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The currently available techniques for the safety evaluation of candidate drugs are usually cost-intensive and time-consuming and are often insufficient to predict human relevant cardiotoxicity. The purpose of this study was to develop an in vitro repeated exposure toxicity methodology allowing the identification of predictive genomics biomarkers of functional relevance for drug-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The hiPSC-CMs were incubated with 156 nM doxorubicin, which is a well-characterized cardiotoxicant, for 2 or 6 days followed by washout of the test compound and further incubation in compound-free culture medium until day 14 after the onset of exposure. An xCELLigence Real-Time Cell Analyser was used to monitor doxorubicin-induced cytotoxicity while also monitoring functional alterations of cardiomyocytes by counting of the beating frequency of cardiomyocytes. Unlike single exposure, repeated doxorubicin exposure resulted in long-term arrhythmic beating in hiPSC-CMs accompanied by significant cytotoxicity. Global gene expression changes were studied using microarrays and bioinformatics tools. Analysis of the transcriptomic data revealed early expression signatures of genes involved in formation of sarcomeric structures, regulation of ion homeostasis and induction of apoptosis. Eighty-four significantly deregulated genes related to cardiac functions, stress and apoptosis were validated using real-time PCR. The expression of the 84 genes was further studied by real-time PCR in hiPSC-CMs incubated with daunorubicin and mitoxantrone, further anthracycline family members that are also known to induce cardiotoxicity. A panel of 35 genes was deregulated by all three anthracycline family members and can therefore be expected to predict the cardiotoxicity of compounds acting by similar mechanisms as doxorubicin, daunorubicin or mitoxantrone. The identified gene panel can be applied in the safety assessment of novel drug candidates as well as available therapeutics to identify compounds that may cause cardiotoxicity.

Cultivated hepatocytes represent a well-established in vitro system. However, the applicability of hepatocytes in toxicogenomics is still controversially discussed. Recently, an in vivo/in vitro discrepancy has been described, whereby the non-genotoxic rat liver carcinogen methapyrilene alters the expression of the metabolizing genes SULT1A1 and ABAT, as well as the DNA damage response gene GADD34 in vitro, but not in vivo. If the collagen sandwich cultures of hepatocytes really produce false-positive data, this would compromise its application in toxicogenomics. To revisit the putative in vivo/in vitro discrepancy, we first analyzed and modeled methapyrilene concentrations in the portal vein of rats. The relatively short half-life of 2.8 h implies a rapid decrease in orally administered methapyrilene in vivo below concentrations that can cause gene expression alterations. This corresponded to the time-dependent alteration levels of GADD34, ABAT and SULT1A1 RNA in the liver: RNA levels are altered 1, 6 and 12 h after methapyrilene administration, but return to control levels after 24 and 72 h. In contrast, methapyrilene concentrations in the culture medium supernatant of primary rat hepatocyte cultures decreased slowly. This explains why GADD34, ABAT and SULT1A1 were still deregulated after 24 h exposure in vitro, but not in vivo. It should also be considered that the earliest analyzed time point in the previous in vivo studies was 24 h after methapyrilene administration. In conclusion, previously observed in vitro/in vivo discrepancy can be explained by different pharmacokinetics present in vitro and in vivo. When the in vivo half-life is short, levels of some initially altered genes may have returned to control levels already 24 h after administration.

The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.

Although shorter chain homologues and other types of fluorinated chemicals are currently used as alternatives to long-chain perfluoroalkyl substances (PFASs), their safety information remains unclear and urgently needed. Here, the cytotoxicity of several fluorinated alternatives (i.e., 6:2 fluorotelomer carboxylic acid (6:2 FTCA), 6:2 fluorotelomer sulfonic acid (6:2 FTSA), 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA), and hexafluoropropylene oxide (HFPO) homologues) to human liver HL-7702 cell line were measured and compared with perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Their binding mode and affinity to human liver fatty acid binding protein (hL-FABP) were also determined. Compared with PFOA and PFOS, 6:2 Cl-PFESA, HFPO trimer acid (HFPO-TA), HFPO tetramer acid (HFPO-TeA), and 6:2 FTSA showed greater toxic effects on cell viabilities. At low exposure doses, these alternatives induced cell proliferation with similar mechanism which was different from that of PFOA and PFOS. Furthermore, binding affinity to hL-FABP decreased in the order of 6:2 FTCA < 6:2 FTSA < HFPO dimer acid (HFPO-DA) < PFOA < PFOS/6:2 Cl-PFESA/HFPO-TA. Due to their distinctive structure, 6:2 Cl-PFESA and HFPO homologues were bound to the hL-FABP inner pocket with unique binding modes and higher binding energy compared with PFOA and PFOS. This research enhances our understanding of the toxicity of PFAS alternatives during usage and provides useful evidence for the development of new alternatives.

The skin is the largest organ of the human body and the one mostly exposed to outdoor contaminants. To evaluate the biological mechanisms underlying skin damage caused by fine particulate matter (PM2.5), we analyzed the effects of PM2.5 on cultured human keratinocytes and the skin of experimental animals. PM2.5 was applied to human HaCaT keratinocytes at 50 µg/mL for 24 h and to mouse skin at 100 µg/mL for 7 days. The results indicate that PM2.5 induced oxidative stress by generating reactive oxygen species both in vitro and in vivo, which led to DNA damage, lipid peroxidation, and protein carbonylation. As a result, PM2.5 induced endoplasmic reticulum stress, mitochondrial swelling, and autophagy, and caused apoptosis in HaCaT cells and mouse skin tissue. The PM2.5-induced cell damage was attenuated by antioxidant N-acetyl cysteine, confirming that PM2.5 cellular toxicity was due to oxidative stress. These findings contribute to understanding of the pathophysiological mechanisms triggered in the skin by PM2.5, among which oxidative stress may play a major role.

The risk of drug-induced liver injury (DILI) poses a major challenge for development of natural products derived from traditional Chinese medicines (NP-TCMs). It is urgent to find a new method for the safety assessment of the NP-TCMs. Recent study has reported an in vitro/in silico method to estimate the acceptable daily intake of hepatotoxic compounds using support vector machine (SVM) classifier and physiologically based pharmacokinetic (PBPK) modeling. However, this method is not suitable for estimating the dosing schedule of compounds which are administered in multiple daily doses. Thus, in this study, the method mentioned above was in particular optimized, and used to estimate the hepatotoxic plasma concentrations of 17 NP-TCMs. Additionally, the oral dosing schedules of the triptolide, emodin, matrine and oxymatrine were also predicted by the SVM classifier and PBPK modeling. The optimization included that: (1) in vitro cytotoxicity data of 28 training set compounds was optimized using benchmark concentrations (BMC) modeling; (2) AUC of the training set compound was used as the in vivo metric instead of Cmax to better reflect the total daily exposure of compounds which are administered in multiple daily doses; (3) using the mean AUC in plasma as in vivo metric and BMC value as in vitro metric could achieve the better toxicity separation index (0.962 vs. 0.938); (4) The TSI for Cmax and BMC values was 0.985 calculated in this study, and the results indicated that BMC modeling improved the separation performance. This optimized in vitro–in vivo extrapolation (IVIVE) workflow could extrapolate in vitro BMC to blood concentrations and the oral dosing schedule which are corresponding to certain risk of hepatotoxicity. The estimated safe dosing schedule of oxymatrine by this optimized method was close to the clinical recommended dosing regimen. The results indicate that the optimized method could be used to predict the dosing schedule of compounds administered in multiple daily doses, and our optimized workflow could be helpful for the safety assessment as well as the research and development on NP-TCMs.

Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC 50 values. Regardless of comparing IC 50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α -GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery.

Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants with various adverse health effects in humans including disruption of lipid metabolism. Aim of the present study was to elucidate the molecular mechanisms of PFAS-mediated effects on lipid metabolism in human cells. Here, we examined the impact of a number of PFAS (PFOS, PFOA, PFNA, PFDA, PFHxA, PFBA, PFHxS, PFBS, HFPO-DA, and PMPP) and of some exposure-relevant PFAS mixtures being composed of PFOS, PFOA, PFNA and PFHxS on lipid metabolism in human HepaRG cells, an in vitro model for human hepatocytes. At near cytotoxic concentrations, the selected PFAS and PFAS mixtures induced triglyceride accumulation in HepaRG cells and consistently affected the expression of marker genes for steatosis, as well as PPARα target genes and genes related to lipid and cholesterol metabolism, pointing to common molecular mechanisms of PFAS in disrupting cellular lipid and cholesterol homeostasis. PPARα activation was examined by a transactivation assay in HEK293T cells, and synergistic effects were observed for the selected PFAS mixtures at sum concentrations higher than 25 µM, whereas additivity was observed at sum concentrations lower than 25 µM. Of note, any effect observed in the in vitro assays occurred at PFAS concentrations that were at least four to five magnitudes above real-life internal exposure levels of the general population.

2-Benzylbenzimidazole ‘nitazene’ opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on μ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure–activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by ‘ring’ substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), ‘desnitazene’ analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (β-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that ‘ring’ modifications overall yield highly active drugs. With the exception of 4′-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs’ high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.