The effects measured with in vitro cell-based bioassays are typically reported as nominal effect concentrations (C nom), but the freely dissolved concentration in the exposure medium (C w) and the total cellular concentration (C cell) are considered more quantitative dose metrics that allow extrapolation to the whole-organism level. To predict C w and C cell, 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 cell/w) 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 cell/w 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 w because they do not account for saturable protein binding and overestimate C cell for organic acids, if BSA is used as surrogate for cellular proteins.

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Exposure assessment in in vitro cell-based bioassays is challenging for ionizable organic chemicals (IOCs), because they are present as more than one chemical species in the bioassay medium. Furthermore, compared to neutral organic chemicals, their binding to medium proteins and lipids is driven by more complex molecular interactions. Total medium concentrations (C total,medium ) and/or freely dissolved medium concentrations (C free,medium ) were determined for one neutral chemical and 14 IOCs (acids, bases, multifunctional) at concentrations relevant for determination of cytotoxicity and effect. C free,medium was measured in two in vitro bioassays at the time of dosing and after 24 h of incubation using solid-phase microextraction. C free,medium was maximally 1.7 times lower than the nominal concentrations (C nom ) for the hydrophilic chemicals (caffeine and lamotrigine). For the organic acids (naproxen, ibuprofen, warfarin, and diclofenac), C free,medium was by a factor of 4 lower than C nom at high concentrations, but the ratio was much higher at low concentrations, indicating a nonlinear binding behavior. The experimental C free,medium was also compared with C free,medium predicted with a mass balance model accounting for binding to medium proteins and lipids. The mass balance model performed well for five of the test chemicals (within a factor of 10), but it underestimated C free,medium by up to a factor of 1200 for chemicals that showed nonlinear binding to medium components. These findings emphasize that experimental exposure assessment is required for improved understanding of in vitro toxicity data.

The aim of the current study was to understand and develop models to predict the pH-dependent toxicity of ionizable pharmaceuticals in embryos of the zebrafish Danio rerio. We found a higher uptake and toxicity with increasing neutral fraction of acids (diclofenac, genistein, naproxen, torasemide, and warfarin) and bases (metoprolol and propranolol). Simple mass balance models accounting for the partitioning to lipids and proteins in the zebrafish embryo were found to be suitable to predict the bioconcentration after 96 h of exposure if pH values did not differ much from the internal pH of 7.55. For other pH values, a kinetic ion-trap model for the zebrafish embryo explained the pH dependence of biouptake and toxicity. The total internal lethal concentrations killing 50% of the zebrafish embryos (ILC50) were calculated from the measured BCF and LC50. The resulting ILC50 were independent of external pH. Critical membrane concentrations were deduced by an internal mass balance model, and apart from diclofenac, whose specific toxicity in fish had already been established, all pharmaceuticals were confirmed to act as baseline toxicants in zebrafish.

High-throughput in vitro 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[a]pyrene (BaP), an AhR ligand known to upregulate cytochrome P450 in vitro and in vivo. We combined fluorescence microscopy with chemical analysis, real-time PCR, and ethoxyresorufin-O-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 CYP1A1 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 in vitro and can improve the interpretation of in vitro reporter gene effect data and extrapolation to in vivo human exposure.