ToxProfiler: A novel human-based reporter assay for in vitro chemical safety assessment.

In vitro chemical safety assessment often relies on simple and general cytotoxicity endpoint measurements and fails to adequately predict human toxicity. To improve the in vitro chemical safety assessment, it is important to understand the underlying mechanisms of toxicity. Here we introduce ToxProfiler, a novel human-based reporter assay that quantifies the chemical-induced stress responses at a single-cell level and reveals the toxicological mode-of-action (MoA) of novel drugs and chemicals.

Mapping the dynamics of Nrf2 antioxidant and NFκB inflammatory responses by soft electrophilic chemicals in human liver cells defines the transition from adaptive to adverse responses.

A comprehensive understanding of the dynamic activation and crosstalk between different cellular stress response pathways that drive cell adversity is crucial in chemical safety assessment. Various chemicals have electrophilic properties that drive cell injury responses in particular oxidative stress signaling and inflammatory signaling. Here we used bacterial artificial chromosome-based GFP cellular stress reporters with live cell confocal imaging, to systematically monitor the differential modulation of the dynamics of stress pathway activation by six different soft electrophiles: sulforaphane, andrographolide, diethyl maleate, CDDO-Me, ethacrynic acid and tert-butyl hydroquinone.

Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene.

Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters.

In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging.

The in vitro assessment of the toxicity of volatile, oxidisable, redox-cycling compounds: phenols as an example.

Phenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it.

Squaramide-Based Supramolecular Materials Drive HepG2 Spheroid Differentiation.

A major challenge in the use of HepG2 cell culture models for drug toxicity screening is their lack of maturity in 2D culture. 3D culture in Matrigel promotes the formation of spheroids that express liver-relevant markers, yet they still lack various primary hepatocyte functions. Therefore, alternative matrices where chemical composition and materials properties are controlled to steer maturation of HepG2 spheroids remain desired.

Systematic transcriptome-based comparison of cellular adaptive stress response activation networks in hepatic stem cell-derived progeny and primary human hepatocytes.

Various adaptive cellular stress response pathways are critical in the pathophysiology of liver disease and drug-induced liver injury. Human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs) provide a promising tool to study cellular stress response pathways, but in this context there is limited insight on how HLCs compare to other in vitro liver models. Here, we systematically compared the transcriptomic profiles upon chemical activation in HLCs, hiPSC, primary human hepatocytes (PHH) and HepG2 liver cancer cells.

Comparing in vitro human liver models to in vivo human liver using RNA-Seq.

The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues.

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes.

System Microscopy of Stress Response Pathways in Cholestasis Research.

Exposure to oxidative radical species and cytokine-mediated inflammatory stress are established contributors to hepatocyte cell death during cholestasis. Cellular counter measures against those stressors are called adaptive stress response pathways. While in early stages of the disease adaptive stress pathways protect the hepatocytes, in later stages during prolonged stressed conditions they fail.

A systematic analysis of Nrf2 pathway activation dynamics during repeated xenobiotic exposure.

Oxidative stress leads to the activation of the Nuclear factor-erythroid-2-related factor 2 (Nrf2) pathway. While most studies have focused on the activation of the Nrf2 pathway after single chemical treatment, little is known about the dynamic regulation of the Nrf2 pathway in the context of repeated exposure scenarios. Here we employed single cell live imaging to quantitatively monitor the dynamics of the Nrf2 pathway during repeated exposure, making advantage of two HepG2 fluorescent protein reporter cell lines, expressing GFP tagged Nrf2 or sulfiredoxin 1 (Srxn1), a direct downstream target of Nrf2.

The association of diabetes mellitus and insulin treatment with expression of insulin-related proteins in breast tumors.

Background: The insulin receptor (INSR) and the insulin growth factor 1 receptor (IGF1R) play important roles in the etiology of both diabetes mellitus and breast cancer. We aimed to evaluate the expression of hormone and insulin-related proteins within or related to the PI3K and MAPK pathway in breast tumors of women with or without diabetes mellitus, treated with or without insulin (analogues).

Methods: Immunohistochemistry was performed on tumor tissue of 312 women with invasive breast cancer, with or without pre-existing diabetes mellitus, diagnosed in 2000-2010, who were randomly selected from a Danish breast cancer cohort.

Insulin-like growth factor 1 receptor activation promotes mammary gland tumor development by increasing glycolysis and promoting biomass production.

Background: The insulin-like growth factor 1 (IGF1) signaling axis plays a major role in tumorigenesis. In a previous experiment, we chronically treated mice with several agonists of the IGF1 receptor (IGF1R). We found that chronic treatment with insulin analogues with high affinity towards the IGF1R (IGF1 and X10) decreased the mammary gland tumor latency time in a p53WAPCre mouse model.

Treatment with insulin (analogues) and breast cancer risk in diabetics; a systematic review and meta-analysis of in vitro, animal and human evidence.

Introduction: Several studies have suggested that anti-diabetic insulin analogue treatment might increase cancer risk. The aim of this study was to review the postulated association between insulin and insulin analogue treatment and breast cancer development, and plausible mechanisms.

Method: A systematic literature search was performed on breast cell-line, animal and human studies using the key words 'insulin analogue' and 'breast neoplasia' in MEDLINE at PubMed, EMBASE, and ISI Web of Science databases.

Alternative signaling network activation through different insulin receptor family members caused by pro-mitogenic antidiabetic insulin analogues in human mammary epithelial cells.

Introduction: Insulin analogues are designed to have improved pharmacokinetic parameters compared to regular human insulin. This provides a sustained control of blood glucose levels in diabetic patients. All novel insulin analogues are tested for their mitogenic side effects, however these assays do not take into account the molecular mode of action of different insulin analogues.

Mammary gland tumor promotion by chronic administration of IGF1 and the insulin analogue AspB10 in the p53R270H/⁺WAPCre mouse model.

Introduction: Insulin analogues are structurally modified molecules with altered pharmaco-kinetic and -dynamic properties compared to regular human insulin used by diabetic patients. While these compounds are tested for undesired mitogenic effects, an epidemiological discussion is ongoing regarding an association between insulin analogue therapy and increased cancer incidence, including breast cancer. Standard in vivo rodent carcinogenesis assays do not pick up this possible increased carcinogenic potential.

Exposure to bacterial signals does not alter pea aphids' survival upon a second challenge or investment in production of winged offspring.

Pea aphids have an obligate nutritional symbiosis with the bacteria Buchneraaphidicola and frequently also harbor one or more facultative symbionts. Aphids are also susceptible to bacterial pathogen infections, and it has been suggested that aphids have a limited immune response towards such pathogen infections compared to other, more well-studied insects. However, aphids do possess at least some of the genes known to be involved in bacterial immune responses in other insects, and immune-competent hemocytes.

Impact of plasmid quality on lipoplex-mediated transfection.

This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models.

Escherichia coli K-12 pathogenicity in the pea aphid, Acyrthosiphon pisum, reveals reduced antibacterial defense in aphids.

To better understand the molecular basis underlying aphid immune tolerance to beneficial bacteria and immune defense to pathogenic bacteria, we characterized how the pea aphid Acyrthosiphon pisum responds to Escherichia coli K-12 infections. E. coli bacteria, usually cleared in the hemolymph of other insect species, were capable of growing exponentially and killing aphids within a few days.