The cell stress panel is a combination of endpoints that delivers a toxicity profile for novel therapeutics providing evidence of the toxicological mode of action. It consists of 36 biomarkers that represent cellular stress signalling pathways, organelle health and cellular cytotoxicity.
Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.
Next Generation Risk Assessment (NGRA) is defined as an exposure-led, hypothesis driven risk assessment approach that integrates in silico, in chemico and in vitro approaches.
1Dent M et al., (2018) Comput toxicol 7; 20-26
|Cell Line||HepG2 (human hepatoblastoma) cell line*|
|Analysis Platform||Confocal Cellomics ArrayScan® (Thermo Scientific)
Seahorse XFe96 extracellular flux analyser
Intellicyt iQue® Screener PLUS.
|Test Compound Concentrations||8 point dose response curve with top concentration based on 100x Cmax or solubility limit. 3 replicates per concentration.*|
|Compound Requirements||200 μL of a solution to achieve 100x Cmax (200 x top concentration to maintain 0.5% DMSO) or equivalent amount in solid compound.|
|Time Points||24 hour*|
|Quality Controls||Negative control: 0.5% DMSO (vehicle)
Positive control: 2 compounds per mechanism
|Data Delivery||Dose response curve, MEC and AC50 value for each measured parameter.
Cell count, nuclear size, DNA structure, ER stress panel 1 (ER integrity, BiP, XBP1), ER stress panel 2 (ATF4, PERK, CHOP), AhR translocation, DNA damage (pH2AX, p53), inflammation & pH (ICAM-1, intracellular pH, HIF1α, IL-1β, IL-6, IL-8, IFN-γ), mitochondrial oxidative stress (mitochondrial ROS, PGC1α, TNFAIP3), oxidative stress (NRF2, HMOX1, SRXN1), osmotic & heat shock (NFAT, hsp70), metal stress (metallothionein, MTF1), apoptosis & necrosis (caspase 3/7, NFkB, cell membrane permeability), phospholipidosis & steatosis, glutathione (GSH) content, reactive oxygen species (ROS), mitochondrial membrane potential, mitochondrial mass, cellular ATP & LDH release, Seahorse assay (oxygen consumption rate (OCR), extracellular acidification rate (ECAR), reserve capacity)*
* other options available on request.
Cell stress panel overview summarising the 12 cell stress mechanisms and their associated molecular markers detected within the cell stress panel assay.
Metallothionein protein is regulated by MTF1 in order to bind and sequester toxic heavy metals, therefore an increase in this molecular marker indicates metal stress (figure 6a & 6b).
Hatherell et al (2020) describe the initial validation of the cell stress panel whereby this predominately high content imaging (HCI) strategy has the potential to improve our understanding of chemical exposure outcomes using point of departure (PoD) in relation to Cmax with a set of 13 benchmark substances5.
In combination with other cellular assays and in silico approaches this panel could provide a powerful NGRA tool to use in non-animal safety decision making.
1 Dent M et al. (2018) Principles underpinning the use of new methodologies in the risk assessment of cosmetic ingredients. Comput Toxicol 7: 20-26
2 Middleton A et al (2017) Case studies in cellular stress: defining adversity/adaptation tipping points. Appl In Vitro Toxicol 3(2): 199-210
3 Campbell JL et al. (2012) Physiologically based pharmacokinetic/toxicokinetic modeling. Methods Mol Biol 929: 439-499
4 Moxon TE et al. (2020) Application of phisiologically based kinetic (PBK) modelling in the next generation risk assessment of dermally applied consumer products. Toxicol In Vitro 63: 104746
5 Hatherell S et al. (2020) Identifying and characterizing stress pathways of concern for customer safety in next generation risk assessment. Toxicol Sci 10.1093/toxsci/kfaa054