Difference between revisions of "Event:227"
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The transactivation assay provides the most applicable OECD Level 2 assay (i.e. In vitro assays providing mechanistic data) aimed at identifying the initiating event leading to adverse outcome (LeBlanc, Norris, and Kloas 2011). Currently no internationally validated assays are available. | The transactivation assay provides the most applicable OECD Level 2 assay (i.e. In vitro assays providing mechanistic data) aimed at identifying the initiating event leading to adverse outcome (LeBlanc, Norris, and Kloas 2011). Currently no internationally validated assays are available. | ||
− | Transactivation assays are performed using transient or stably transfected cells with the PPAR expression plasmid and a reporter plasmid, correspondingly. There are also other methods that have been used to measure PPAR activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARα transcription factor assay kits. | + | Transactivation assays are performed using transient or stably transfected cells with the PPAR expression plasmid and a reporter plasmid, correspondingly. There are also other methods that have been used to measure PPAR activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARα transcription factor assay kits, for details see table 1. |
+ | |||
+ | {| {{wikitable}} border="1" style="border-collapse:collapse;font-size:75%" | ||
+ | |- | ||
+ | | | ||
+ | Biological level of organisation | ||
+ | |||
+ | | colspan="7" | | ||
+ | Molecular | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | Key event | ||
+ | |||
+ | | colspan="7" | | ||
+ | PPARα activation | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | What is measured? | ||
+ | |||
+ | | colspan="2" | | ||
+ | <center>Ligand Binding</center> | ||
+ | |||
+ | <center></center> | ||
+ | |||
+ | | colspan="5" | | ||
+ | <center>Transcriptional activity</center> | ||
+ | |||
+ | <center></center> | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | Method/test category | ||
+ | |||
+ | | | ||
+ | <center>molecular modelling</center> | ||
+ | |||
+ | | | ||
+ | <center>binding assay</center> | ||
+ | |||
+ | | colspan="3" | | ||
+ | <center></center> | ||
+ | |||
+ | <center>transactivation reporter gene assay</center> | ||
+ | |||
+ | | colspan="2" | | ||
+ | <center>transcription factor assay</center> | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | Method/test | ||
+ | |||
+ | name | ||
+ | |||
+ | | | ||
+ | molecular modelling; docking | ||
+ | |||
+ | | | ||
+ | Scintillation proximity binding assay | ||
+ | |||
+ | | colspan="3" | | ||
+ | <center></center> | ||
+ | |||
+ | <center>luciferase reporter gene assay</center> | ||
+ | |||
+ | | | ||
+ | PPARα (mouse/rat) Reporter Assay Kit | ||
+ | |||
+ | | | ||
+ | Electrophoretic Mobility Shift Assay (EMSA) | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | Test environment | ||
+ | |||
+ | | | ||
+ | <center>In silico</center> | ||
+ | |||
+ | | | ||
+ | <center>In vitro</center> | ||
+ | |||
+ | | colspan="3" | | ||
+ | <center>In vitro</center> | ||
+ | |||
+ | | colspan="2" | | ||
+ | <center>In vitro, ex vivo</center> | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | 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 | ||
+ | |||
+ | | colspan="3" | | ||
+ | Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in PPAR functional activity. | ||
+ | |||
+ | | colspan="2" | | ||
+ | PPARα 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 | ||
+ | |||
+ | | | ||
+ | Assesses the ability of compounds to bind to human PPARα. Identifies the modulators of PPARα. | ||
+ | |||
+ | | colspan="3" | | ||
+ | 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. | ||
+ | |||
+ | | colspan="2" | | ||
+ | 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 PPARs. The type of beads that are involved in the SPA are microscopic in size and within the beads itself, there is a scintillant which emits light when it is stimulated. Stimulation occurs when radio-labelled molecules interact and bind to the surface of the bead and trigger the bead to emit light. | ||
+ | |||
+ | | | ||
+ | PPARα/γ COS-1cell transactivation assay (transient transfection with human or mouse PPARα/γ expression plasmid and pHD(x3)-Luc reporter plasmid | ||
+ | |||
+ | | | ||
+ | (PPRE)3- luciferase reporter construct C2C12 | ||
+ | |||
+ | | | ||
+ | Proprietary rodent cell line expressing the mouse/rat PPARα | ||
+ | |||
+ | | | ||
+ | Transcriptional activity of PPARα can be assessed using commercially available kits like e.g. PPAR-α transcription factor assay kit. | ||
+ | |||
+ | | | ||
+ | Gene regulation and determining protein: DNA interactions are 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 gene upstream regulatory region. EMSA can also be utilized quantitatively to measure thermodynamic and kinetic parameters. | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | Assay type | ||
+ | |||
+ | | | ||
+ | <center>Quantitative</center> | ||
+ | |||
+ | | | ||
+ | <center>Qualitative </center> | ||
+ | |||
+ | | | ||
+ | <center>Quantitative</center> | ||
+ | |||
+ | | | ||
+ | <center>Quantitative</center> | ||
+ | |||
+ | | | ||
+ | <center>Quantitative</center> | ||
+ | |||
+ | | | ||
+ | Quantitative | ||
+ | |||
+ | | | ||
+ | <center>Quantitative</center> | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | Application domain | ||
+ | |||
+ | | | ||
+ | Virtual screening | ||
+ | |||
+ | | | ||
+ | In vitro screening | ||
+ | |||
+ | | | ||
+ | In vitro Screening, functional studies activity (reported use: agonist) | ||
+ | |||
+ | | | ||
+ | | ||
+ | |||
+ | | | ||
+ | In vitro Screening functional activity (antagonist/agonist) | ||
+ | |||
+ | | | ||
+ | | ||
+ | |||
+ | | | ||
+ | | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | Source | ||
+ | |||
+ | | | ||
+ | Research/ | ||
+ | |||
+ | commercial | ||
+ | |||
+ | | | ||
+ | Research | ||
+ | |||
+ | | | ||
+ | Research | ||
+ | |||
+ | | | ||
+ | Research | ||
+ | |||
+ | | | ||
+ | commercial | ||
+ | |||
+ | | | ||
+ | commercial | ||
+ | |||
+ | | | ||
+ | Research/commercial | ||
+ | |||
+ | |- | ||
+ | | | ||
+ | Ref | ||
+ | |||
+ | | | ||
+ | <center>(Feige et al. 2007), (Kaya et al. 2006)</center> | ||
+ | |||
+ | | | ||
+ | <center>(Lapinskas et al. 2005), (Wu, Gao, and Wang 2005)</center> | ||
+ | |||
+ | | | ||
+ | <center>(Maloney and Waxman 1999)</center> | ||
+ | |||
+ | | | ||
+ | <center>(Feige et al. 2007)</center> | ||
+ | |||
+ | | | ||
+ | <center>[http://indigobiosciences.com/products/ppar-products/mouse-ppar-alpha-mpparα-nr1c1/ Indigobiosciences]</center> | ||
+ | |||
+ | | | ||
+ | <center>[http://www.abcam.com/ppar-alpha-transcription-factor-assay-kit-ab133107.html Abcam] </center> | ||
+ | |||
+ | <center></center> | ||
+ | |||
+ | | | ||
+ | <center></center> | ||
+ | |||
+ | |} | ||
== Evidence Supporting Taxonomic Applicability == | == Evidence Supporting Taxonomic Applicability == |
Revision as of 16:45, 24 March 2015
Contents
Event Title
Key Event Overview
Please follow link to widget page to edit this section.
AOPs Including This Key Event
Chemical Initiators
The following are chemical initiators that operate through this AOP:
Taxonomic Applicability
Name | Scientific Name | Evidence | Links |
---|---|---|---|
rat | Rattus sp. | Strong | NCBI |
mouse | Mus musculus | Weak | NCBI |
human | Homo sapiens | Moderate | NCBI |
Level of Biological Organization
Biological Organization |
---|
Molecular |
How this Key Event works
Biological state
The Peroxisome Proliferator Activated receptor α (PPAR α) belongs to Peroxisome Proliferator Activated receptors (PPARs; NR1C) steroid/thyroid/retinoid receptor superfamily of transcription factors.
Biological compartments
PPARα is expressed in high levels in tissues that perform significant catabolism of fatty acids (FAs), such as brown adipose tissue, liver, heart, kidney, and intestine (Michalik et al., 2006). The receptor is present in also in skeletal muscle, intestine, pancreas, lung, placenta (Mukherjee et al., 1997).
General role in biology
PPARs are activated by fatty acids and their derivatives; they are sensors of dietary lipids and are involved in lipid and carbohydrate metabolism; immune response and peroxisome proliferation (Wahli and Desvergne 1999), (Evans, Barish, & Wang, 2004). PPARα is a also a target of hypothalamic hormone signalling and was found to play a role in embryonic development (Yessoufou and Wahli 2010).
Fibrates, activators of PPAR𝛼, are commonly used to treat hypertriglyceridemia and other dyslipidemic states as they have been shown to decrease circulating lipid levels (Lefebvre et al. 2006).
How it is Measured or Detected
Binding of ligands to PPARα is measured using binding assays in vitro and in silico, whereas the information about functional activation is derived from the transactivation assays (e.g. reporter assay with 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 and Reddy 2007) (Viswakarma et al. 2010). Transactivation assays are performed using transient or stably transfected cells with the PPAR expression plasmid and a reporter plasmid, correspondingly.
The transactivation assay provides the most applicable OECD Level 2 assay (i.e. In vitro assays providing mechanistic data) aimed at identifying the initiating event leading to adverse outcome (LeBlanc, Norris, and Kloas 2011). Currently no internationally validated assays are available.
Transactivation assays are performed using transient or stably transfected cells with the PPAR expression plasmid and a reporter plasmid, correspondingly. There are also other methods that have been used to measure PPAR activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARα transcription factor assay kits, for details see table 1.
Biological level of organisation |
Molecular | ||||||
Key event |
PPARα activation | ||||||
What is measured? |
|
| |||||
Method/test category |
|
|
|
| |||
Method/test name |
molecular modelling; docking |
Scintillation proximity binding assay |
|
PPARα (mouse/rat) Reporter Assay Kit |
Electrophoretic Mobility Shift Assay (EMSA) | ||
Test environment |
|
|
|
| |||
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 |
Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in PPAR functional activity. |
PPARα 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 |
Assesses the ability of compounds to bind to human PPARα. Identifies the modulators of PPARα. |
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 PPARs. The type of beads that are involved in the SPA are microscopic in size and within the beads itself, there is a scintillant which emits light when it is stimulated. Stimulation occurs when radio-labelled molecules interact and bind to the surface of the bead and trigger the bead to emit light. |
PPARα/γ COS-1cell transactivation assay (transient transfection with human or mouse PPARα/γ expression plasmid and pHD(x3)-Luc reporter plasmid |
(PPRE)3- luciferase reporter construct C2C12 |
Proprietary rodent cell line expressing the mouse/rat PPARα |
Transcriptional activity of PPARα can be assessed using commercially available kits like e.g. PPAR-α transcription factor assay kit. |
Gene regulation and determining protein: DNA interactions are 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 gene upstream regulatory region. EMSA can also be utilized quantitatively to measure thermodynamic and kinetic parameters. |
Assay type |
|
|
|
|
|
Quantitative |
|
Application domain |
Virtual screening |
In vitro screening |
In vitro Screening, functional studies activity (reported use: agonist) |
|
In vitro Screening functional activity (antagonist/agonist) |
|
|
Source |
Research/ commercial |
Research |
Research |
Research |
commercial |
commercial |
Research/commercial |
Ref |
|
|
|
|
|
|
|
Evidence Supporting Taxonomic Applicability
PPARα have been identified in frog (Xenopus laevis), mouse, identified in human, rat, fish, hamster and chicken (reviewed in (Wahli and Desvergne 1999)).
Evidence for Chemical Initiation of this Molecular Initiating Event
Phthalates
MHEP (CAS 4376-20-9) directly binds in vitro to PPARα (Lapinskas et al. 2005) and activates this receptor in transactivation assays PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999), (Hurst and Waxman 2003), (Bility et al. 2004), (Lampen, Zimnik, and Nau 2003), (Venkata et al. 2006) ]. DEHP (CAS 117-81-7) has not been found to bind and activate PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999). However, the recent studies shown activation of PPARα by DEHP (ToxCast).
Notably, PPARα are responsive to DEHP in vitro as they are translocated to the nucleus (in primary Sertoli cells) (Dufour et al. 2003), (Bhattacharya et al. 2005).
Expression of PPARα [mRNA and protein] have been also reported to be upregulated in vivo upon DEHP treatment (Xu et al. 2010).
Organotin
Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009)