ToxReporter: Cell-Based Toxicity ELISA

An innovative platform to measure toxicity levels in a variety of pathways using a single ELISA

Press Release
The Science of ToxReporter
Assay Performance Characteristics
Example: AP1 ToxReporter Assay

ToxReporter Advantages
Ordering ToxReporter Products
Links to Other Millipore Toxicity Products
Download Product Selection Guide

Historically, the most common cause of drug failures was poor PK/PD and ADME properties. Because Pharma have been able to address this specific area with new technologies, poor ADME will diminish as a reason for drug failure in the coming years, although lack of efficacy and commercial reasons will always continue to be an issue. Consequently, now and in the future the areas of major concern are toxicity and adverse clinical effects.

These are areas where new solutions are required – solutions that allow the identification of “bad” compounds and either “fail” these drugs as soon as practically possible in the drug discovery continuum or be aware of potential risks and "flag" compounds as they move down the pipeline. Identifying potential toxicity issues earlier will save significant amounts of money, as well as lives and the avoidance of serious side effects.

Millipore, in collaboration with CXR Biosciences, is pleased to offer ToxReporter: nine unique cell-based toxicity assays that address the in vitro market segment and allow the analysis of many of the common cellular events that lead to compound failure in the clinic or during clinical development, including:

	Oxidative stress Cellular stress DNA damage Immunosuppression Heavy metal toxicity

Using the nine cell lines and toxicity ELISA developed by Millipore, it is now possible to identify a potential toxicity issue early in the drug development process and then include that assay in on-going SAR studies. This will result in a better understanding of both the structural features that drive toxicity pathways and the features driving potency and selectivity.

The Science of ToxReporter

Virtually all toxic responses are preceded by the transcriptional activation of stress response pathways with many therapeutic responses involving downstream transcriptional events. Consequently, the promoters of genes activated in this manner have the potential to be used as early biomarkers of toxicity response.

ToxReporter was designed to provide in vitro cell lines in which a series of promoters were used to drive the expression of a common reporter molecule. This reporter molecule is easily detected in blood, urine and tissue culture medium using an ELISA. ToxReporter cell lines allow the detection of early biomarkers of toxicity and cellular stress. Examples of stressful stimuli include compounds in drug discovery and organic or inorganic molecules potentially requiring environmental monitoring.

ToxReporter currently consists of five cell lines containing unique reporter gene constructs. Each contains toxicological pathway specific response elements downstream of a common reporter biomarker, as shown above.

AP-1 ToxReporter Cell Line: stress response induced by toxic agents leads to the dimerization of c-fos and c-jun (AP-1 complex) that leads to the activation the AP-1 promoter
GRE ToxReporter Cell Line: stress response induced by toxic agents leads to the activation of the GRE promoter
HSP70 ToxReporter Cell Line: stress response induced by toxic agents leads to the activation of the HSP70 promoter
NFκB ToxReporter Cell Line: stress response induced by toxic agents leads to the activation and translocation of NFkB from cytoplasm to the nucleus, leading to the activation the NFkB promoter
p21^waf1 ToxReporter Cell Line: stress response induced by toxic agents leads to the activation of the p21 promoter
ARE ToxReporter Cell Line: stress response induced by toxic agents leads to the activation of nuclear factor erythoid 2-related factor (Nf2) which binds and activates the ARE promoter
Hmox1 ToxReporter Cell Line: stress response induced by toxic agents leads to the activation of various transcription factors which then bind and activate the Hmox1 promoter
HRE ToxReporter Cell Line: stress response induced by toxic agents leads to the activation of hypoxia-induced factor 1 (HIF-1) which then binds and activates the HRE promoter
XRE ToxReporter Cell Line: stress response induced by toxic agents leads to the activation of aryl hydrocarbon receptor (AH) which then binds and activates the XRE promoter

Specific Response Element Potential Toxicity Mechanism Comment

AP-1
Oxidative stress, DNA damage and Apoptosis Activator Protein-1 is a redox sensitive transcription factor associated with oxidative stress, DNA damage and apoptosis. Effect is down regulated by activated GR (Glucocorticoid receptor).

GRE
Immunosuppressant Glucocorticoid Response Element regulates immunosuppressive and anti-inflammatory activities in multiple physiological systems.

HSP70
Cellular stress, Heavy metal toxicity Heat Shock Protein 70 is a chaperone protein associated with heat shock, metal toxicity and cellular stress responses.

NFκB
Oxidative stress, DNA damage A redox sensitive transcription factor associated with oxidative stress, DNA damage and apoptosis. Effect is down regulated by activated GR (Glucocorticoid receptor).

p21^waf1
DNA damage p21^waf1 is associated with DNA damage

ARE
Oxidative stress Anti-Oxidant Response Element leads to the expression of a number of phase II detoxification genes in response to electrophilic compounds or oxidants.

Hmox1
Oxidative stress Hemoxygenase I associated with oxidative stress response.

HRE
Mitochondrial DNA damage Hypoxia Response Element associated with hypoxic stress, DNA damage, especially mitochondrial.

XRE
Induction of CYP450s Xenobiotic Response Element is associated with the AR receptor (Aryl hydrocarbon Receptor) which induces the expression of cytochrome P450s (1A1)

Specific Response Element	Potential Toxicity Mechanism	Comment
AP-1	Oxidative stress, DNA damage and Apoptosis	Activator Protein-1 is a redox sensitive transcription factor associated with oxidative stress, DNA damage and apoptosis. Effect is down regulated by activated GR (Glucocorticoid receptor).
GRE	Immunosuppressant	Glucocorticoid Response Element regulates immunosuppressive and anti-inflammatory activities in multiple physiological systems.
HSP70	Cellular stress, Heavy metal toxicity	Heat Shock Protein 70 is a chaperone protein associated with heat shock, metal toxicity and cellular stress responses.
NFκB	Oxidative stress, DNA damage	A redox sensitive transcription factor associated with oxidative stress, DNA damage and apoptosis. Effect is down regulated by activated GR (Glucocorticoid receptor).
p21^waf1	DNA damage	p21^waf1 is associated with DNA damage
ARE	Oxidative stress	Anti-Oxidant Response Element leads to the expression of a number of phase II detoxification genes in response to electrophilic compounds or oxidants.
Hmox1	Oxidative stress	Hemoxygenase I associated with oxidative stress response.
HRE	Mitochondrial DNA damage	Hypoxia Response Element associated with hypoxic stress, DNA damage, especially mitochondrial.
XRE	Induction of CYP450s	Xenobiotic Response Element is associated with the AR receptor (Aryl hydrocarbon Receptor) which induces the expression of cytochrome P450s (1A1)

Assay Performance Characteristics

EXAMPLE: AP1 ToxReporter Assay

Z'-Factor
Untreated (n=16) and TPA treated (n=16) cells were assayed for ToxReporter levels using ToxReporter ELISA kit. This is representative of one independent experiment.

Specific Response Element Stimulant
(Positive Control) Number Treated Number Untreated Z'-Factor

AP-1
TPA
16
16
0.6

GRE
Dexamethasone
16
16
0.7

HSP70
CuSO₄
16
16
0.6

NFκB
TNFα
16
16
0.6

p21^waf-1
Etoposide
16
16
0.6

ARE
TBHQ
24
24
0.5

Hmox1
Hemin
24
24
0.5

HRE
COCL₂
24
24
0.6

XRE
3-MC
24
24
0.5

Specific Response Element	Stimulant (Positive Control)	Number Treated	Number Untreated	Z'-Factor
AP-1	TPA	16	16	0.6
GRE	Dexamethasone	16	16	0.7
HSP70	CuSO₄	16	16	0.6
NFκB	TNFα	16	16	0.6
p21^waf-1	Etoposide	16	16	0.6
ARE	TBHQ	24	24	0.5
Hmox1	Hemin	24	24	0.5
HRE	COCL₂	24	24	0.6
XRE	3-MC	24	24	0.5

Dose Response Curves

ToxReporter cells were cultured on a 96-well microtiter plate as quadruplicate samples for 2 days. Cells were stimulated with the appropriate dilutions of stimulant for 24 hours. ToxReporter levels were assayed using ToxReporter ELISA kit. For EC₅₀ calculations, non-linear best-fit dose response was used (GraphPad PRISM 5.0 software).

AP-1 Dose Response

EC₅₀ for TPA = 1.5 nM

Time Course Curves for Dose Response

ToxReporter cells were cultured on a 96-well microtiter plate as quadruplicate samples for 2 days. Cells were treated with appropriate dilutions of stimulant for 8, 16 and 24 hours. At each time point, 5 mL of media from each well was removed and frozen at -20 °C. ToxReporter levels were assayed when samples from all time points were collected. The asterisk represents the optimal time and stimulant concentration.

AP-1 Time Course

ToxReporter Advantages

Advantages of Millipore’s ToxReporter technology include:

A quantitative vs. a qualitative assay: the ELISA can be used to generate a standard curve while other cell-based toxicity assays can only establish a ratio
Flexibility of experimental design: In one assay (ELISA) you can test supernatants from more than one cell line or test one cell line with various compounds – and at various time points
A stable reporter biomarker: you can collect, freeze and store samples to run later on one ELISA plate
The ability to collect 1 – 10 µL samples across multiple time points from a single treated well
The only instrument needed is a plate reader capable of reading ELISA plates
A greatly diminished risk of test compound fluorescence being an issue

Ordering ToxReporter Products

» View ToxReporter in the Product Catalogue

Consult the following chart when ordering the ToxReporter products you need to run your assays. Click to enlarge.

Other Millipore Toxicity Products

Download our
product selection guide:
ToxReporter ELISA: Cell-based Toxicity

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