Lycopersicum L.) Extracts.” Journal of Agricultural and Food Chemistry 61 (14) (April 10): 3419–27. doi:10.1021/jf304790a.

Issemann, I., & Green, S. (1990). Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature, 347(6294), 645–650.

Kaya, T., Mohr, S. C., Waxman, D. J., & Vajda, S. (2006). Computational screening of phthalate monoesters for binding to PPARgamma. Chemical Research in Toxicology, 19(8), 999–1009. doi:10.1021/tx050301s

Lapinskas, P. J., Brown, S., Leesnitzer, L. M., Blanchard, S., Swanson, C., Cattley, R. C., & Corton, J. C. (2005). Role of PPARα in mediating the effects of phthalates and metabolites in the liver. Toxicology, 207(1), 149–163.

Le Maire, A., Grimaldi, M., Roecklin, D., Dagnino, S., Vivat-Hannah, V., Balaguer, P., & Bourguet, W. (2009). Activation of RXR-PPAR heterodimers by organotin environmental endocrine disruptors. EMBO Reports, 10(4), 367–73. doi:10.1038/embor.2009.8

LeBlanc, G., Norris, D., & Kloas, W. (2011). Detailed Review Paper State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors, (178).

Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., & Kliewer, S. A. (1995). An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-activated Receptor (PPAR ). Journal of Biological Chemistry, 270(22), 12953–12956. doi:10.1074/jbc.270.22.12953

Maloney, E. K., & Waxman, D. J. (1999). trans-Activation of PPARα and PPARγ by Structurally Diverse Environmental Chemicals. Toxicology and Applied Pharmacology, 161(2), 209–218.

Michalik, L., Zoete, V., Krey, G., Grosdidier, A., Gelman, L., Chodanowski, P., … Michielin, O. (2007). Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior. The Journal of Biological Chemistry, 282(13), 9666–77. doi:10.1074/jbc.M610523200

Morán-Salvador, E., López-Parra, M., García-Alonso, V., Titos, E., Martínez-Clemente, M., González-Périz, A., … Clària, J. (2011). Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 25(8), 2538–50. doi:10.1096/fj.10-173716

Pereira-Fernandes, A., Demaegdt, H., Vandermeiren, K., Hectors, T. L. M., Jorens, P. G., Blust, R., & Vanparys, C. (2013). Evaluation of a screening system for obesogenic compounds: screening of endocrine disrupting compounds and evaluation of the PPAR dependency of the effect. PloS One, 8(10), e77481. doi:10.1371/journal.pone.0077481

ToxCastTM Data, US Environmental Protection Agency. http://www.epa.gov/ncct/toxcast/data.html.

Vanden Heuvel, J. P. (1999). Peroxisome proliferator-activated receptors (PPARS) and carcinogenesis. Toxicological Sciences : An Official Journal of the Society of Toxicology, 47(1), 1–8.

Viswakarma, N., Jia, Y., Bai, L., Vluggens, A., Borensztajn, J., Xu, J., & Reddy, J. K. (2010). Coactivators in PPAR-Regulated Gene Expression. PPAR Research, 2010. doi:10.1155/2010/250126

Wahli, W., & Desvergne, B. (1999). Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews, 20(5), 649–88.

Wu, B., Gao, J., & Wang, M. (2005). Development of a complex scintillation proximity assay for high-throughput screening of PPARgamma modulators. Acta Pharmacologica Sinica, 26(3), 339–44. doi:10.1111/j.1745-7254.2005.00040.x

Yu, S., & Reddy, J. K. (2007). Transcription coactivators for peroxisome proliferator-activated receptors. Biochimica et Biophysica Acta, 1771(8), 936–51. doi:10.1016/j.bbalip.2007.01.008