Metabolic Depletion of Atp by Fructose Inversely Controls Cd95- and Tumor Necrosis Factor Receptor 1–Mediated Hepatic Apoptosis

Hepatocyte apoptosis is crucial in several forms of liver disease. Here, we examined in different models of murine liver injury whether and how metabolically induced alterations of hepatocyte ATP levels control receptor-mediated apoptosis. ATP was depleted either in primary hepatocytes or in vivo by various phosphate-trapping carbohydrates such as fructose. After the activation of the tumor necrosis factor (TNF) receptor or CD95, the extent of hepatocyte apoptosis and liver damage was quantified. TNF-induced cell death was completely blocked in ATP-depleted hepatocyte cultures, whereas apoptosis mediated by CD95 was enhanced. Similarly, acute TNF-induced liver injury in mice was entirely inhibited by ATP depletion with ketohexoses, whereas CD95-mediated hepatotoxicity was enhanced. ATP depletion prevented mitochondrial cytochrome c release, loss of mitochondrial membrane potential, activation of type II caspases, DNA fragmentation, and cell lysis after exposure to TNF. The extent of apoptosis inhibition correlated with the severity of ATP depletion, and TNF-induced apoptosis was restored when ATP was repleted by increasing the extracellular phosphate concentration. Our study demonstrates that TNF-induced hepatic apoptosis can be selectively and reversibly blocked upstream of mitochondrial dysfunction by ketohexose-mediated ATP depletion

Chemical Concentrations in Cell Culture Compartments (C⁵) – Concentration Definitions

Some laboratory issues are taken for granted as they seem to be simple and not worth much thought. This applies to "concentrations of a chemical tested for bioactivity/toxicity". Can there be any issue about weighing a compound, diluting it in culture medium and calculating the final mass (or particle number)-to-volume ratio? We discuss here some basic concepts about concentrations and their units, addressing also differences between "dose" and "concentration". The problem of calculated nominal concentrations not necessarily corresponding to local concentrations (relevant for biological effects of a chemical) is highlighted. We present and exemplify different concentration measures, for instance those relying on weight, volume, or particle number of the test compound in a given volume; we also include normalizations to the mass, protein content, or cell number of the reference system. Interconversion is discussed as a major, often unresolved, issue. We put this into the context of the overall objective of defining concentrations, i.e., the determination of threshold values of bioactivity (e.g., an EC50). As standard approach for data display, the negative decadic logarithm of the molar concentrations (-log(M)) is recommended here, but arguments are also presented for exceptions from such a rule. These basic definitions are meant as a foundation for follow-up articles that examine the concepts of nominal, free, and intracellular concentrations to provide guidance on how to relate in vitro concentrations to in vivo doses by in vitro-to-in vivo extrapolation (IVIVE) in order to advance the use of new approach methods (NAM) in regulatory decision making.publishe

The sensitivity of the zebrafish embryo coiling assay for the detection of neurotoxicity by compounds with diverse modes of action

Open Access via the Springer Agreement Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 681002 (EU-ToxRisk).Peer reviewedPublisher PD

Botulinum neurotoxin C initiates two different programs for neurite degeneration and neuronal apoptosis

Clostridial neurotoxins are bacterial endopeptidases that cleave the major SNARE proteins in peripheral motorneurons. Here, we show that disruption of synaptic architecture by botulinum neurotoxin C1 (BoNT/C) in central nervous system neurons activates distinct neurodegenerative programs in the axo-dendritic network and in the cell bodies. Neurites degenerate at an early stage by an active caspase-independent fragmentation characterized by segregation of energy competent mitochondria. Later, the cell body mitochondria release cytochrome c, which is followed by caspase activation, apoptotic nuclear condensation, loss of membrane potential, and, finally, cell swelling and lysis. Recognition and scavenging of dying processes by glia also precede the removal of apoptotic cell bodies, in line with a temporal and spatial segregation of different degenerative processes. Our results suggest that, in response to widespread synaptic damage, neurons first dismantle their connections and finally undergo apoptosis, when their spatial relationships are lost

Cathepsin B Acts as a Dominant Execution Protease in Tumor Cell Apoptosis Induced by Tumor Necrosis Factor

Death receptors can trigger cell demise dependent or independent of caspases. In WEHI-S fibrosarcoma cells, tumor necrosis factor (TNF) induced an increase in cytosolic cathepsin B activity followed by death with apoptotic features. Surprisingly, this process was enhanced by low, but effectively inhibiting, concentrations of pan-caspase inhibitors. Contrary to caspase inhibitors, a panel of pharmacological cathepsin B inhibitors, the endogenous cathepsin inhibitor cystatin A as well as antisense-mediated depletion of cathepsin B rescued WEHI-S cells from apoptosis triggered by TNF or TNF-related apoptosis-inducing ligand. Thus, cathepsin B can take over the role of the dominant execution protease in death receptor-induced apoptosis. The conservation of this alternative execution pathway was further examined in other tumor cell lines. Here, cathepsin B acted as an essential downstream mediator of TNF-triggered and caspase-initiated apoptosis cascade, whereas apoptosis of primary cells was only minimally dependent on cathepsin B. These data imply that cathepsin B, which is commonly overexpressed in human primary tumors, may have two opposing roles in malignancy, reducing it by its proapoptotic features and enhancing it by its known facilitation of invasion

Markers of Murine Embryonic and Neural Stem Cells, Neurons and Astrocytes: Reference Points for Developmental Neurotoxicity Testing

Developmental neurotoxicity (DNT) is a serious concern for environmental chemicals, as well as for food and drug constituents. Animal-based DNT models have relatively low sensitivity, and they are limited by high work-load, cost and animal ethics. Murine embryonic stem cells (mESC) recapitulate several critical processes involved in the development of the nervous system if they are induced to differentiate into neural cells. They therefore represent an alternative toxicological model to predict human hazard. In this review, we discuss how mESC can be used for DNT assays. We have compiled a list of mRNA markers that define undifferentiated mESC (n = 42); neural stem cells (n = 73), astrocytes (n = 25) and the pattern of different neuronal and non-neuronal cell types generated (n = 57). We propose that transcriptional profiling can be used as a sensitive endpoint in toxicity assays to distinguish neural differentiation states during normal and disturbed development. Importantly, we believe that it can be scaled up to relatively high throughput whilst still providing rich information on disturbances affecting small cell subpopulations. Moreover, this approach can provide insight into underlying mechanisms and pathways of toxicity. We broadly discuss the methodological basis of marker lists and DNT assay design. The discussion is put in the context of a new generation of alternative assays (embryonic stem cell based DNT testing = ESDNT V2.0), that may later include human induced pluripotent stem cells, and that are not designed for 1:1 replacement of animal experiments, but are rather intended to improve human risk assessment by using independent scientific principles.JRC.I.2-Validation of Alternative Method

Kinetic modeling of stem cell transcriptome dynamics to identify regulatory modules of normal and disturbed neuroectodermal differentiation

Thousands of transcriptome data sets are available, but approaches for their use in dynamic cell response modelling are few, especially for processes affected simultaneously by two orthogonal influencing variables. We approached this problem for neuroepithelial development of human pluripotent stem cells (differentiation variable), in the presence or absence of valproic acid (signaling variable). Using few basic assumptions (sequential differentiation states of cells; discrete on/off states for individual genes in these states), and time-resolved transcriptome data, a comprehensive model of spontaneous and perturbed gene expression dynamics was developed. The model made reliable predictions (average correlation of 0.85 between predicted and subsequently tested expression values). Even regulations predicted to be non-monotonic were successfully validated by PCR in new sets of experiments. Transient patterns of gene regulation were identified from model predictions. They pointed towards activation of Wnt signaling as a candidate pathway leading to a redirection of differentiation away from neuroepithelial cells towards neural crest. Intervention experiments, using a Wnt/beta-catenin antagonist, led to a phenotypic rescue of this disturbed differentiation. Thus, our broadly applicable model allows the analysis of transcriptome changes in complex time/perturbation matrices

The Biological and Ethical Basis of the Use of Human Embryonic Stem Cells for In Vitro Test Systems or Cell Therapy

Human embryonic stem cells (hESC) are now routinely cultured in many laboratories, and differentiation protocols are available to generate a large variety of cell types. In an ongoing ethical debate opinions of different groups are based on varying sets of religious, historical, cultural and scientific arguments as well as on widely differing levels of general information. We here give an overview of the biological background for non-specialists, and address all issues of the current stem cell debate that are of concern in different cultures and states. Thirty-five chapters address embryo definition, potential killing and the beginning of human life, in addition to matters of human dignity, patenting, commercialisation, and potential alternatives for the future, such as induced pluripotent (reprogrammed) stem cells. All arguments are compiled in a synopsis, and compromise solutions, e.g. for the definition of the beginning of personhood and for assigning dignity to embryos, are suggested. Until recently, the major application of hESC was thought to be transplantation of cells derived from hESC for therapeutic use. We discuss here that the most likely immediate uses will rather be in vitro test systems and disease models. Major and minor pharmaceutical companies have entered this field, and the European Union is sponsoring academic research into hESC-based innovative test systems. This development is supported by new testing strategies in Europe and the USA focussing on human cell-based in vitro systems for safety evaluations, and shifting the focus of toxicology away from classical animal experiments towards a more mechanistic understanding.JRC.I.3-In-vitro method