Effects of Solvents and Dosing Procedure on Chemical Toxicity in Cell-Based <i>in Vitro</i> Assays

Tanneberger, Katrin; Rico-Rico, Ángeles; Kramer, Nynke I.; Busser, F; Hermens, Joop L. M.; Schirmer, Kristin

doi:10.1021/es100045y

Cited by 60 publications

(64 citation statements)

References 32 publications

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“…In order to have a more precise in vitro-in vivo extrapolation, the use of toxicokinetics data as well as physiologically based toxicokinetics modeling (PBTK) is recommended (Coecke et al, 2013). The nominal concentrations applied to the culture systems may deviate from the actual concentration of the compound due to the occurrence of multiple possible events, such as accumulation, evaporation, binding to plastic and/ or medium components, uptake and metabolism (Blaauboer, 2010;Tanneberger et al, 2010). The combination of toxicodynamic and toxicokinetic approaches has been extensively investigated in the EU funded 7th Framework Project Predict-IV.…”

Section: Discussionmentioning

confidence: 99%

Determination of liver specific toxicities in rat hepatocytes by high content imaging during 2-week multiple treatment

Germano

Uteng

Philippe

et al. 2015

Toxicology in Vitro

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DILI is a major safety issue during drug development and one of the leading causes for market withdrawal. Despite many efforts made in the past, the prediction of DILI using in vitro models remains very unreliable. In the present study, the well-established hepatocyte Collagen I-Matrigel™ sandwich culture was used, mimicking chronic drug treatment after multiple incubations for 14 days. Ten drugs associated with different types of specific preclinical and clinical liver injury were evaluated at non-cytotoxic concentrations. Mrp2-mediated transport, intracellular accumulation of neutral lipids and phospholipids were selected as functional endpoints by using Cellomics™ Arrayscan® technology and assessed at five timepoints (day 1, 3, 7, 10, 14). Liver specific functional impairments after drug treatment were enhanced over time and could be monitored by HCI already after few days and before cytotoxicity. Phospholipidosis-inducing drugs Chlorpromazine and Amiodarone displayed the same response as in vivo. Cyclosporin A, Chlorpromazine, and Troglitazone inhibited Mrp2-mediated biliary transport, correlating with in vivo findings. Steatosis remained difficult to be reproduced under the current in vitro testing conditions, resulting into false negative and positive responses. The present results suggest that the repeated long-term treatment of rat hepatocytes in the Collagen I-Matrigel™ sandwich configuration might be a suitable tool for safety profiling of the potential to induce phospholipidosis and impair Mrp2-mediated transport processes, but not to predict steatosis.

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Section: Discussionmentioning

confidence: 99%

Determination of liver specific toxicities in rat hepatocytes by high content imaging during 2-week multiple treatment

Germano

Uteng

Philippe

et al. 2015

Toxicology in Vitro

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“…intended) chemical concentrations. However, recent studies show that measurements of external exposure are more appropriate than nominal concentrations due to the number of competing processes occurring in the culture well, like sorption to various compartments in a well or evaporation [3], [6], [8]. Yet, external concentrations as dose metric are still only a surrogate which may impede interpretation and extrapolation of toxicological effects because internal concentrations are thought to give rise to the biologically effective dose [9], [10].…”

Section: Introductionmentioning

confidence: 99%

Measured and Modeled Toxicokinetics in Cultured Fish Cells and Application to In Vitro - In Vivo Toxicity Extrapolation

et al. 2014

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Effect concentrations in the toxicity assessment of chemicals with fish and fish cells are generally based on external exposure concentrations. External concentrations as dose metrics, may, however, hamper interpretation and extrapolation of toxicological effects because it is the internal concentration that gives rise to the biological effective dose. Thus, we need to understand the relationship between the external and internal concentrations of chemicals. The objectives of this study were to: (i) elucidate the time-course of the concentration of chemicals with a wide range of physicochemical properties in the compartments of an in vitro test system, (ii) derive a predictive model for toxicokinetics in the in vitro test system, (iii) test the hypothesis that internal effect concentrations in fish (in vivo) and fish cell lines (in vitro) correlate, and (iv) develop a quantitative in vitro to in vivo toxicity extrapolation method for fish acute toxicity. To achieve these goals, time-dependent amounts of organic chemicals were measured in medium, cells (RTgill-W1) and the plastic of exposure wells. Then, the relation between uptake, elimination rate constants, and log KOW was investigated for cells in order to develop a toxicokinetic model. This model was used to predict internal effect concentrations in cells, which were compared with internal effect concentrations in fish gills predicted by a Physiologically Based Toxicokinetic model. Our model could predict concentrations of non-volatile organic chemicals with log KOW between 0.5 and 7 in cells. The correlation of the log ratio of internal effect concentrations in fish gills and the fish gill cell line with the log KOW was significant (r>0.85, p = 0.0008, F-test). This ratio can be predicted from the log KOW of the chemical (77% of variance explained), comprising a promising model to predict lethal effects on fish based on in vitro data.

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“…Indeed, hydrophobic and volatile chemicals have proven to be difficult to dose and obtain reliable results for in cell-based assays (5). One possible reason for this is that the free concentration of a test chemical can be much lower than the nominal concentration and can vary considerably between assays and over time.…”

Section: Introductionmentioning

confidence: 99%

Development of a Partition-Controlled Dosing System for Cell Assays

Kramer

Busser

Oosterwijk

et al. 2010

Chem. Res. Toxicol.

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Hydrophobic and volatile chemicals have proven to be difficult to dose in cell assays. Cosolvents are often needed to dissolve these chemicals in cell culture medium. Moreover, the free concentration of these chemicals in culture medium may diminish over time due to metabolism, evaporation, and nonspecific binding to well plate surfaces and serum constituents. The aim of this study was to develop a partitioncontrolled dosing system to maintain constant concentrations of benzo(a)pyrene, 1,2-dichlorobenzene, and 1,2,4-trichlorobenzene in an ethoxyresorufin-O-deethylase (EROD) assay and a cytotoxicity assay with the rainbow trout (Oncorhynchus mykiss) cell lines RTL-W1 and RTgill-W1. Polydimethylsiloxane (PDMS) sheets were loaded with test chemicals in a spiked methanol/water solution and placed in the wells, filled with culture medium, of a 24-well culture plate. Cells were grown on inserts and were subsequently added to the wells with the PDMS sheets. The system reached equilibrium within 24 h, even for the very hydrophobic chemical benzo(a)pyrene. The reservoir of test chemical in PDMS was large enough to compensate for the loss of >95% of the test chemical from the culture medium. The PDMS sheets maintained medium concentrations constant for >72 h. Nominal median effect concentrations (EC 50 ) were 1.3-7.0 times lower in the partition-controlled dosing systems than in conventional assays spiked using dimethyl sulfoxide (DMSO) as a carrier solvent, thus indicating that the apparent sensitivity of the bioassay increased when controlled and constant exposure conditions could be assured. The EC 50 values of the test chemicals based on free concentrations were estimated in the partition-controlled dosing systems using measured PDMS-bare culture medium partition coefficients. Results indicated that 61, 70, and 99.8% of 1,2-diclorobenzene, 1,2,4-trichlorobenzene, and benzo(a)pyrene were bound to serum constituents in the culture medium.

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Effects of Solvents and Dosing Procedure on Chemical Toxicity in Cell-Based in Vitro Assays

Cited by 60 publications

References 32 publications

Determination of liver specific toxicities in rat hepatocytes by high content imaging during 2-week multiple treatment

Determination of liver specific toxicities in rat hepatocytes by high content imaging during 2-week multiple treatment

Measured and Modeled Toxicokinetics in Cultured Fish Cells and Application to In Vitro - In Vivo Toxicity Extrapolation

Development of a Partition-Controlled Dosing System for Cell Assays

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