L10 In Vitro Toxicity Testing PDF 2024

6/7/2024 L10 In vitro toxicity testing Timo Hamers 1 Toxicity testing in general  What type of information is requested? DESCRIPTIVE In vitro...

6/7/2024 L10 In vitro toxicity testing Timo Hamers 1 Toxicity testing in general  What type of information is requested? DESCRIPTIVE In vitro > WHAT are the effects of a compound at what dose? > In other words: what is the HAZARD? MECHANISTIC In vitro > HOW does the compound cause this type of toxicity? > From mechanism of action (molecular) to mode of action (biochemical, physiological, individual)  For what purpose is the information used? To make a PROGNOSIS In vitro > If I have this dose/concentration, what is the risk? > Threshold values can be derived To make a DIAGNOSIS In vitro > Is my sample toxic or not? > How polluted or clean is my sample? 2 1 2  6/7/2024 Set-up of this lecture  Protein-based bioassays Binding assays Enzyme inhibition assays  Cell-based techniques Primary cells Cell line Immortalized cell line Reporter gene assays Differentiation models Complex models 3 3 Protein based bioassays  Source of protein Isolated from organism Recombinant (expressed in transformed bacteria or transfected cells)  Two types of assays will be addressed: Binding assays > Ligand binding assays – Radiolabeled ligand (RLBA) – Fluorescent-labeled ligand (FLBA) Enzyme inhibition assay > Measure reaction rate – Decrease in substrate – Increase in product 4 2 4  6/7/2024 Estrogen receptor (ER) transduction pathway OH (xeno-)estrogens 17ß-estradiol CELL OH HO NUCLEUS 17ß-ethynyl-estradiol ERE gene Binding to HO Receptors mRNA bisphenol-A HO OH 4-nonylphenol OH Protein O O di(2-ethylhexyl)phthalate O O Feminizing effects 5 5 Estrogen receptor (ER) binding assay Competitive radioligand binding assay Murk et al. 2002 6 3 6  6/7/2024 Principle of acetylcholinesterase inhibitors neurotransmission acetylcholine acetylcholinesterase (ACh) (AChE) neuron 1 neuron 2 + ACh + AChE + Inhibitor + AChE 7 7 Principle of Ellman’s AChE inhibition assay + +DTNB ATCh + AChE A + TCh + AChE disulfide + TNB O O + N + AChE N + + AChE S OH + HS acetylthiocholine acetate thiocholine HOOC COOH + O2N S S NO 2 HOOC DTNB O2N S COOH S + + N HS NO 2 disulfide TNB Ellman et al. 1961 8 4 8  6/7/2024 Set-up of this lecture  Protein-based bioassays Binding assays Enzyme inhibition assays  Cell-based techniques Primary cells Cell line Immortalized cell line Reporter gene assays Differentiation models Complex models 9 9 Cell based bioassays From thermofischer.com 10 5 10  6/7/2024 Primary cell culture Proteases limited, cell-type specific number of cell doublings http://www.cloud-clone.com/topic/Primary-cell-culture.html 11 11 Cell lines passaging passaging senescence finite number subcultures 12 6 12  6/7/2024 Immortal cell line = Continuous cell line “transformation” viral or chemical passaging induced mutation passaging “Immortal” Infinite number of passages subcultures tumor tissue 13 13 Examples of immortal cell lines  Liver HepG2 (human) H4IIE (rat)  Lung A549 (human)  Fibroblast 3T3 (mouse)  Kidney HEK 293 (human embryo) MDCK (dog)  Breast MCF7 (human) T47D (rat)  Cervix HeLa (human) Henrietta Lacks; 1951 14 7 14  6/7/2024 Examples of cell-based toxicity assays  Cell viability (cytotoxicity) Mitochondrial functioning, membrane leakage, energy levels  Cell growth  Uptake, biotransformation, elimination  Cell metabolism  Cell-type dependent functioning  Omics Transcriptomics: gene expression (mRNA) Proteomics: protein profile Metabolomics: products and intermediates of metabolism (L14)  Reporter gene assays  Differentiation 15 15 Reporter gene assay reporter-gene Receptor responsive gene reporter gene mRNA Receptor Corresponding ligand mRNA Corresponding Reporter protein protein Easy read-out, e.g. reporter protein Is fluorescent Converts substrate into colored product Converts substrate into luminescent product See also L16 Effect-based monitoring 16 8 16  6/7/2024 Use of differentiation models In toxicology  To study effects on cell differentiation as such Developmental toxicity  To “create” differentiated cells in vitro for further toxicity testing 17 17 Stem cell differentiation https://www.yourgenome.org/facts/what-is-a-stem-cell 18 9 18  6/7/2024 Stem cell differentiation – Cell potency Berdasco & Esteller, 2011 19 19 Stem cell differentiation – Cell potency  Cell potency is a continuum Totipotent: present in fertilized egg (zygote) Pluripotent cells derived from human blastocyst (pre- implementation embryo): cells can give rise to any type of cell, except extra-embryonic cells (e.g. placenta) Multipotent cells: germ layers endoderm, mesoderm, ectoderm  Stem cells can divide indefinitely while remaining undifferentiated  Sources of human stem cells Pluripotent from amniotic fluid? Multipotent from adults, umbilical cord blood, bone marrow, etc. Human embryos (ethical issues) 21 10 21  6/7/2024 Embryonic stem cell differentiation model  Embryonic stem cell line  Not an immortalized cell line No mutation!  Not a primary cell culture Can divide indefinitely! Inner cell mass  Human Embryonic Stem Cells (hESC) Raises ethical issues NL allows use of > Already establish hESC lines > Use of “surplus embryos” from IVF to establish new hESC lines Landry & Zucker, 2004 22 22 Reprogramming mature, differentiated cells into pluripotent stem cells https://www.bellavista.ngo/en/2017/08/01/nobel-prize-goes-to-stem-cell-pioneers- john-b-gurdon-and-shinya-yamanaka/ 23 11 23  6/7/2024 Induced pluripotent stem cell (iPSC) differentiation https://beyondthedish.wordpress.com/2015/08/08/new-york-stem-cell-foundation-invents- robotic-platform-for-making-induced-pluripotent-stem-cells// 24 24 Use of differentiation models In toxicology  To study effects on cell differentiation as such Developmental toxicity  To “create” differentiated cells in vitro for further toxicity testing In medicine  To create “disease in a dish” models To develop new drugs To study the disease  Cell replacement therapy, using Genetically engineered stem cells from the patient Healthy stem cells from a donor 25 12 25  6/7/2024 Use of iPSC in medicine https://www.singularityweblog.com/stem-cells-ips-cells/ 26 26 Transdifferentiation models  Transdifferentation (lineage reprogramming) Mature somatic cell transforms into another mature somatic cell type without undergoing an intermediate pluripotent state or progenitor cell type  Direct reprogramming of somatic cells vs. direct programming of pluripotent stem cells  Fibroblasts (produce collagen and extracellular matrix, structural framework in animal tissue, wound healing, most common cell type in connective tissue) 29 13 29  6/7/2024 3T3 transdifferentation of fibroblast into adipocyte Pre-adipocyte Phase Lipid Stain Contrast Day 0 Adipogenic cocktail Undifferentiated Cells Differentiated DMSO Day 8 Differentiated Mature Adipocyte TZD or EDC 30 30 (Quantitative) in vitro – in vivo extrapolation (Q)IVIVE  Trend in toxicology to use less animal experiments  How predictive are in vitro results?  Increase the complexity of the in vitro system, taking into account Cell-Cell interactions Extracellular matrix (ECM) – Cell interactions Physiology and inter-tissue communications  Use of modeling (In silico) Physiologically Based Pharmacokinetics (PBPK) Pharmacodynamics (PD) 31 14 31  6/7/2024 Increased complexity in in vitro test systems  From 2D monolayer to 3D culturing More realistic type of cell growth, including cell-cell interactions, polarization, differentiation, extracellular matrix, etc. Many different techniques to grow a spheroid (clump) of cells Slide from Jha, 2014 32 32 Increased complexity in in vitro test systems  Cell co-culturing Cenni et al. 2011 33 15 33  6/7/2024 Increased complexity in in vitro test systems  Organ-on-a-chip: multichannel 3D microfluid cell culture chip “artificial organ” https://www.deingenieur.nl/artikel/organs-on-chips-versnellen-ontwikkeling-medicijnen See also video-clip at this site 34 34 Increased complexity in in vitro test systems  Body-on-a-chip Multi-compartmental perfused systems Huh et al. 2011 35 16 35  6/7/2024 Goals: after this lecture, you can…  Explain the principle of protein-based bioassays  Distinguish between primary cells, cell lines, and immortal cell lines  Name several types of cell-based toxicity assays  Explain the principle of a reporter gene bioassay  Distinguish between embryonic stem cell differentiation models, iPSC models, and transdifferentiation models  Classify stem cells as pluripotent and multipotent  Provide illustrative examples of more complex in vitro models 47 47 17