Publications by authors named "Fabian C Fischer"

9 Publications

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Effective exposure of chemicals in in vitro cell systems: A review of chemical distribution models.

Toxicol In Vitro 2021 Mar 1;73:105133. Epub 2021 Mar 1.

Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands.

Nominal effect concentrations from in vitro toxicity assays may lead to inaccurate estimations of in vivo toxic doses because the nominal concentration poorly reflects the concentration at the molecular target in cells in vitro, which is responsible for initiating effects and can be referred to as the biologically effective dose. Chemicals can differentially distribute between in vitro assay compartments, including serum constituents in exposure medium, microtitre plate plastic, headspace and extracellular matrices. The partitioning of test chemicals to these extracellular compartments reduces the concentration at the molecular target. Free concentrations in medium and cell-associated concentrations are considered better proxies of the biologically effective dose. This paper reviews the mechanisms by which test chemicals distribute between in vitro assay compartments, and also lists the physicochemical properties driving the extent of this distribution. The mechanisms and physicochemical properties driving the distribution of test chemical in vitro help explain the makeup of mass balance models that estimate free concentrations and cell-associated concentrations in in vitro toxicity assays. A thorough understanding of the distribution processes and assumptions underlying these mass balance models helps define chemical and biological applicability domains of individual models, as well as provide a perspective on how to improve model predictivity and quantitative in vitro-in vivo extrapolations.
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http://dx.doi.org/10.1016/j.tiv.2021.105133DOI Listing
March 2021

Cytotoxicity Burst? Differentiating Specific from Nonspecific Effects in Tox21 Reporter Gene Assays.

Environ Health Perspect 2020 07 23;128(7):77007. Epub 2020 Jul 23.

Department of Cell Toxicology, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany.

Background: High-throughput screening of chemicals with reporter gene assays in Tox21 has produced a large database on cytotoxicity and specific modes of action. However, the validity of some of the reported activities is questionable due to the "cytotoxicity burst," which refers to the supposition that many stress responses are activated in a nonspecific way at concentrations close to cell death.

Objectives: We propose a pragmatic method to identify whether reporter gene activation is specific or cytotoxicity-triggered by comparing the measured effects with baseline toxicity.

Methods: Baseline toxicity, also termed narcosis, is the minimal toxicity any chemical causes. Quantitative structure-activity relationships (QSARs) developed for baseline toxicity in mammalian reporter gene cell lines served as anchors to define the chemical-specific threshold for the cytotoxicity burst and to evaluate the degree of specificity of the reporter gene activation. Measured 10% effect concentrations were related to measured or QSAR-predicted 10% cytotoxicity concentrations yielding specificity ratios (SR). We applied this approach to our own experimental data and to chemicals that were tested in six of the high-throughput Tox21 reporter gene assays.

Results: Confirmed baseline toxicants activated reporter gene activity around cytotoxic concentrations triggered by the cytotoxicity burst. In six Tox21 assays, 37%-87% of the active hits were presumably caused by the cytotoxicity burst () and only 2%-14% were specific with against experimental cytotoxicity but 75%-97% were specific against baseline toxicity. This difference was caused by a large fraction of chemicals showing excess cytotoxicity.

Conclusions: The specificity analysis for measured effects identified whether a cytotoxicity burst had likely occurred. The SR-analysis not only prevented false positives, but it may also serve as measure for relative effect potency and can be used for quantitative extrapolation and risk assessment of chemicals. https://doi.org/10.1289/EHP6664.
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http://dx.doi.org/10.1289/EHP6664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377237PMC
July 2020

Cellular Metabolism in High-Throughput Reporter Gene Assays and Implications for the Quantitative - Extrapolation.

Chem Res Toxicol 2020 07 13;33(7):1770-1779. Epub 2020 Apr 13.

Department Cell Toxicology, Helmholtz Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany.

High-throughput reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell lines provide stable readouts of the activation of cellular receptors or transcription factors of interest, but such reporter gene assays have been criticized for not capturing cellular metabolism. We characterized the metabolic activity of the widely applied AREc32 (human breast cancer MCF-7), ARE-bla (human liver cancer HepG2), and GR-bla (human embryonic kidney HEK293) reporter gene cells in the absence and in the presence of benzo[]pyrene (BaP), an AhR ligand known to upregulate cytochrome P450 and . We combined fluorescence microscopy with chemical analysis, real-time PCR, and ethoxyresorufin--deethylase activity measurements to track temporal changes in BaP and its metabolites in the cells and surrounding medium over time in relation to the expression and activity of metabolic enzymes. Decreasing BaP concentrations and formation of metabolites agreed with the high basal CYP1 activity of ARE-bla and the strong mRNA induction in AREc32, whereas BaP concentrations were constant in GR-bla, in which neither metabolites nor CYP1 induction was detected. The study emphasizes that differences in sensitivity between reporter gene assays may be caused not only by different reporter constructs but also by a varying biotransformation rate of the evaluated parent chemical. The basal metabolic capacity of reporter gene cells in the absence of chemicals is not a clear indication because we demonstrated that the metabolic activity can be upregulated by AhR ligands during the assay. The combination of methods presented here is suitable to characterize the metabolic activity of cells and can improve the interpretation of reporter gene effect data and extrapolation to human exposure.
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http://dx.doi.org/10.1021/acs.chemrestox.0c00037DOI Listing
July 2020

How To Improve the Dosing of Chemicals in High-Throughput Mammalian Cell Assays.

Chem Res Toxicol 2019 08 2;32(8):1462-1468. Epub 2019 Aug 2.

Department Cell Toxicology , Helmholtz Centre for Environmental Research - UFZ , Permoserstraße 15 , 04318 Leipzig , Germany.

Controlling the exposure of chemicals in mammalian cell assays is an important prerequisite for the application of methods in risk and hazard assessment of chemicals. Existing models require numerous physicochemical and system parameters to quantify the effective concentration in the assay. Synthesizing these studies, this article briefly communicates how the protein-rich supplement in the medium can be utilized to adjust constant and quantifiable exposure concentrations without the need for measurements and complex modeling. We present a simplified mass balance equation based on chemical properties and system parameters from openly accessible databases, which can be used to adjust the dose of chemicals in the exposure medium, leading to defined and stable freely dissolved concentrations (). The proposed framework prevents experimental artifacts associated with the use of cosolvents and medium oversaturation and enables the conversion of effect data to freely dissolved effect concentrations (EC), which can directly be applied in quantitative to extrapolation models and compared to other exposure scenarios.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00167DOI Listing
August 2019

Quantification of freely dissolved effect concentrations in in vitro cell-based bioassays.

Arch Toxicol 2019 08 22;93(8):2295-2305. Epub 2019 Jun 22.

Department of Cell Toxicology, Helmholtz Centre for Environmental Research (UFZ), Permoserstr. 15, 04318, Leipzig, Germany.

Improved understanding of chemical exposure in in vitro bioassays is required for quantitative in vitro-in vivo extrapolation (QIVIVE). In this study, we quantified freely dissolved concentrations in medium sampled from in vitro cell-based bioassays (C) for nine chemicals with different hydrophobicity and speciation at the time point of dosing and after an incubation period of 24 h using solid-phase microextraction. The chemicals were tested in two reporter gene assays, the AREc32 assay indicative of the oxidative stress response and the PPARγ-GeneBLAzer assay that responds to chemicals which bind to the peroxisome proliferator-activated receptor gamma. For seven of the nine chemicals, C did not change significantly over time in both assays and the experimentally determined C generally agreed well with predictions of a mass balance model that describes the partitioning between proteinaceous and lipidous medium constituents, cells and the aqueous phase. Two chemicals showed a decrease of C in the AREc32 assay over time that was probably caused by cellular metabolism. Furthermore, C of the acidic chemical diclofenac deviated from the model predictions by more than a factor of 10 at higher concentrations, which indicates nonlinear binding and saturation of the medium proteins. Bioassay results are typically reported as nominal effect concentrations (EC), although it is established that freely dissolved effect concentrations (EC) are a better measure for the bioavailable dose and the method developed here provides a simple experimental approach to measure and model EC in in vitro bioassay for improved QIVIVE models.
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http://dx.doi.org/10.1007/s00204-019-02498-3DOI Listing
August 2019

C18-Coated Solid-Phase Microextraction Fibers for the Quantification of Partitioning of Organic Acids to Proteins, Lipids, and Cells.

Chem Res Toxicol 2019 01 10;32(1):168-178. Epub 2019 Jan 10.

Department of Cell Toxicology , Helmholtz Centre for Environmental Research - UFZ , Permoserstr. 15 , 04318 Leipzig , Germany.

The effects measured with in vitro cell-based bioassays are typically reported as nominal effect concentrations ( C), but the freely dissolved concentration in the exposure medium ( C) and the total cellular concentration ( C) are considered more quantitative dose metrics that allow extrapolation to the whole-organism level. To predict C and C, the partitioning of the test chemicals to medium proteins and lipids and cells has to be known. In this study, we developed a solid-phase microextraction (SPME) method based on C18-coated fibers to quantify the partitioning of diclofenac, 2,4-dichlorophenoxyacetic acid (2,4-D), ibuprofen, naproxen, torasemide, warfarin, and genistein to bovine serum albumin (BSA), phospholipid liposomes, fetal bovine serum (FBS), and cells. For ibuprofen, 2,4-D, naproxen, and warfarin, the partitioning to the SPME fibers was found to be concentration dependent, which had to be considered for the calculation of distribution ratios to biological materials. The sorption isotherms to FBS were nonlinear for diclofenac, 2,4-D, ibuprofen, naproxen, and warfarin. The FBS isotherms could be described by assuming that the total amount of chemical bound to FBS is the sum of the amount specifically bound to the binding sites of albumin and nonspecifically bound to all medium proteins and lipids. The determined cell-water distribution ratios ( D) differed considerably between four different cell lines (up to 1.83 log-units) and also between different batches of the same cell line (up to 0.48 log-units). The relative importance of protein and lipid content for D was evaluated with a mass balance model and different types of cellular proteins and lipids as input parameters. Existing in vitro mass balance models may underestimate C because they do not account for saturable protein binding and overestimate C for organic acids, if BSA is used as surrogate for cellular proteins.
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http://dx.doi.org/10.1021/acs.chemrestox.8b00249DOI Listing
January 2019

Application of Experimental Polystyrene Partition Constants and Diffusion Coefficients to Predict the Sorption of Neutral Organic Chemicals to Multiwell Plates in in Vivo and in Vitro Bioassays.

Environ Sci Technol 2018 11 31;52(22):13511-13522. Epub 2018 Oct 31.

Helmholtz Centre for Environmental Research - UFZ , Department Cell Toxicology , Permoserstraße 15 , 04318 Leipzig , Germany.

Sorption to the polystyrene (PS) of multiwell plates can affect the exposure to organic chemicals over time in in vitro and in vivo bioassays. Experimentally determined diffusion coefficients in PS ( D) were in a narrow range of 1.25 to 8.0 · 10 m s and PS-water partition constants ( K) ranged from 0.04 to 5.10 log-units for 22 neutral organic chemicals. A kinetic model, which explicitly accounts for diffusion in the plastic, was applied to predict the depletion of neutral organic chemicals from different bioassay media by sorption to various multiwell plate formats. For chemicals with log K > 3, the medium concentrations decreased rapidly and considerably in the fish embryo toxicity assay but medium concentrations remained relatively constant in the cell-based bioassays with medium containing 10% fetal bovine serum (FBS), emphasizing the ability of the protein- and lipid-rich medium to compensate for losses by multiwell plate sorption. The PS sorption data may serve not only for exposure assessment in bioassays but also to model the contaminant uptake by and release from plastic packaging material and the chemical transport by PS particles in the environment.
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http://dx.doi.org/10.1021/acs.est.8b04246DOI Listing
November 2018

Cellular Uptake Kinetics of Neutral and Charged Chemicals in in Vitro Assays Measured by Fluorescence Microscopy.

Chem Res Toxicol 2018 08 16;31(8):646-657. Epub 2018 Jul 16.

Department of Cell Toxicology , Helmholtz Centre for Environmental Research - UFZ , Permoserstraße 15 , 04318 Leipzig , Germany.

Cellular uptake kinetics are key for understanding time-dependent chemical exposure in in vitro cell assays. Slow cellular uptake kinetics in relation to the total exposure time can considerably reduce the biologically effective dose. In this study, fluorescence microscopy combined with automated image analysis was applied for time-resolved quantification of cellular uptake of 10 neutral, anionic, cationic, and zwitterionic fluorophores in two reporter gene assays. The chemical fluorescence in the medium remained relatively constant during the 24-h assay duration, emphasizing that the proteins and lipids in the fetal bovine serum (FBS) supplemented to the assay medium represent a large reservoir of reversibly bound chemicals with the potential to compensate for chemical depletion by cell uptake, growth, and sorption to well materials. Hence FBS plays a role in stabilizing the cellular dose in a similar way as polymer-based passive dosing, here we term this process as serum-mediated passive dosing (SMPD). Neutral chemicals accumulated in the cells up to 12 times faster than charged chemicals. Increasing medium FBS concentrations accelerated uptake due to FBS-facilitated transport but led to lower cellular concentrations as a result of increased sorption to medium proteins and lipids. In vitro cell exposure results from the interaction of several extra- and intracellular processes, leading to variable and time-dependent exposure between different chemicals and assay setups. The medium FBS plays a crucial role for the thermodynamic equilibria as well as for the cellular uptake kinetics, hence influencing exposure. However, quantification of cellular exposure by an area under the curve (AUC) analysis illustrated that, for the evaluated bioassay setup, current in vitro exposure models that assume instantaneous equilibrium between medium and cells still reflect a realistic exposure because the AUC was typically reduced less than 20% compared to the cellular dose that would result from instantaneous equilibrium.
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http://dx.doi.org/10.1021/acs.chemrestox.8b00019DOI Listing
August 2018

Modeling Exposure in the Tox21 in Vitro Bioassays.

Chem Res Toxicol 2017 05 24;30(5):1197-1208. Epub 2017 Apr 24.

Department Cell Toxicology, Helmholtz Centre for Environmental Research - UFZ , Permoserstraße 15, 04318 Leipzig, Germany.

High-throughput in vitro bioassays are becoming increasingly important in the risk characterization of anthropogenic chemicals. Large databases gather nominal effect concentrations (C) for diverse modes of action. However, the biologically effective concentration can substantially deviate due to differences in chemical partitioning. In this study, we modeled freely dissolved (C), cellular (C), and membrane concentrations (C) in the Tox21 GeneBLAzer bioassays for a set of neutral and ionogenic organic chemicals covering a large physicochemical space. Cells and medium constituents were experimentally characterized for their lipid and protein content, and partition constants were either collected from the literature or predicted by mechanistic models. The chemicals exhibited multifaceted partitioning to proteins and lipids with distribution ratios spanning over 8 orders of magnitude. Modeled C deviated over 5 orders of magnitude from C and can be compared to in vivo effect data, environmental concentrations, and the unbound fraction in plasma, which is needed for the in vitro to in vivo extrapolation. C was relatively constant for chemicals with membrane lipid-water distribution ratios of 1000 or higher and proportional to C. Representing a sum parameter for exposure that integrates the entire dose from intracellular partitioning, C is particularly suitable for the effect characterization of chemicals with multiple target sites and the calculation of their relative effect potencies. Effective membrane concentrations indicated that the specific effects of very hydrophobic chemicals in multiple bioassays are occurring at concentrations close to baseline toxicity. The equilibrium partitioning model including all relevant system parameters and a generic bioassay setup is attached as an excel workbook to this paper and can readily be applied to diverse in vitro bioassays.
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http://dx.doi.org/10.1021/acs.chemrestox.7b00023DOI Listing
May 2017