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 &nbsp;
 +
 
 +
|-
 +
|
 +
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)
 +
 
 +
|
 +
&nbsp;
 +
 
 +
|
 +
In vitro Screening functional activity (antagonist/agonist)
 +
 
 +
|
 +
&nbsp;
 +
 
 +
|
 +
&nbsp;
 +
 
 +
|-
 +
|
 +
Source
 +
 
 +
|
 +
Research/
 +
 
 +
commercial
 +
 
 +
|
 +
&nbsp;Research
 +
 
 +
|
 +
Research
 +
 
 +
|
 +
Research
 +
 
 +
|
 +
commercial
 +
 
 +
|
 +
&nbsp;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



Event Title

PPARα, Activation
Short name: PPARα, Activation

Key Event Overview

Please follow link to widget page to edit this section.

AOPs Including This Key Event

AOP Name Event Type Essentiality
PPAR alpha activation leading to decreased fertility upon utero exposure in rodent males MIE Weak
PPARα activation leading to impaired fertility in adult male rodents MIE Weak
NFE2L2/FXR activation leading to hepatic steatosis KE

Chemical Initiators

The following are chemical initiators that operate through this AOP:

  1. Di(2-ethylhexyl) phthalate
  2. Mono(2-ethylhexyl) phthalate


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?

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

PPARα (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

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
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)

 

 

Source

Research/

commercial

 Research

Research

Research

commercial

 commercial

Research/commercial

Ref

(Feige et al. 2007), (Kaya et al. 2006)
(Lapinskas et al. 2005), (Wu, Gao, and Wang 2005)
(Maloney and Waxman 1999)
(Feige et al. 2007)
Indigobiosciences
Abcam

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)

References