Experimental exposure assessment of designed chemical mixtures in cell-based in vitro bioassays

Cell-based bioassays are useful tools for the effect assessment of complex mixtures, but so far exposure assessment has not been performed for mixtures of chemicals. In the present study, cytotoxicity and activation of oxidative stress response were measured for three designed chemical mixtures with up to twelve components. The measurements of biological responses were complemented by concentration measurements using solid-phase microextraction to derive the freely dissolved concentrations of the mixtures (Cfree,mix). The tested mixtures showed slightly higher cytotoxic effects than predicted by the concentration addition model. Nominal and freely dissolved effect concentrations of the mixtures were very similar (within a factor of 1.5), but nominal concentrations (Cnom) and Cfree of the individual mixture components were only similar for the hydrophilic chemicals (e.g., caffeine, coumarin, lamotrigine). For hydrophobic (e.g., fluoranthene) and acidic chemicals (e.g., diclofenac, naproxen) Cfree was up to 648 times lower than Cnom. Chemicals were dosed in equipotent nominal concentration ratios and therefore contributed equally to the detected effects. Hydrophilic chemicals with low potency dominated Cnom,mix (up to 95%) and Cfree,mix (up to 99%). Several mixture components (e.g., diclofenac, ibuprofen, naproxen and warfarin) showed increasing free fractions with increasing Cnom,mix and therefore also a concentration-dependent contribution to Cfree,mix. Based on the findings of this study, we concluded that Cnom,mix will be sufficient for evaluating the toxicity of mixtures that contain chemicals with diverse physicochemical properties at low concentration levels. In contrast, for risk assessment purposes and quantitative in vitro to in vivo extrapolations, Cfree,mix is a better parameter because the in vitro responses can be related to freely dissolved concentrations in human plasma

How to improve the dosing of chemicals in high-throughput in vitro mammalian cell assays

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

C18-coated solid-phase microextraction fibers for the quantification of partitioning of organic acids to proteins, lipids, and cells

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

Partitioning of Organic Ions to Muscle Protein: Experimental Data, Modeling, and Implications for in Vivo Distribution of Organic Ions

The in vivo partitioning behavior of ionogenic organic chemicals (IOCs) is of paramount importance for their toxicokinetics and bioaccumulation. Among other proteins, structural proteins including muscle proteins could be an important sorption phase for IOCs, because of their high quantity in the human and other animals’ body and their polar nature. Binding data for IOCs to structural proteins are, however, severely limited. Therefore, in this study muscle protein–water partition coefficients (<i>K</i>_MP/w) of 51 systematically selected organic anions and cations were determined experimentally. A comparison of the measured <i>K</i>_MP/w with bovine serum albumin (BSA)–water partition coefficients showed that anionic chemicals sorb more strongly to BSA than to muscle protein (by up to 3.5 orders of magnitude), while cations sorb similarly to both proteins. Sorption isotherms of selected IOCs to muscle protein are linear (i.e., <i>K</i>_MP/w is concentration independent), and <i>K</i>_MP/w is only marginally influenced by pH value and salt concentration. Using the obtained data set of <i>K</i>_MP/w a polyparameter linear free energy relationship (PP-LFER) model was established. The derived equation fits the data well (<i>R</i>² = 0.89, RMSE = 0.29). Finally, it was demonstrated that the in vitro measured <i>K</i>_MP/w values of this study have the potential to be used to evaluate tissue-plasma partitioning of IOCs in vivo

Role of bioavailability and protein binding of four anionic perfluoroalkyl substances in cell-based bioassays for quantitative in vitro to in vivo extrapolations

Perfluoroalkyl substances (PFAS) are persistent and pose a risk to human health. High throughput screening (HTS) cell-based bioassays may inform risk assessment of PFAS provided that quantitative in vitro to in vivo extrapolation (QIVIVE) can be developed. The QIVIVE ratio is the ratio of nominal (Cnom) or freely dissolved concentration (Cfree) in human blood to Cnom or Cfree in the bioassays. Considering that the concentrations of PFAS in human plasma and in vitro bioassays may vary by orders of magnitude, we tested the hypothesis that anionic PFAS bind to proteins concentration-dependently and therefore the binding differs substantially between human plasma and bioassays, which has an impact on QIVIVE. Solid phase microextraction (SPME) with C18-coated fibers served to quantify the Cfree of four anionic PFAS (perfluorobutanoate (PFBA), perfluorooctanoate (PFOA), perfluorohexane sulfonate (PFHxS) and perfluorooctane sulfonate (PFOS)) in the presence of proteins and lipid, medium components, cells and human plasma over five orders of magnitude in concentrations. The C18-SPME method was used to quantify the non-linear binding to proteins, human plasma and medium, and the partition constants to cells. These binding parameters were used to predict Cfree of PFAS in cell bioassays and human plasma by a concentration-dependent mass balance model (MBM). The approach was illustrated with a reporter gene assay indicating activation of the peroxisome proliferator-activated receptor gamma (PPARγ-GeneBLAzer). Blood plasma levels were collected from literature for occupational exposure and the general population. The QIVIVEnom ratios were higher than the QIVIVEfree ratios due to the strong affinity to proteins and large differences in protein contents between human blood and bioassays. For human health risk assessment, the QIVIVEfree ratios of many in vitro assays need to be combined to cover all health relevant endpoints. If Cfree cannot be measured, they can be estimated with the MBM and concentration-dependent distribution ratios

Experimental exposure assessment of ionizable organic chemicals in in vitro cell-based bioassays

Exposure assessment in 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 () and/or freely dissolved medium concentrations () were determined for one neutral chemical and 14 IOCs (acids, bases, multifunctional) at concentrations relevant for determination of cytotoxicity and effect. was measured in two bioassays at the time of dosing and after 24 h of incubation using solid-phase microextraction. was maximally 1.7 times lower than the nominal concentrations () for the hydrophilic chemicals (caffeine and lamotrigine). For the organic acids (naproxen, ibuprofen, warfarin, and diclofenac), was by a factor of 4 lower than at high concentrations, but the ratio was much higher at low concentrations, indicating a nonlinear binding behavior. The experimental was also compared with 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 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 toxicity data

Equilibrium Sorption of Structurally Diverse Organic Ions to Bovine Serum Albumin

Reliable partitioning data are essential for assessing the bioaccumulation potential and the toxicity of chemicals. In contrast to neutral organic chemicals, the partitioning behavior of ionogenic organic chemicals (IOCs) is still a black box for environmental scientists. Partitioning to serum albumin, the major protein in blood plasma, strongly influences the freely dissolved concentration of many chemicals (including IOCs), which affects their transport and distribution in the body. Because consistent data sets for partitioning of IOCs are rarely available, bovine serum albumin-water partition coefficients (<i>K</i>_BSA/w) were measured in this study for 45 anionic and 4 cationic organic chemicals, including various substituted benzoic and naphthoic acids, sulfonates and several pesticides and pharmaceuticals. The results of this study suggest that binding to BSA is substantially influenced by the three-dimensional structure of the chemicals and the position of substitutions on the sorbing molecules. For example, we found a difference of >1.5 log units between isomeric chemicals such as 3,4-dichlorobenzoic acid and 2,6-dichlorobenzoic acid, and 1-naphthoic acid and 2-naphthoic acid. Conventional modeling approaches (e.g., based on octanol–water partition coefficients) poorly predict log <i>K</i>_BSA/w of organic ions (<i>R</i>² ≤ 0.5), partially because they do not capture the observed steric effects. Hence, alternative modeling strategies will be required for accurate prediction of serum albumin-water partition coefficients of organic ions

Combined ion-trapping and mass balance models to describe the pH-dependent uptake and toxicity of acidic and basic pharmaceuticals in zebrafish embryos (Danio rerio)

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