ERRATUM.

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

Development of an In Vitro Human Thyroid Microtissue Model for Chemical Screening.

Thyroid hormones (TH) are essential for regulating a number of diverse physiological processes required for normal growth, development, and metabolism. The US EPA Endocrine Disruptor Screening Program (EDSP) has identified several molecular thyroid targets relevant to hormone synthesis dynamics that have been adapted to high-throughput screening (HTS) assays to rapidly evaluate the ToxCast/Tox21 chemical inventories for potential thyroid disrupting chemicals (TDCs). The uncertainty surrounding the specificity of active chemicals identified in these screens and the relevance to phenotypic effects on in vivo human TH synthesis are notable data gaps for hazard identification of TDCs. The objective of this study was to develop a medium-throughput organotypic screening assay comprised of reconstructed human thyroid microtissues to quantitatively evaluate the disruptive effects of chemicals on TH production and secretion. Primary human thyroid cells procured from qualified euthyroid donors were analyzed for retention of NK2 homeobox 1 (NKX2-1), Keratin 7 (KRT7), and Thyroglobulin (TG) protein expression by high-content image analysis to verify enrichment of follicular epithelial cells. A direct comparison of 2-dimensional (2D) and 3-dimensional (3D) 96-well culture formats was employed to characterize the morphology, differential gene expression, TG production, and TH synthesis over the course of 20 days. The results indicate that modeling human thyroid cells in the 3D format was sufficient to restore TH synthesis not observed in the 2D culture format. Inhibition of TH synthesis in an optimized 3D culture format was demonstrated with reference chemicals for key molecular targets within the thyroid gland. Implementation of the assay may prove useful for interpreting phenotypic effects of candidate TDCs identified by HTS efforts currently underway in the EDSP.

Identifying qualitative differences in PPARα signaling networks in human and rat hepatocytes and their significance for next generation chemical risk assessment methods.

In this paper, we evaluate the PPARα signaling network in rats, examining transcriptional responses in primary hepatocytes exposed to a PPARα specific ligand, GW7647. These transcriptomic studies were complemented with ChIP-seq studies of PPARα binding and transcription binding motif identification for PPARα responsive genes. We also conducted a limited study of GW7647 dosing the in intact rat to examine differences in transcriptional responses for primary hepatocytes in vitro and in the intact liver. The rat network has a much larger number of down-regulated genes and pathways than we had found in the human and the PPARα binding motifs in rat differed for upregulated and down regulated genes. Based on these results and comparison with our previous work with the human PPARα signaling network, we identified qualitative differences in the transcriptional networks controlled by PPARα activation in the two species that provide an explanation of the interspecies differences in the responses of humans and rodents to GW7647 and likely to other PPARα agonists. These studies also allow some observations on the manner in which in vitro, fit-for-purpose assays in human hepatocytes could form the basis for risk assessment without recourse to in-life studies in rodents or other test species.

Long-Term Engineered Cultures of Primary Mouse Hepatocytes for Strain and Species Comparison Studies During Drug Development.

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

Comparative analysis of metabolism of trichloroethylene and tetrachloroethylene among mouse tissues and strains.

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are structurally similar chemicals that are metabolized through oxidation and glutathione conjugation pathways. Both chemicals have been shown to elicit liver and kidney toxicity in rodents and humans; however, TCE has been studied much more extensively in terms of both metabolism and toxicity. Despite their qualitative similarities, quantitative comparison of tissue- and strain-specific metabolism of TCE and PCE has not been performed. To fill this gap, we conducted a comparative toxicokinetic study where equimolar single oral doses of TCE (800 mg/kg) or PCE (1000 mg/kg) were administered to male mice of C57BL/6J, B6C3F1/J, and NZW/LacJ strains. Samples of liver, kidney, serum, brain, and lung were obtained for up to 36 h after dosing. For each tissue, concentrations of parent compounds, as well as their oxidative and glutathione conjugation metabolites were measured and concentration-time profiles constructed. A multi-compartment toxicokinetic model was developed to quantitatively compare TCE and PCE metabolism. As expected, the flux through oxidation metabolism pathway predominated over that through conjugation across all mouse strains examined, it is 1,200-3,800 fold higher for TCE and 26-34 fold higher for PCE. However, the flux through glutathione conjugation, albeit a minor metabolic pathway, was 21-fold higher for PCE as compared to TCE. The degree of inter-strain variability was greatest for oxidative metabolites in TCE-treated and for glutathione conjugation metabolites in PCE-treated mice. This study provides critical data for quantitative comparisons of TCE and PCE metabolism, and may explain the differences in organ-specific toxicity between these structurally similar chemicals.

Editor's Highlight: Comparative Dose-Response Analysis of Liver and Kidney Transcriptomic Effects of Trichloroethylene and Tetrachloroethylene in B6C3F1 Mouse.

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are ubiquitous environmental contaminants and occupational health hazards. Recent health assessments of these agents identified several critical data gaps, including lack of comparative analysis of their effects. This study examined liver and kidney effects of TCE and PCE in a dose-response study design. Equimolar doses of TCE (24, 80, 240, and 800 mg/kg) or PCE (30, 100, 300, and 1000 mg/kg) were administered by gavage in aqueous vehicle to male B6C3F1/J mice. Tissues were collected 24 h after exposure. Trichloroacetic acid (TCA), a major oxidative metabolite of both compounds, was measured and RNA sequencing was performed. PCE had a stronger effect on liver and kidney transcriptomes, as well as greater concentrations of TCA. Most dose-responsive pathways were common among chemicals/tissues, with the strongest effect on peroxisomal ß-oxidation. Effects on liver and kidney mitochondria-related pathways were notably unique to PCE. We performed dose-response modeling of the transcriptomic data and compared the resulting points of departure (PODs) to those for apical endpoints derived from long-term studies with these chemicals in rats, mice, and humans, converting to human equivalent doses using tissue-specific dosimetry models. Tissue-specific acute transcriptional effects of TCE and PCE occurred at human equivalent doses comparable to those for apical effects. These data are relevant for human health assessments of TCE and PCE as they provide data for dose-response analysis of the toxicity mechanisms. Additionally, they provide further evidence that transcriptomic data can be useful surrogates for in vivo PODs, especially when toxicokinetic differences are taken into account.

Dose-response in a high density three-dimensional liver device with real-time bioenergetic and metabolic flux quantification.

Real-time dose-response curves for fructose have been non-invasively determined in primary rat hepatocyte alginate spheroids cultured in a NMR-compatible fluidized-bed bioreactor. Using 13C-labeled glucose and glycine culture medium, fructose dose was compared to glucose uptake and glycogen synthesis rate using 13C NMR spectroscopy, and to ATP and fructose-1-phosphate concentration using 31P NMR spectroscopy. A highly efficient multicoaxial perfusion system maintains high density 3-D hepatocyte cultures, permitting 13C and 31P NMR spectral time courses with 1min time points. The perfusion system was turned off to demonstrate its efficiency and effect on the metabolites. Within 16min, glycogen plummeted, lactate became the largest 13C-glucose metabolite via anaerobic glycolysis, while glutathione was the largest 13C-glycine metabolite. ATP depletion and fructose-1-phosphate formation demonstrated a dose response with a 3h EC50 of 19mM±8.9mM and 17.4mM±3.7mM, respectively. Computational modeling of mass transfer corroborated experimental results and helped determine the optimal bioreactor loading densities, oxygen concentration, and perfusion rates to maintain physiologically-relevant nutrient levels. The total bioreactor plus perfusion loop has a dead volume of 2ml, and contains 5 million hepatocytes. Due to the non-invasive measurements, there is a reduction of animal tissue by an order-of-magnitude, depending on the number of time points in an experiment. This dynamic flux approach may have generic utility for dose-response studies monitoring multiple metabolic reactions in other primary mammalian cells, such as human, that have strict oxygen demands.

Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.

Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 µg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species.

In vitro models for liver toxicity testing.

Over the years, various liver-derived in vitro model systems have been developed to enable investigation of the potential adverse effects of chemicals and drugs. Liver tissue slices, isolated microsomes, perfused liver, immortalized cell lines, and primary hepatocytes have been used extensively. Immortalized cell lines and primary isolated liver cells are currently most widely used in vitro models for liver toxicity testing. Limited throughput, loss of viability, and decreases in liver-specific functionality and gene expression are common shortcomings of these models. Recent developments in the field of in vitro hepatotoxicity include three-dimensional tissue constructs and bioartificial livers, co-cultures of various cell types with hepatocytes, and differentiation of stem cells into hepatic lineage-like cells. In an attempt to provide a more physiological environment for cultured liver cells, some of the novel cell culture systems incorporate fluid flow, micro-circulation, and other forms of organotypic microenvironments. Co-cultures aim to preserve liver-specific morphology and functionality beyond those provided by cultures of pure parenchymal cells. Stem cells, both embryonic- and adult tissue-derived, may provide a limitless supply of hepatocytes from multiple individuals to improve reproducibility and enable testing of the individual-specific toxicity. This review describes various traditional and novel in vitro liver models and provides a perspective on the challenges and opportunities afforded by each individual test system.

Evaluation of an in vitro toxicogenetic mouse model for hepatotoxicity.

Numerous studies support the fact that a genetically diverse mouse population may be useful as an animal model to understand and predict toxicity in humans. We hypothesized that cultures of hepatocytes obtained from a large panel of inbred mouse strains can produce data indicative of inter-individual differences in in vivo responses to hepato-toxicants. In order to test this hypothesis and establish whether in vitro studies using cultured hepatocytes from genetically distinct mouse strains are feasible, we aimed to determine whether viable cells may be isolated from different mouse inbred strains, evaluate the reproducibility of cell yield, viability and functionality over subsequent isolations, and assess the utility of the model for toxicity screening. Hepatocytes were isolated from 15 strains of mice (A/J, B6C3F1, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, BALB/cByJ, AKR/J, MRL/MpJ, NOD/LtJ, NZW/LacJ, PWD/PhJ and WSB/EiJ males) and cultured for up to 7 days in traditional 2-dimensional culture. Cells from B6C3F1, C57BL/6J, and NOD/LtJ strains were treated with acetaminophen, WY-14,643 or rifampin and concentration-response effects on viability and function were established. Our data suggest that high yield and viability can be achieved across a panel of strains. Cell function and expression of key liver-specific genes of hepatocytes isolated from different strains and cultured under standardized conditions are comparable. Strain-specific responses to toxicant exposure have been observed in cultured hepatocytes and these experiments open new opportunities for further developments of in vitro models of hepatotoxicity in a genetically diverse population.