The goal of the present study was to develop an online web-based toolbox that contains generic physiologically based kinetic (PBK) models for rats and humans, including underlying calculation tools to predict plasma protein binding and tissue:plasma distribution, to be used for quantitative in-vitro-to-in-vivo extrapolations (QIVIVE). The PBK models within the toolbox allow first estimations of internal plasma and tissue concentrations of chemicals to be made, based on the logP and p K a of the chemicals and values for intestinal uptake and intrinsic hepatic clearance. As a case study, the toolbox was used to predict oral equivalent doses of in vitro ToxCast bioactivity data for the food additives methylparaben, propyl gallate, octyl gallate, and dodecyl gallate. These oral equivalent doses were subsequently compared with human exposure estimates, as a low tier assessment allowing prioritization for further assessment. The results revealed that daily intake levels of especially propyl gallate can lead to internal plasma concentrations that are close to in vitro biological effect concentrations, particularly with respect to the inhibition of human thyroid peroxidase (TPO). Estrogenic effects were not considered likely to be induced by the food additives, as daily exposure levels of the different compounds remained 2 orders of magnitude below the oral equivalent doses for in vitro estrogen receptor activation. Overall, the results of the study show how the toolbox, which is freely accessible through , can be used to obtain initial internal dose estimates of chemicals and to prioritize chemicals for further assessment, based on the comparison of oral equivalent doses of in vitro biological activity data with human exposure levels.
The goal of the present study was to assess the predictive performance of a minimal generic rat physiologically based kinetic (PBK) model based on in vitro and in silico input data to predict peak plasma concentrations (Cmax) upon single oral dosing. To this purpose, a dataset was generated of 3960 Cmax predictions for 44 compounds, applying different combinations of in vitro and in silico approaches for chemical parameterization, and comparison of the predictions to reported in vivo data. Best performance was obtained when i) the hepatic clearance was parameterized based on in vitro measured intrinsic clearance values, ii) the method of Rodgers and Rowland for calculating partition coefficients, and iii) in silico calculated fraction unbound plasma and Papp values (the latter especially for very lipophilic compounds). Based on these input data, the median Cmax of 32 compounds could be predicted within 10-fold of the observed Cmax, with 21 out of these 32 compounds being predicted within 5-fold, and 8 compounds within 2-fold. Overestimations of more than 10-fold were observed for 12 compounds, whereas no underestimations of more than 10-fold occurred. Median Cmax predictions were frequently found to be within 10-fold of the observed Cmax when the scaled unbound hepatic intrinsic clearance (Clint,u) was either higher than 20 L/h or lower than 1 L/h. Similar findings were obtained with a test set of five in-house BASF compounds. Overall, this study provides relevant insights in the predictive performance of a minimal PBK model based on in vitro and in silico input data.
Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse effects, including hepatotoxicity, developmental toxicity and immunotoxicity. So far, little information is available about the mechanisms underlying the toxicity of PFASs, including those related to their immunotoxicity. Reported immunotoxic effects of PFASs include decreased antibody responses in experimental animals and humans, indicating that PFASs may, among others, affect B cell function. In the present study, we first assessed the effects of PFOA on the transcriptome of the human Namalwa B cell line using RNA seq analysis. Gene expression changes, analyzed using Ingenuity Pathway Analysis, pointed to various cellular processes affected by PFOA, including ‘B cell development’ and ‘Primary immunodeficiency signaling’. Interestingly, PFOA decreased the expression of RAG1 and RAG2, genes involved in immunoglobulin and T cell receptor V(D)J recombination. As a next step, time- and concentration-dependent changes in the expression of RAG1 and RAG2 upon exposure to PFOA, PFNA, PFHxS and PFOS were studied through RT-qPCR analysis. Analysis with the concentration–response modeling software PROAST resulted in the following potency ranking: PFNA > PFOA > PFOS > PFHxS. Altogether, the present in vitro study provides insights into the effects of selected PFASs on B cells, identifying RAG1 and RAG2 expression as possible relevant targets that may play a role in the immunotoxicity of PFASs.
Flow conditions have been shown to be important in improving longevity and functionality of primary hepatocytes, but the impact of flow on HepaRG cells is largely unknown. We studied the expression of genes encoding CYP enzymes and transporter proteins and CYP1 and CYP3A4 activity during 8 weeks of culture in HepaRG cells cultured under static conditions (conventional 24-/96-well plate culture with common bicarbonate/CO2 buffering) and under flow conditions in an organ-on-chip (OOC) device. Since the OOC-device is a closed system, bicarbonate/CO2 buffering was not possible, requiring application of another buffering agent, such as HEPES. In order to disentangle the effects of HEPES from the effects of flow, we also applied HEPES-supplemented medium in static cultures and studied gene expression and CYP activity. We found that cells cultured under flow conditions in the OOC-device, as well as cells cultured under static conditions with HEPES-supplemented medium, showed more stable gene expression levels. Furthermore, only cells cultured in the OOC-device showed relatively high baseline CYP1 activity, and their gene expression levels of selected CYPs and transporters were most similar to gene expression levels in human primary hepatocytes. However, there was a decrease in baseline CYP3A4 activity under flow conditions compared to HepaRG cells cultured under static conditions. Altogether, the present study shows that HepaRG cells cultured in the OOC-device were more stable than in static cultures, being a promising in vitro model to study hepatoxicity of chemicals upon chronic exposure.
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