Chemical exposure can lead to cell injury that can lead to cell death and disease. Often the cell injury initiates various cell stress response signalling pathways. Strong activation of these pathways reflects the onset of toxicity. ToxProfiler can be applied to accurately quantify the chemically induced stress pathways to reveal the toxicological mode-of-action of novel medicines, (agro)chemicals, cosmetics and food ingredients. Automated live-cell confocal microscopy and image segmentation pipelines are established in this assay to generate a fingerprint of the cellular stress responses that are activated upon exposure of the cells to a compound.
Unique biomarkers to generate a toxicity profile
ToxProfiler consists of 7 stable genetically engineered human liver HepG2 cell lines. Each cell line contains a fluorescent reporter that is associated with a specific cellular stress response signal transduction pathway as detailed below.
Oxidative stress (SRXN1-GFP)
Oxidative stress is the imbalance between the reactive oxygen species that are produced and the cell’s ability to neutralise these formed reactive intermediates. Chemicals can interfere with the ROS balance which might eventually lead to several pathologies like cancer, heart disease, drug-induced liver injury (DILI) or Parkinson’s disease. SRXN1 is an antioxidant produced by the cell to counteract oxidative stress and serves as a reliable biomarker for this stress response.
Cell cycle stress (P21-GFP)
Cell cycle stress can be caused by various toxicological events, including DNA damage, microtubule disruption and inhibition of various cell cycle kinases. Cell cycle disruption will impact cell proliferation and can cause genome instability and apoptosis. The p53 tumour suppressor plays a pivotal role in cell cycle control. Cell cycle inhibition is detected in ToxProfiler by measuring the nuclear expression of the p53-associated p21-GFP reporter.
ER stress (CHOP-GFP)
Endoplasmic reticulum (ER) stress occurs after disjunction of the ER, a cellular organelle that is critical for the protein folding and secretion, calcium homeostasis and lipid biosynthesis. Chemicals can interfere with proper ER function and by doing so cause several pathologies like drug-induced liver injury (DILI), cancer, ischemia or insulin resistance. CHOP is a well-known PERK/ATF4 dependent pro-apoptotic master regulator and biomarker of ER stress.
Autophagy is a natural cellular mechanism that degrades redundant or defective macromolecules. This process allows for the regulated degradation and recycling of cellular components. Autophagy is often induced after starvation, hypoxia or (chemically induced) cell damage. LC3 is a key player in the autophagy process. The number of LC3 positive autophagosomes are indicative of the activation of the autophagy process.
Ion stress (MT1X-GFP)
Ion stress is the imbalance of the cellular ion homeostasis. Both physiological as well as xenobiotic ions can disrupt this balance. Ions (heavy metals) can bind to oxygen, nitrogen and sulfhydryl groups in proteins. This can alter enzymatic activity and increase the ROS or RNS stress, resulting in a variety of pathologies. MT1X is a metallothionein which is strongly upregulated upon ion stress and capable of binding ions and detoxify them.
Protein stress (HSPA1B-GFP)
Protein stress is the imbalance of protein homeostasis, a condition that is broader then unfolded protein response/ER stress. Protein stress is induced after increased temperatures, oxidative stress, ER stress and heavy metal stress. Unfolded or misfolded proteins can cause several pathologies like cancer, drug-induced liver injury (DILI) and several neurogenerative diseases. HSPA1B is a chaperone involved in protein folding and strongly induced after protein stress.
Inflammation is a protective response to harmful stimuli such as pathogens, damaged cells or irritants. Chemicals can affect this process directly, causing the irritation or damage or by inhibiting the inflammation response. Both can lead to a variety of pathologies like drug-induced liver injury (DILI), atherosclerosis, fever. ICAM1 is a protein involved in leucocyte requirement/binding and is induced after cytokine-induced inflammation responses.