The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.Toxicolog

Stimulation of de novo glutathione synthesis by nitrofurantoin for enhanced resilience of hepatocytes

Toxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG-132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could thus be a promising strategy for a transient and targeted activation of the endogenous antioxidant machinery. Graphical abstract.Toxicolog

Histone deacetylase 5 regulates interleukin 6 secretion and insulin action in skeletal muscle.

Objective: Physical exercise training is associated with increased glucose uptake in skeletal muscle and improved glycemic control. HDAC5, a class IIa histone deacetylase, has been shown to regulate transcription of the insulin-responsive glucose transporter GLUT4 in cultured muscle cells. In this study, we analyzed the contribution of HDAC5 to the transcriptional network in muscle and the beneficial effect of muscle contraction and regular exercise on glucose metabolism. Methods: HDAC5 knockout mice (KO) and wild-type (WT) littermates were trained for 8 weeks on treadmills, metabolically phenotyped, and compared to sedentary controls. Hdac5-deficient skeletal muscle and cultured Hdac5-knockdown (KD) C2C12 myotubes were utilized for studies of gene expression and glucose metabolism. Chromatin immunoprecipitation (ChIP) studies were conducted to analyze Il6 promoter activity using H3K9ac and HDAC5 antibodies. Results: Global transcriptome analysis of Hdac5 KO gastrocnemius muscle demonstrated activation of the IL-6 signaling pathway. Accordingly, knockdown of Hdac5 in C2C12 myotubes led to higher expression and secretion of IL-6 with enhanced insulin-stimulated activation of AKT that was reversed by Il6 knockdown. Moreover, Hdac5-deficient myotubes exhibited enhanced glucose uptake, glycogen synthesis, and elevated expression levels of the glucose transporter GLUT4. Transcription of Il6 was further enhanced by electrical pulse stimulation in Hdac5-deficient C2C12 myotubes. ChIP identified a ∼1 kb fragment of the Il6 promoter that interacts with HDAC5 and demonstrated increased activation-associated histone marker AcH3K9 in Hdac5-deficient muscle cells. Exercise intervention of HDAC5 KO mice resulted in improved systemic glucose tolerance as compared to WT controls. Conclusions: We identified HDAC5 as a negative epigenetic regulator of IL-6 synthesis and release in skeletal muscle. HDAC5 may exert beneficial effects through two different mechanisms, transcriptional control of genes required for glucose disposal and utilization, and HDAC5-dependent IL-6 signaling cross-talk to improve glucose uptake in muscle in response to exercise

Comparing in vitro human liver models to in vivo human liver using RNA-Seq

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.Toxicolog

In vitro/in silico prediction of drug induced steatosis in relation to oral doses and blood concentrations by the Nile Red assay.

The accumulation of lipid droplets in hepatocytes is a key feature of drug-induced liver injury (DILI) and can be induced by a subset of hepatotoxic compounds. In the present study, we optimized and evaluated an in vitro technique based on the fluorescent dye Nile Red, further named Nile Red assay to quantify lipid droplets induced by the exposure to chemicals. The Nile Red assay and a cytotoxicity test (CTB assay) were then performed on cells exposed concentration-dependently to 60 different compounds. Of these, 31 were known to induce hepatotoxicity in humans, and 13 were reported to also cause steatosis. In order to compare in vivo relevant blood concentrations, pharmacokinetic models were established for all compounds to simulate the maximal blood concentrations (Cmax) at therapeutic doses. The results showed that several hepatotoxic compounds induced an increase in lipid droplets at sub-cytotoxic concentrations. To compare how well (1) the cytotoxicity test alone, (2) the Nile Red assay alone, and (3) the combination of the cytotoxicity test and the Nile Red assay (based on the lower EC10 of both assays) allow the differentiation between hepatotoxic and non-hepatotoxic compounds, a previously established performance metric, the Toxicity Separation Index (TSI) was calculated. In addition, the Toxicity Estimation Index (TEI) was calculated to determine how well blood concentrations that cause an increased DILI risk can be estimated for hepatotoxic compounds. Our findings indicate that the combination of both assays improved the TSI and TEI compared to each assay alone. In conclusion, the study demonstrates that inclusion of the Nile Red assay into in vitro test batteries may improve the prediction of DILI compounds

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.Genome Instability and Cance