Comparative Analysis of the Physiological and Transport Functions of Various Sources of Renal Proximal Tubule Cells Under Static and Fluidic Conditions in PhysioMimix{trade mark, serif} T12 Platform.

In vitro models that can faithfully replicate critical aspects of kidney tubule function such as directional drug transport are in high demand in pharmacology and toxicology. Accordingly, development and validation of new models is underway. The objective of this study was to characterize physiological and transport functions of various sources of human renal proximal tubule epithelial cells (RPTECs). We tested TERT1-immortalized RPTEC, including OAT1-, OCT2- or OAT3-overexpressing variants, and primary RPTECs. Cells were cultured on transwell membranes in static (24-well transwells) and fluidic (transwells in PhysioMimix{trade mark, serif} T12 organ-on-chip with 2 mL/s flow) conditions. Barrier formation, transport, and gene expression were evaluated. We show that two commercially available primary RPTECs were not suitable for studies of directional transport on transwells because they formed a substandard barrier even though they exhibited higher expression of transporters, especially under flow. TERT1-parent, -OAT1 and -OAT3 cells formed robust barriers, but were unaffected by flow. TERT1-OAT1 cells exhibited inhibitable para-aminohippurate transport, it was enhanced by flow. However, efficient tenofovir secretion and perfluorooctanoic acid reabsorption by TERT1-OAT1 cells were not modulated by flow. Gene expression showed that TERT1 and TERT1-OAT1 cells were most correlated with human kidney than other cell lines, but that flow did not have noticeable effects. Overall, our data show that addition of flow to in vitro studies of the renal proximal tubule may afford benefits in some aspects of modeling kidney function, but that careful consideration of the impact such adaptations would have on the cost and throughput of the experiments is needed. Significance Statement The topic of reproducibility and robustness of the complex microphysiological systems is looming large in the field of biomedical research; therefore, the uptake of these new models by the end-users is slow. This study systematically compared various RPTEC sources and experimental conditions, aiming to identify the level of model complexity needed for testing renal tubule transport. We demonstrate that while tissue chips may afford some benefits, their throughput and complexity need careful consideration in each context of use.

Evaluating scientific confidence in the concordance of in vitro and in vivo protective points of departure.

To fulfil the promise of reducing reliance on mammalian in vivo laboratory animal studies, new approach methods (NAMs) need to provide a confident basis for regulatory decision-making. However, previous attempts to develop in vitro NAMs-based points of departure (PODs) have yielded mixed results, with PODs from U.S. EPA's ToxCast, for instance, appearing more conservative (protective) but poorly correlated with traditional in vivo studies. Here, we aimed to address this discordance by reducing the heterogeneity of in vivo PODs, accounting for species differences, and enhancing the biological relevance of in vitro PODs. However, we only found improved in vitro-to-in vivo concordance when combining the use of Bayesian model averaging-based benchmark dose modeling for in vivo PODs, allometric scaling for interspecies adjustments, and human-relevant in vitro assays with multiple induced pluripotent stem cell-derived models. Moreover, the available sample size was only 15 chemicals, and the resulting level of concordance was only fair, with correlation coefficients <0.5 and prediction intervals spanning several orders of magnitude. Overall, while this study suggests several ways to enhance concordance and thereby increase scientific confidence in vitro NAMs-based PODs, it also highlights challenges in their predictive accuracy and precision for use in regulatory decision making.

Risk-based prioritization of PFAS using phenotypic and transcriptomic data from human induced pluripotent stem cell-derived hepatocytes and cardiomyocytes.

Per- and polyfluoroalkyl substances (PFAS) are chemicals with important applications; they are persistent in the environment and may pose human health hazards. Regulatory agencies are considering restrictions and bans of PFAS; however, little data exists for informed decisions. Several prioritization strategies were proposed for evaluation of potential hazards of PFAS. Structure-based grouping could expedite the selection of PFAS for testing; still, the hypothesis that structure-effect relationships exist for PFAS requires confirmation. We tested 26 structurally diverse PFAS from 8 groups using human-induced pluripotent stem cell-derived hepatocytes and cardiomyocytes, and tested concentration-response effects on cell function and gene expression. Few phenotypic effects were observed in hepatocytes, but negative chronotropy was observed for 8 of the 26 PFAS. Substance- and cell type-dependent transcriptomic changes were more prominent but lacked substantial group-specific effects. In hepatocytes, we found up-regulation of stress-related and extracellular matrix organization pathways, and down-regulation of fat metabolism. In cardiomyocytes, contractility-related pathways were most affected. We derived phenotypic and transcriptomic points of departure and compared them to predicted PFAS exposures. The conservative estimates for bioactivity and exposure were used to derive bioactivity-to-exposure ratio (BER) for each PFAS, most (23 of 26) PFAS had BER > 1. Overall, these data suggests that structure-based grouping of PFAS may not be sufficient to predict their biological effects. Testing of individual PFAS may be needed for scientific-based decision-making. Our proposed strategy of using two human cell types and considering phenotypic and transcriptomic effects, combined with dose-response analysis and calculation of BER, may be used for PFAS prioritization.

Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals used in many products. However, most of these substances have not been tested for safety, and concerns exist that they may be harmful to human health and/or the environment. This study aimed to use human cell-based models to investigate if some of the PFAS may exhibit hazardous properties and if similarities among substances are observed. Few effects were observed in liver cells, but a decrease in beating frequency was observed in heart cells for some PFAS. Gene expression changes were substance- and cell type-dependent. We did not find convincing structure-based similarities among PFAS; this suggests that testing of individual PFAS may be necessary in the future to inform health decisions. Overall, this study showed that a test strategy of using two human cell types, from liver and heart, may inform PFAS prioritization without a need for testing in animals.

Hazard and risk characterization of 56 structurally diverse PFAS using a targeted battery of broad coverage assays using six human cell types.

Per- and poly-fluoroalkyl substances (PFAS) are extensively used in commerce leading to their prevalence in the environment. Due to their chemical stability, PFAS are considered to be persistent and bioaccumulative; they are frequently detected in both the environment and humans. Because of this, PFAS as a class (composed of hundreds to thousands of chemicals) are contaminants of very high concern. Little information is available for the vast majority of PFAS, and regulatory agencies lack safety data to determine whether exposure limits or restrictions are needed. Cell-based assays are a pragmatic approach to inform decision-makers on potential health hazards; therefore, we hypothesized that a targeted battery of human in vitro assays can be used to determine whether there are structure-bioactivity relationships for PFAS, and to characterize potential risks by comparing bioactivity (points of departure) to exposure estimates. We tested 56 PFAS from 8 structure-based subclasses in concentration response (0.1-100 µM) using six human cell types selected from target organs with suggested adverse effects of PFAS - human induced pluripotent stem cell (iPSC)-derived hepatocytes, neurons, and cardiomyocytes, primary human hepatocytes, endothelial and HepG2 cells. While many compounds were without effect; certain PFAS demonstrated cell-specific activity highlighting the necessity of using a compendium of in vitro models to identify potential hazards. No class-specific groupings were evident except for some chain length- and structure-related trends. In addition, margins of exposure (MOE) were derived using empirical and predicted exposure data. Conservative MOE calculations showed that most tested PFAS had a MOE in the 1-100 range; ∼20% of PFAS had MOE<1, providing tiered priorities for further studies. Overall, we show that a compendium of human cell-based models can be used to derive bioactivity estimates for a range of PFAS, enabling comparisons with human biomonitoring data. Furthermore, we emphasize that establishing structure-bioactivity relationships may be challenging for the tested PFAS.

Analysis of reproducibility and robustness of a renal proximal tubule microphysiological system OrganoPlate 3-lane 40 for in vitro studies of drug transport and toxicity.

Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir, and perfluorooctanoic acid) versus large molecules (fluorescent dextrans, 60-150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate 3-lane 40 as compared with multiwell cultures are the presence of media flow, albeit intermittent, and increased throughput compared with other microfluidic models. However, OrganoPlate 3-lane 40 model appeared to offer only limited (eg, MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared with the multiwell plate culture conditions. Although OrganoPlate 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multiwell culture and microfluidic OrganoPlate 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule.

Integrative Chemical-Biological Grouping of Complex High Production Volume Substances from Lower Olefin Manufacturing Streams.

Human cell-based test methods can be used to evaluate potential hazards of mixtures and products of petroleum refining ("unknown or variable composition, complex reaction products, or biological materials" substances, UVCBs). Analyses of bioactivity and detailed chemical characterization of petroleum UVCBs were used separately for grouping these substances; a combination of the approaches has not been undertaken. Therefore, we used a case example of representative high production volume categories of petroleum UVCBs, 25 lower olefin substances from low benzene naphtha and resin oils categories, to determine whether existing manufacturing-based category grouping can be supported. We collected two types of data: nontarget ion mobility spectrometry-mass spectrometry of both neat substances and their organic extracts and in vitro bioactivity of the organic extracts in five human cell types: umbilical vein endothelial cells and induced pluripotent stem cell-derived hepatocytes, endothelial cells, neurons, and cardiomyocytes. We found that while similarity in composition and bioactivity can be observed for some substances, existing categories are largely heterogeneous. Strong relationships between composition and bioactivity were observed, and individual constituents that determine these associations were identified. Overall, this study showed a promising approach that combines chemical composition and bioactivity data to better characterize the variability within manufacturing categories of petroleum UVCBs.

Cumulative Risk Meets Inter-Individual Variability: Probabilistic Concentration Addition of Complex Mixture Exposures in a Population-Based Human In Vitro Model.

Although humans are continuously exposed to complex chemical mixtures in the environment, it has been extremely challenging to investigate the resulting cumulative risks and impacts. Recent studies proposed the use of "new approach methods," in particular in vitro assays, for hazard and dose−response evaluation of mixtures. We previously found, using five human cell-based assays, that concentration addition (CA), the usual default approach to calculate cumulative risk, is mostly accurate to within an order of magnitude. Here, we extend these findings to further investigate how cell-based data can be used to quantify inter-individual variability in CA. Utilizing data from testing 42 Superfund priority chemicals separately and in 8 defined mixtures in a human cell-based population-wide in vitro model, we applied CA to predict effective concentrations for cytotoxicity for each individual, for "typical" (median) and "sensitive" (first percentile) members of the population, and for the median-to-sensitive individual ratio (defined as the toxicodynamic variability factor, TDVF). We quantified the accuracy of CA with the Loewe Additivity Index (LAI). We found that LAI varies more between different mixtures than between different individuals, and that predictions of the population median are generally more accurate than predictions for the "sensitive" individual or the TDVF. Moreover, LAI values were generally <1, indicating that the mixtures were more potent than predicted by CA. Together with our previous studies, we posit that new approach methods data from human cell-based in vitro assays, including multiple phenotypes in diverse cell types and studies in a population-wide model, can fill critical data gaps in cumulative risk assessment, but more sophisticated models of in vitro mixture additivity and bioavailability may be needed. In the meantime, because simple CA models may underestimate potency by an order of magnitude or more, either whole-mixture testing in vitro or, alternatively, more stringent benchmarks of cumulative risk indices (e.g., lower hazard index) may be needed to ensure public health protection.

A Population-Based Human In Vitro Approach to Quantify Inter-Individual Variability in Responses to Chemical Mixtures.

Human cell-based population-wide in vitro models have been proposed as a strategy to derive chemical-specific estimates of inter-individual variability; however, the utility of this approach has not yet been tested for cumulative exposures in mixtures. This study aimed to test defined mixtures and their individual components and determine whether adverse effects of the mixtures were likely to be more variable in a population than those of the individual chemicals. The in vitro model comprised 146 human lymphoblastoid cell lines from four diverse subpopulations of European and African descent. Cells were exposed, in concentration−response, to 42 chemicals from diverse classes of environmental pollutants; in addition, eight defined mixtures were prepared from these chemicals using several exposure- or hazard-based scenarios. Points of departure for cytotoxicity were derived using Bayesian concentration−response modeling and population variability was quantified in the form of a toxicodynamic variability factor (TDVF). We found that 28 chemicals and all mixtures exhibited concentration−response cytotoxicity, enabling calculation of the TDVF. The median TDVF across test substances, for both individual chemicals or defined mixtures, ranged from a default assumption (101/2) of toxicodynamic variability in human population to >10. The data also provide a proof of principle for single-variant genome-wide association mapping for toxicity of the chemicals and mixtures, although replication would be necessary due to statistical power limitations with the current sample size. This study demonstrates the feasibility of using a set of human lymphoblastoid cell lines as an in vitro model to quantify the extent of inter-individual variability in hazardous properties of both individual chemicals and mixtures. The data show that population variability of the mixtures is unlikely to exceed that of the most variable component, and that similarity in genome-wide associations among components may be used to accrue additional evidence for grouping of constituents in a mixture for cumulative assessments.

Development of Duration-Based Non-Mutagenic Thresholds of Toxicological Concern (TTCs) Relevant to Parenteral Extractables and Leachables (E&Ls).

The threshold of toxicological concern (TTC), i.e., the dose of a compound lacking sufficient experimental toxicity data that is unlikely to result in an adverse health effect in humans, is important for evaluating extractables and leachables (E&Ls) as it guides analytical testing and minimizes the use of animal studies. The Extractables and Leachables Safety Information Exchange (ELSIE) consortium, which consists of member companies that span biotechnology, pharmaceutical, and medical device industries, brought together subject matter expert toxicologists to derive TTC values for organic, non-mutagenic E&L substances when administered parenterally. A total of 488 E&L compounds from the ELSIE database were analyzed and parenteral point of departure (PPOD) estimates were derived for 252 compounds. The PPOD estimates were adjusted to extrapolate to subacute, subchronic, and chronic durations of nonclinical exposure and the lower fifth percentiles were calculated. An additional 100-fold adjustment factor to account for nonclinical species and human variability was subsequently applied to derive the parenteral TTC values for E&Ls. The resulting parenteral TTC values are 35, 110, and 180 µg/day for human exposures of >10 years to lifetime, >1-10 years, and ≤1 year, respectively. These parenteral TTCs are expected to be conservative for E&Ls that are considered non-mutagenic per ICH M7(R1) guidelines.

Dosing Methods to Enable Cell-Based In Vitro Testing of Complex Substances: A Case Study with a PAH Mixture.

Cell-based testing of multi-constituent substances and mixtures for their potential adverse health effects is difficult due to their complex composition and physical-chemical characteristics. Various extraction methods are typically used to enable studies in vitro; however, a limited number of solvents are biocompatible with in vitro studies and the extracts may not fully represent the original test article's composition. While the methods for dosing with "difficult-to-test" substances in aquatic toxicity studies are well defined and widely used, they are largely unsuited for small-volume (100 microliters or less) in vitro studies with mammalian cells. Therefore, we aimed to evaluate suitability of various scaled-down dosing methods for high-throughput in vitro testing by using a mixture of polycyclic aromatic hydrocarbons (PAH). Specifically, we compared passive dosing via silicone micro-O-rings, cell culture media-accommodated fraction, and traditional solvent (dimethyl sulfoxide) extraction procedures. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was used to evaluate kinetics of PAH absorption to micro-O-rings, as well as recovery of PAH and the extent of protein binding in cell culture media with and without cells for each dosing method. Bioavailability of the mixture from different dosing methods was also evaluated by characterizing in vitro cytotoxicity of the PAH mixture using EA.hy926 and HepG2 human cell lines. Of the tested dosing methods, media accommodated fraction (MAF) was determined to be the most appropriate method for cell-based studies of PAH-containing complex substances and mixtures. This conclusion is based on the observation that the highest fraction of the starting materials can be delivered using media accommodated fraction approach into cell culture media and thus enable concentration-response in vitro testing.