Validation of gene expression profiles from cholestatic hepatotoxicants in vitro against human in vivo cholestasis.

Drug-induced liver injury remains the most common cause of acute liver failure and a frequently indicated reason for withdrawal of drugs. For the purpose of evaluating the relevance of liver cell models for assessing hepatotoxic risks in intact humans, we here aimed to benchmark 'omics-derived mechanistic data from three in vitro models for parenchymal liver function, intended for the investigation of drug-induced cholestasis, against 'omics data from cholestatic patients. Transcriptomic changes in HepG2 cells, primary mouse hepatocytes and primary human hepatocytes exposed to known cholestatic compounds were analyzed using microarrays.

Exploiting microRNA and mRNA profiles generated in vitro from carcinogen-exposed primary mouse hepatocytes for predicting in vivo genotoxicity and carcinogenicity.

The well-defined battery of in vitro systems applied within chemical cancer risk assessment is often characterised by a high false-positive rate, thus repeatedly failing to correctly predict the in vivo genotoxic and carcinogenic properties of test compounds. Toxicogenomics, i.e.

Evaluating microRNA profiles reveals discriminative responses following genotoxic or non-genotoxic carcinogen exposure in primary mouse hepatocytes.

Chemical carcinogenesis can be induced by genotoxic (GTX) or non-genotoxic (NGTX) carcinogens. GTX carcinogens have a well-described mode of action. However, the complex mechanisms by which NGTX carcinogens act are less clear and may result in conflicting results between species [e.

Integrating multiple omics to unravel mechanisms of Cyclosporin A induced hepatotoxicity in vitro.

In order to improve attrition rates of candidate-drugs there is a need for a better understanding of the mechanisms underlying drug-induced hepatotoxicity. We aim to further unravel the toxicological response of hepatocytes to a prototypical cholestatic compound by integrating transcriptomic and metabonomic profiling of HepG2 cells exposed to Cyclosporin A. Cyclosporin A exposure induced intracellular cholesterol accumulation and diminished intracellular bile acid levels.

Epigenetic mechanisms underlying arsenic-associated lung carcinogenesis.

Arsenic is an established human carcinogen, but the mechanisms through which it contributes to for instance lung cancer development are still unclear. As arsenic is methylated during its metabolism, it may interfere with the DNA methylation process, and is therefore considered to be an epigenetic carcinogen. In the present study, we hypothesize that arsenic is able to induce DNA methylation changes, which lead to changes in specific gene expression, in pathways associated with lung cancer promotion and progression.

Integrative cross-omics analysis in primary mouse hepatocytes unravels mechanisms of cyclosporin A-induced hepatotoxicity.

The liver is responsible for drug metabolism and drug-induced hepatotoxicity is the most frequent reason for drug withdrawal, indicating that better pre-clinical toxicity tests are needed. In order to bypass animal models for toxicity screening, we exposed primary mouse hepatocytes for exploring the prototypical hepatotoxicant cyclosporin A. To elucidate the mechanisms underlying cyclosporin A-induced hepatotoxicity, we analyzed expression levels of proteins, mRNAs, microRNAs and metabolites.

Elimination of heparin interference during microarray processing of fresh and biobank-archived blood samples.

In the context of environmental health research, biobank blood samples have recently been identified as suitable for high-throughput omics analyses enabling the identification of new biomarkers of exposure and disease. However, blood samples containing the anti-coagulant heparin could complicate transcriptomic analysis because heparin may inhibit RNA polymerase causing inefficient cRNA synthesis and fluorophore labelling. We investigated the inhibitory effect of heparin and the influence of storage conditions (0 or 3 hr bench times, storage at room temperature or -80°C) on fluorophore labelling in heparinized fresh human buffy coat and whole blood biobank samples during the mRNA work-up protocol for microarray analysis.

Classification of hepatotoxicants using HepG2 cells: A proof of principle study.

With the number of new drug candidates increasing every year, there is a need for high-throughput human toxicity screenings. As the liver is the most important organ in drug metabolism and thus capable of generating relatively high levels of toxic metabolites, it is important to find a reliable strategy to screen for drug-induced hepatotoxicity. Microarray-based transcriptomics is a well-established technique in toxicogenomics research and is an ideal approach to screen for drug-induced injury at an early stage.

Characterisation of cisplatin-induced transcriptomics responses in primary mouse hepatocytes, HepG2 cells and mouse embryonic stem cells shows conservation of regulating transcription factor networks.

The toxic mechanisms of cisplatin have been frequently studied in many species and in vitro cell models. The Netherlands Toxicogenomics Centre focuses on developing in vitro alternatives using genomics technologies for animal-based assays on, e.g.

RNA-Seq provides new insights in the transcriptome responses induced by the carcinogen benzo[a]pyrene.

Whole-genome transcriptome measurements are pivotal for characterizing molecular mechanisms of chemicals and predicting toxic classes, such as genotoxicity and carcinogenicity, from in vitro and in vivo assays. In recent years, deep sequencing technologies have been developed that hold the promise of measuring the transcriptome in a more complete and unbiased manner than DNA microarrays. Here, we applied this RNA-seq technology for the characterization of the transcriptomic responses in HepG2 cells upon exposure to benzo[a]pyrene (BaP), a well-known DNA damaging human carcinogen.

Benzo[a]pyrene-induced changes in microRNA-mRNA networks.

Toxicological studies assessing the safety of compounds for humans frequently use in vitro systems to characterize toxic responses in combination with transcriptomic analyses. Thus far, changes have mostly been investigated at the mRNA level. Recently, microRNAs have attracted attention because they are powerful negative regulators of mRNA levels and, thus, may be responsible for the modulation of important mRNA networks implicated in toxicity.

Time-series analysis of gene expression profiles induced by nitrosamides and nitrosamines elucidates modes of action underlying their genotoxicity in human colon cells.

N-nitroso compounds (NOCs) may represent a carcinogenic risk to humans following endogenous colonic nitrosation processes. We used the colon adenocarcinoma cell line Caco-2 to investigate transcriptomic changes at three time points (1, 6, 24 h) following exposure to genotoxic concentrations of six different NOCs (two nitrosamides, four nitrosamines) with the purpose of identifying biological processes that may play a part in the carcinogenicity of these compounds. This is especially important for nitrosamide exposure where, in light of their high reactivity, important gene expression modifications may take place early in the exposure.

Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures.

The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.

Evaluation of developmental toxicant identification using gene expression profiling in embryonic stem cell differentiation cultures.

The murine embryonic stem cell test (EST) is an alternative testing method designed to assess potential developmental toxicity of compounds. The implementation of transcriptomics in the EST has been shown to reduce the culture duration and improve endpoint evaluation and is expected to result in an enhanced predictability and definition of the applicability domain. We evaluated the identification of developmental toxicity in the EST using two gene sets ("Van_Dartel_heartdiff_24h" and "EST biomarker genes") defined in our earlier studies.

Gene expression profiling in primary mouse hepatocytes discriminates true from false-positive genotoxic compounds.

Well-established in vitro methods for testing the genotoxic potency of chemicals--such as the Ames/Salmonella test, the mouse lymphoma assay, the micronucleus test and the chromosomal aberration test--show a high false-positive rate for predicting in vivo genotoxicity and carcinogenicity. Thus, there is a need for more reliable in vitro assays. We investigated whether gene expression profiling in metabolically competent primary mouse hepatocytes is capable of discriminating true genotoxic (GTX) compounds from false-positive genotoxic (FP-GTX) compounds.

Time series analysis of benzo[A]pyrene-induced transcriptome changes suggests that a network of transcription factors regulates the effects on functional gene sets.

Chemical carcinogens may cause a multitude of effects inside cells, thereby affecting transcript levels of genes by direct activation of transcription factors (TF) or indirectly through the formation of DNA damage. As the temporal profiles of these responses may be profoundly different, examining time-dependent changes may provide new insights in TF networks related to cellular responses to chemical carcinogens. Therefore, we investigated in human hepatoma cells gene expression changes caused by benzo[a]pyrene at 12 time points after exposure, in relation to DNA adduct and cell cycle.

Inhibition of acute pulmonary and systemic inflammation by 1,7-dimethylxanthine.

The nuclear enzyme poly(ADP-ribose) polymerse-1 (PARP-1) has previously been reported to play an important role in lipopolysaccharide (LPS)-induced pulmonary inflammation and is highly activated in COPD patients. In the present study, the anti-inflammatory efficacy of a previously identified poly(ADP-ribose) polymerase-1 (PARP-1) inhibiting caffeine metabolite, 1,7-dimethylxanthine, was both in vivo as well as ex vivo evaluated. Orally administered 1,7-dimethylxanthine significantly attenuated lung myeloperoxidase-levels, transcription of IL-6, TNF-alpha, MIP1alpha and MIP2 genes as well as PAR-polymer formation in a mouse model of intratracheally LPS-induced acute pulmonary inflammation.

Inhibition of LPS-induced pulmonary inflammation by specific flavonoids.

In the present study, the anti-inflammatory effects of the flavonoids flavone, fisetin and tricetin were evaluated in a mouse model of LPS-induced acute pulmonary inflammation. The flavonoid fisetin significantly reduced lung myeloperoxidase-levels and gene-expression of inflammatory mediators such as IL-6, TNF-alpha, IL-1beta, MIP-1alpha and MIP-2. The LPS-induced gene transcription of HO-1 and SOD2 was also significantly reduced by fisetin.

Assessing the metabolic competence of sandwich-cultured mouse primary hepatocytes.

Primary human and rat hepatocyte cultures are well established in vitro systems used in toxicological studies. However, whereas transgenic mouse models provide an opportunity for studying mechanisms of toxicity, mouse primary hepatocyte cultures are less well described. The potential usefulness of a mouse hepatocyte-based in vitro model was assessed in this study by investigating time-dependent competence for xenobiotic metabolism and gene expression profiles.

Dietary flavones and flavonoles are inhibitors of poly(ADP-ribose)polymerase-1 in pulmonary epithelial cells.

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1), which was initially known to be highly activated by oxidative stress-induced DNA strand breaks, has been shown to be involved in the pathophysiology of acute and chronic inflammatory diseases. PARP-1 deficiency in mice led to the discovery of its coactivating function in the nuclear factor-kappa B-mediated gene expression and in addition, pharmaceutical inhibition of PARP-1 was shown to reduce the production of inflammatory mediators. In this study, the in vitro PARP-1-inhibiting effect of various flavonoids was investigated.

Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair.

There is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacities. Therefore, a modification of the alkaline comet assay was developed to determine the ability of human lymphocyte extracts to perform the initial steps of the nucleotide excision repair (NER) process, i.e.