Modeling Exposure in the Tox21 <i>in Vitro</i> Bioassays

High-throughput <i>in vitro</i> bioassays are becoming increasingly important in the risk characterization of anthropogenic chemicals. Large databases gather nominal effect concentrations (<i>C</i>_nom) 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 (<i>C</i>_free), cellular (<i>C</i>_cell), and membrane concentrations (<i>C</i>_mem) 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 <i>C</i>_free deviated over 5 orders of magnitude from <i>C</i>_nom and can be compared to <i>in vivo</i> effect data, environmental concentrations, and the unbound fraction in plasma, which is needed for the <i>in vitro</i> to <i>in vivo</i> extrapolation. <i>C</i>_cell was relatively constant for chemicals with membrane lipid–water distribution ratios of 1000 or higher and proportional to <i>C</i>_nom. Representing a sum parameter for exposure that integrates the entire dose from intracellular partitioning, <i>C</i>_cell 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 <i>in vitro</i> bioassays

Modeling Exposure in the Tox21 <i>in Vitro</i> Bioassays

High-throughput <i>in vitro</i> bioassays are becoming increasingly important in the risk characterization of anthropogenic chemicals. Large databases gather nominal effect concentrations (<i>C</i>_nom) 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 (<i>C</i>_free), cellular (<i>C</i>_cell), and membrane concentrations (<i>C</i>_mem) 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 <i>C</i>_free deviated over 5 orders of magnitude from <i>C</i>_nom and can be compared to <i>in vivo</i> effect data, environmental concentrations, and the unbound fraction in plasma, which is needed for the <i>in vitro</i> to <i>in vivo</i> extrapolation. <i>C</i>_cell was relatively constant for chemicals with membrane lipid–water distribution ratios of 1000 or higher and proportional to <i>C</i>_nom. Representing a sum parameter for exposure that integrates the entire dose from intracellular partitioning, <i>C</i>_cell 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 <i>in vitro</i> bioassays

Cellular Uptake Kinetics of Neutral and Charged Chemicals in <i>in Vitro</i> Assays Measured by Fluorescence Microscopy

Cellular uptake kinetics are key for understanding time-dependent chemical exposure in <i>in vitro</i> 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. <i>In vitro</i> 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 <i>in vitro</i> 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

Paired Liver:Plasma PFAS Concentration Ratios from Adolescents in the Teen-LABS Study and Derivation of Empirical and Mass Balance Models to Predict and Explain Liver PFAS Accumulation

Animal studies have pointed at the liver as a hotspot for per- and polyfluoroalkyl substances (PFAS) accumulation and toxicity; however, these findings have not been replicated in human populations. We measured concentrations of seven PFAS in matched liver and plasma samples collected at the time of bariatric surgery from 64 adolescents in the Teen-Longitudinal Assessment of Bariatric Surgery (Teen-LABS) study. Liver:plasma concentration ratios were perfectly explained (r2 > 0.99) in a multilinear regression (MLR) model based on toxicokinetic (TK) descriptors consisting of binding to tissue constituents and membrane permeabilities. Of the seven matched plasma and liver PFAS concentrations compared in this study, the liver:plasma concentration ratio of perfluoroheptanoic acid (PFHpA) was considerably higher than the liver:plasma concentration ratio of other PFAS congeners. Comparing the MLR model with an equilibrium mass balance model (MBM) suggested that complex kinetic transport processes are driving the unexpectedly high liver:plasma concentration ratio of PFHpA. Intratissue MBM modeling pointed to membrane lipids as the tissue constituents that drive the liver accumulation of long-chain, hydrophobic PFAS, whereas albumin binding of hydrophobic PFAS dominated PFAS distribution in plasma. The liver:plasma concentration data set, empirical MLR model, and mechanistic MBM modeling allow the prediction of liver from plasma concentrations measured in human cohort studies. Our study demonstrates that combining biomonitoring data with mechanistic modeling can identify underlying mechanisms of internal distribution and specific target organ toxicity of PFAS in humans