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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. genotoxic hazards. Models such as human hepatocellular carcinoma cell line (HepG2) cells, mouse primary hepatocytes (PMH) and mouse embryonic stem cells (mESC) are used. Our aim was to identify possibly robust conserved mechanisms between these models using cisplatin as model genotoxic agent. Transcriptomic data newly generated from HepG2 cells and PMH exposed to 7 µM cisplatin for 12, 24 and 48h and 24 and 48h, respectively, were compared with published data from mESC exposed to 5 µM cisplatin for 2–24h. Due to differences in response time between models and marginal changes after shorter exposure periods, we focused on 24 and 48h. At gene level, 44 conserved differentially expressed genes (DEG), involved in processes such as apoptosis, cell cycle, DNA damage response and DNA repair, were found. Functional analysis shows that limited numbers of pathways are conserved. Transcription factor (TF) network analysis indicates 12 common TF networks responding among all models and time points. Four TF, HNF4-α, SP1, c-MYC and p53, capable of regulating ±50% of all DEG, seem of equal importance in all models and exposure periods. Here we showed that transcriptomic responses across several in vitro cell models following exposure to cisplatin are mainly determined by a conserved complex network of 4 TFs. These conserved responses are hypothesised to provide most relevant information for human toxicity prediction and may form the basis for new in vitro alternatives of risk assessment. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cholestasis is modeled in mice using cyclosporin A. [•] Progression of serum phenotype is correlated to liver gene expression changes. [•] Phenotype-directed analysis retrieves in vivo cholestasis-related gene sets. [•] In vitro model evaluation for cholestasis can be performed at the gene set level. [•] Cholestasis-related genes should be expected to differ between in vivo and in vitro. [ABSTRACT FROM AUTHOR]

As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens. [ABSTRACT FROM PUBLISHER]

The γH2AX assay has recently been suggested as a new in vitro assay for detecting genotoxic (GTX) properties of chemicals. This assay is based on the phosphorylation of H2AX histone in response to DNA damage [i.e. induction of double-strand breaks (DSBs)]. Quantification of γH2AX foci using flow cytometry can rapidly detect DNA damage induced by chemicals that cause DNA DSBs. Up to now, only few compounds have been tested with this assay. The main goal of this study was to compare the performance of this automated γH2AX assay with that of standard in vitro genotoxicity assays in predicting in vivo genotoxicity. HepG2 cells were exposed to 64 selected compounds with known GTX properties and subsequently analysed for induction of γH2AX foci. The results of this assay were compared with public data from standard in vitro genotoxicity tests. Accuracy, sensitivity and specificity in predicting in vivo genotoxicity, using the γH2AX assay alone or in combinations with conventional assays, were calculated. Both the γH2AX assay and the bacterial mutagenicity test (Ames) were highly specific for in vivo GTX, whereas chromosomal aberration/micronucleus test (CA/MN) resulted in highest sensitivity. The currently widely used in vitro genotoxicity test battery—Ames test, mouse lymphoma assay (MLA) and CA/MN test—resulted in low accuracy (55–65%) to predict in vivo genotoxicity. Interestingly, the inclusion of γH2AX assay in the standard battery, instead of MLA assay, resulted in higher accuracy (62–70%) compared with other combinations. Advantage of the γH2AX assay in HepG2 cells is its high sensitivity to detect DNA-reactive GTX compounds, although the reduced sensitivity for compounds that require metabolic activation needs to be improved. In conclusion, the automated γH2AX assay can be a useful, fast and cost-effective human cell–based tool for early screening of compounds for in vivo genotoxicity. [ABSTRACT FROM PUBLISHER]

Hepatocyte-like cells derived from the differentiation of human embryonic stem cells (hES-Hep) have potential to provide a human relevant in vitro test system in which to evaluate the carcinogenic hazard of chemicals. In this study, we have investigated this potential using a panel of 15 chemicals classified as noncarcinogens, genotoxic carcinogens, and nongenotoxic carcinogens and measured whole-genome transcriptome responses with gene expression microarrays. We applied an ANOVA model that identified 592 genes highly discriminative for the panel of chemicals. Supervised classification with these genes achieved a cross-validation accuracy of > 95%. Moreover, the expression of the response genes in hES-Hep was strongly correlated with that in human primary hepatocytes cultured in vitro. In order to infer mechanistic information on the consequences of chemical exposure in hES-Hep, we developed a computational method that measures the responses of biochemical pathways to the panel of treatments and showed that these responses were discriminative for the three toxicity classes and linked to carcinogenesis through p53, mitogen-activated protein kinases, and apoptosis pathway modules. It could further be shown that the discrimination of toxicity classes was improved when analyzing the microarray data at the pathway level. In summary, our results demonstrate, for the first time, the potential of human embryonic stem cell--derived hepatic cells as an in vitro model for hazard assessment of chemical carcinogenesis, although it should be noted that more compounds are needed to test the robustness of the assay. [ABSTRACT FROM AUTHOR]

The current method to predict carcinogenicity of chemicals or drugs is the chronic 2-year rodent bioassay, which has disadvantages in duration, animal use, and specificity. An attractive alternative is the DNA repair-deficient Xpa−/−p53+/− mouse model that is sensitive to both genotoxic and nongenotoxic carcinogens. A next step in alternative carcinogenicity testing is the development of reliable in vitro systems. We investigated the use of primary hepatocytes, isolated from wild-type (WT) and Xpa−/−p53+/− mice, in combination with transcriptome analyses for their usefulness to predict carcinogenic features of compounds. As a proof of principle, we studied the response of hepatocytes to the genotoxic carcinogen benzo[a]pyrene (B[a]P). Upon treatment, both WT and Xpa−/−p53+/− hepatocytes appeared to be metabolically active. However, Xpa−/−p53+/− hepatocytes were more sensitive than WT hepatocytes to B[a]P treatment in terms of cell survival. In B[a]P-treated WT hepatocytes, DNA repair and cell cycle control genes were transcriptionally activated. Xpa−/−p53+/− hepatocytes were more responsive to B[a]P exposure, resulting in the downregulation of cancer-related pathways. Deregulation of mitogen-activated protein kinase signaling seems to play an essential role in this and might be the underlying reason for the increased susceptibility of Xpa−/−p53+/− mice toward carcinogens. Our conclusion is that primary hepatocytes combined with transcriptomics are promising to identify the carcinogenic features of chemicals. Furthermore, these cells seem suitable to gain further insight into the molecular mechanisms of the increased sensitivity of Xpa−/−p53+/− mice toward both genotoxic and nongenotoxic carcinogens. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was investigated on the microRNA (miR, miRNA) expression profile in cultured primary rat hepatocytes by means of microarray analysis. Simultaneously, albumin secretory capacity and morphological features of the hepatocytes were evaluated throughout the culture time. In total, 25 out of 348 miRNAs were found to be differentially expressed between freshly isolated hepatocytes and 7-day cultured cells. Nineteen of these miRNAs were connected with ‘general metabolism’. miR-21 and miR-126 were shown to be the most up and down regulated miRs upon cultivation and could be linked to the proliferative response triggered in the hepatocytes upon their isolation from the liver. miR-379 and miR-143, on the other hand, were found to be the most up and down regulated miRs upon TSA treatment. Together with the higher expression of miR-122 observed in TSA-treated versus non-treated cultures, we hypothesize that the changes observed for miR-122, miR-143 and miR-379 could be related to the inhibitory effects of TSA on hepatocellular proliferation. [Copyright &y& Elsevier]

Unexpected hepatotoxicity is one of the major reasons of drugs failing in clinical trials. This emphasizes the need for new screening methods that address toxicological hazards early in the drug discovery process. Here, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxic compounds. Drug-induced hepatotoxicity is mainly divided in hepatic steatosis, cholestasis, or necrosis. For each class, a compound was selected, respectively amiodarone, cyclosporin A, and acetaminophen. The changes in protein expressions in HepG2, after exposure to these test compounds, were studied using quantitative two-dimensional differential gel electrophoresis. Identification of differentially expressed proteins was performed by Maldi-TOF/TOF MS and liquid chromatography-tandem mass spectrometry. In this study, 254 differentially expressed protein spots were detected in a two-dimensional proteome map from which 86 were identified, showing that the proteome of HepG2 cells is responsive to hepatotoxic compounds. cyclosporin A treatment was responsible for most differentially expressed proteins and could be discriminated in the hierarchical clustering analysis. The identified differential proteins show that cyclosporin A may induce endoplasmic reticulum (ER) stress and disturbs the ER-Golgi transport, with an altered vesicle-mediated transport and protein secretion as result. Moreover, the differential protein pattern seen after cyclosporin A treatment can be related to cholestatic mechanisms. Therefore, our findings indicate that the HepG2 in vitro cell system has distinctive characteristics enabling the assessment of cholestatic properties of novel compounds at an early stage of drug discovery. [ABSTRACT FROM AUTHOR]

Abstract: 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.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24h to multiple concentrations of flusilazole (0.54–54μM) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the ‘differentiation track’. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing. [Copyright &y& Elsevier]

Abstract: Hepatic systems toxicology is the integrative analysis of toxicogenomic technologies, e.g., transcriptomics, proteomics, and metabolomics, in combination with traditional toxicology measures to improve the understanding of mechanisms of hepatotoxic action. Hepatic toxicology studies that have employed toxicogenomic technologies to date have already provided a proof of principle for the value of hepatic systems toxicology in hazard identification. In the present review, acetaminophen is used as a model compound to discuss the application of toxicogenomics in hepatic systems toxicology for its potential role in the risk assessment process, to progress from hazard identification towards hazard characterization. The toxicogenomics-based parallelogram is used to identify current achievements and limitations of acetaminophen toxicogenomic in vivo and in vitro studies for in vitro-to-in vivo and interspecies comparisons, with the ultimate aim to extrapolate animal studies to humans in vivo. This article provides a model for comparison of more species and more in vitro models enhancing the robustness of common toxicogenomic responses and their relevance to human risk assessment. To progress to quantitative dose–response analysis needed for hazard characterization, in hepatic systems toxicology studies, generation of toxicogenomic data of multiple doses/concentrations and time points is required. Newly developed bioinformatics tools for quantitative analysis of toxicogenomic data can aid in the elucidation of dose-responsive effects. The challenge herein is to assess which toxicogenomic responses are relevant for induction of the apical effect and whether perturbations are sufficient for the induction of downstream events, eventually causing toxicity. [ABSTRACT FROM AUTHOR]

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. Nonexposed embryonic stem cells (ESC) differentiation cultures were sampled 0, 24, and 48 h after initiation of differentiation. Additionally, cultures exposed to 12 diverse well-characterized positive and negative developmental toxicants were isolated 24 h after the onset of exposure. Inhibition of ESC differentiation was evaluated in parallel by morphological scoring on culture day 10. Transcriptomics analysis was conducted using the Affymetrix Gene Chips platform. We applied principal component analysis on the basis of the two predefined gene sets to define the “differentiation track” that represents ESC differentiation. The significance of derivations in the gene expression–based differentiation track because of compound exposures were evaluated to determine developmental toxicity of tested compounds. We successfully predicted developmental toxicity using transcriptomics for 83% (10/12) and 67% (8/12) of the compounds, respectively, using the two predefined gene sets (“Van_Dartel_heartdiff_24h” and “EST biomarker genes”). Our study suggests that the application of transcriptomics may improve the applicability of the EST for the prediction of the developmental toxicity of chemicals. [ABSTRACT FROM AUTHOR]

Investigating the immunotoxic effects of exposure to chemicals usually comprises evaluation of weight and histopathology of lymphoid tissues, various lymphocyte parameters in the circulation, and immune function. Immunotoxicity assessment is time consuming in humans or requires a high number of animals, making it expensive. Furthermore, reducing the use of animals in research is an important ethical and political issue. Immunotoxicogenomics represents a novel approach to investigate immunotoxicity able of overcoming these limitations. The current research, embedded in the European Union project NewGeneris, aimed to retrieve gene expression profiles that are indicative of exposure to immunotoxicants. To this end, whole-genome gene expression was investigated in human peripheral blood mononuclear cells in response to in vitro exposure to a range of immunotoxic chemicals (4-hydroxy-2-nonenal, aflatoxin B1, benzo[a]pyrene, deoxynivalenol, ethanol, malondialdehyde, polychlorinated biphenyl 153, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) and nonimmunotoxic chemicals (acrylamide, dimethylnitrosamine, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine). Using Agilent oligonucleotide microarrays, whole-genome gene expression profiles were generated, which were analyzed using Genedata's Expressionist software. Using Recursive Feature Elimination and Support Vector Machine, a set of 48 genes was identified that distinguishes the immunotoxic from the nonimmunotoxic compounds. Analysis for enrichment of biological processes showed the gene set to be highly biologically and immunologically relevant. We conclude that we have identified a promising transcriptomic profile indicative of immunotoxic exposure. [ABSTRACT FROM AUTHOR]

In recent decades, our knowledge of the genetics and functional genomics of drug-metabolizing enzymes has increased and a wealth of data on drug-related ‘omics’ has become available. Despite the availability of large amounts of biological information on xenobiotic biotransformation, the number of available biotransformation pathway maps that can easily be used for visualization of multiple omics data is limited. Here, we created integrated biotransformation pathway maps suitable for multiple omics analysis using PathVisio. The ease of visualizing data on these maps was demonstrated by using published microarray data from human hepatocyte-like cell models, exemplifying – where a sufficient capacity for metabolizing chemicals is a prerequisite for a suited model – how the biotransformation pathway maps can be used for model selection. [ABSTRACT FROM AUTHOR]

The embryonic stem cell test (EST) has been designed to predict developmental toxicity based upon compound-induced inhibition of embryonic stem cell (ESC) differentiation. The end point scoring, the test duration, and the definition of the predictivity and the applicability domain require improvements to facilitate implementation of the EST into regulatory testing strategies. The use of transcriptomics to study compound-induced differentiation modulation may improve the EST in each of these aspects. ESC differentiation was induced, and cell samples were collected after 0, 24, and 48 h of differentiation. Additionally, samples were collected that were 24 h exposed to one of five developmentally toxic compounds or a nondevelopmentally toxic compound. All samples were hybridized to Affymetrix GeneChips, and analyses revealed that 26 genes were significantly regulated both during ESC differentiation and by exposure to each of the developmentally toxic compounds tested. Using principal component analysis, we defined a “differentiation track” on the basis of this gene list, which represents ESC differentiation. We showed that significant deviation from the differentiation track was in line with the developmental toxic properties of the compounds. The significance of deviation was analyzed using the leave-one-out cross-validation, which showed a favorable prediction of toxicity in the system. Our findings show that gene expression signatures can be used to identify developmental toxicant–induced differentiation modulation. In addition, studying compound-induced effects at an early stage of differentiation combined with transcriptomics leads to increased objectivity in determining differentiation inhibition and to a reduction of the test duration. Furthermore, this approach may improve the predictivity and applicability domain of the EST. [ABSTRACT FROM PUBLISHER]

Direct comparison of the hepatoma cell lines HepG2 and HepaRG has previously been performed by only evaluating a limited set of genes or proteins. In this study, we examined the whole-genome gene expression of both cell lines before and after exposure to the genotoxic (GTX) carcinogens aflatoxin B1 and benzo[a]pyrene and the nongenotoxic (NGTX) carcinogens cyclosporin A, 17β-estradiol, and 2,3,7,8-tetrachlorodibenzo-para-dioxin for 12 and 48 h. Before exposure, this analysis revealed an extensive network of genes and pathways, which were regulated differentially for each cell line. The comparison of the basal gene expression between HepG2, HepaRG, primary human hepatocytes (PHH), and liver clearly showed that HepaRG resembles PHH and liver the most. After exposure to the GTX and NGTX carcinogens, for both cell lines, common pathways were found that are important in carcinogenesis, for example, cell cycle regulation and apoptosis. However, also clear differences between exposed HepG2 and HepaRG were observed, and these are related to common metabolic processes, immune response, and transcription processes. Furthermore, HepG2 performs better in discriminating between GTX and NGTX carcinogens. In conclusion, these results have shown that HepaRG is a more suited in vitro liver model for biological interpretations of the effects of exposure to chemicals, whereas HepG2 is a more promising in vitro liver model for classification studies using the toxicogenomics approach. Although, it should be noted that only five carcinogens were used in this study. [ABSTRACT FROM PUBLISHER]

Assessing the potential carcinogenicity of chemicals for humans represents an ongoing challenge. Chronic rodent bioassays predict human cancer risk at only limited reliability and are simultaneously expensive and long lasting. In order to seek for alternatives, the ability of a transcriptomics-based primary mouse hepatocyte model to classify carcinogens by their modes of action was evaluated. As it is obvious that exposure will induce a cascade of gene expression modifications, in particular, the influence of exposure time in vitro on discriminating genotoxic (GTX) carcinogens from nongenotoxic (NGTX) carcinogens class discrimination was investigated. Primary mouse hepatocytes from male C57Bl6 mice were treated for 12, 24, 36, and 48 h with two GTX and two NGTX carcinogens. For validation, two additional GTX compounds were studied at 24 and 48 h. Immunostaining of γH2AX foci was applied in order to phenotypically verify DNA damage. It confirmed significant induction of DNA damage after treatment with GTX compounds but not with NGTX compounds. Whole-genome gene expression modifications were analyzed by means of Affymetrix microarrays. When using differentially expressed genes from data sets normalized by Robust Multi-array Average, the two classes and various compounds were better separated from each other by hierarchical clustering when increasing the treatment period. Discrimination of GTX and NGTX carcinogens by Prediction Analysis of Microarray improved with time and resulted in correct classification of the validation compounds. The present study shows that gene expression profiling in primary mouse hepatocytes is promising for discriminating GTX from NGTX compounds and that this discrimination improves with increasing treatment period. [ABSTRACT FROM PUBLISHER]

Abstract: A compound for which marked species differences have been reported in laboratory animals and humans is coumarin. In rats, metabolites of coumarin are highly toxic, whereas in humans, the compound is mainly metabolized to non-toxic metabolites. In the present study, a toxicogenomics-based parallelogram approach was used to compare effects of coumarin on gene expression in human hepatocytes relevant for the situation in vivo. To this purpose, gene expression profiling was performed on human hepatocytes treated with coumarin in a pharmacological relevant and proposed toxic concentration and results were compared to a previously performed coumarin in vivo and in vitro rat toxicogenomics study. No cytotoxicity was observed in human hepatocytes at both concentrations, whereas rats showed clear toxic effects in vitro as well as in vivo. In all three systems, coumarin affected genes involved in the blood coagulation pathway; this indicates relevant responses in cases of human exposure. However, no pathways and processes related to hepatotoxicity in rats were observed in human hepatocytes. Still, repression of energy-consuming biochemical pathways and impairment of mitochondrial function were observed in human hepatocytes treated with the highest concentration of coumarin, possibly indicating toxicity. In conclusion, although species differences in response to coumarin are evident in the present results, the toxicogenomics-based parallelogram approach enables clear discrimination between pharmacological responses at pharmacological doses and proposed toxic responses at high (toxic) doses relevant for humans in vivo. [Copyright &y& Elsevier]

BACKGROUND: Human carcinogenesis is known to be initiated and/or promoted by exposure to chemicals that occur in the environment. Molecular cancer epidemiology is used to identify human environmental cancer risks by applying a range of effect biomarkers, which tend to be nonspecific and do not generate insights into underlying modes of action. Toxicogenomic technologies may improve on this by providing the opportunity to identify molecular biomarkers consisting of altered gene expression profiles. OBJECTIVES: The aim of the present study was to monitor the expression of selected genes in a random sample of adults in Flanders selected from specific regions with (presumably) different environmental burdens. Furthermore, associations of gene expression with blood and urinary measures of biomarkers of exposure, early phenotypic effects, and tumor markers were investigated. RESULTS: Individual gene expression of cytochrome p450 1B1, activating transcription factor 4, mitogen-activated protein kinase 14, superoxide dismutase 2 (Mn), chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha), diacylglycerol O-acyltransferase homolog 2 (mouse), tigger transposable element derived 3, and PTEN-induced putative kinase1 were measured by means of quantitative polymerase chain reaction in peripheral blood cells of 398 individuals. After correction for the confounding effect of tobacco smoking, inhabitants of the Olen region showed the highest differences in gene expression levels compared with inhabitants from the Gent and fruit cultivation regions. Importantly, we observed multiple significant correlations of particular gene expressions with blood and urinary measures of various environmental carcinogens. CONCLUSIONS: Considering the observed significant differences between gene expression levels in inhabitants of various regions in Flanders and the associations of gene expression with blood or urinary measures of environmental carcinogens, we conclude... [ABSTRACT FROM AUTHOR]

Abstract: Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the early stages of similar research projects, the information provided in this paper might be extremely valuable. [Copyright &y& Elsevier]

Abstract: Ochratoxin A (OTA), a naturally occurring mycotoxin, is nephrotoxic in all animal species tested and is considered a potent renal carcinogen, particularly in male rats. Its mechanism of toxicity is still unknown, although oxidative stress appears to be a plausible mechanism. Therefore, the objective of this study was to identify the biological pathways that are modulated in vivo by OTA in male F344 rats in order to gain further insight into its mechanism of renal toxicity. Rats were gavaged daily with OTA (500 µg/kg bw) and gene expression profiles in target and non-target organs were analyzed after 7 and 21 days administration. As was expected, a time-dependent increase of OTA concentrations was found in plasma, kidney and liver, with the concentrations found in both tissues being quite similar. However, histopathological examinations only revealed changes in kidney; signs of nephrotoxicity involving single cell necrosis and karyomegalic nuclei were observed in the treated rats. The number of differentially expressed genes in kidney was much higher than in liver (541 versus 11 at both time points). Several similarities were observed with other in vivo gene expression data. However, great differences were found with previous in vitro gene expression data, with the exception of DNA damage response which was not observed at mRNA level in any of our study conditions. Down-regulation was the predominant effect. Oxidative stress response pathway and genes involved in metabolism and transport were inhibited at both time points. RGN (regucalcin) – a gene implicated in calcium homeostasis – was strongly inhibited at both time points and genes implicated in cell survival and proliferation were up-regulated at day 21. Moreover, translation factors and annexin genes were up-regulated at both time points. Apart from oxidative stress, alterations of the calcium homeostasis and cytoskeleton structure may be present at the first events of OTA toxicity. [Copyright &y& Elsevier]

Abstract: This study on the in vitro micronucleus assay, comprising 11 laboratories using human lymphocytes, was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances were assessed for their ability to induce micronuclei in human lymphocytes in vitro, mitomycin C being used as a positive control. Cultures were exposed to the test substances for a short (early or late) time or for a long time, followed by a short or long recovery period, in the presence of cytochalasin B. Each chemical was evaluated, generally in two laboratories, using three treatment schedules at least twice. The data were assessed for acceptability, and then classified as negative, positive or equivocal. Two of seven genotoxic compounds, namely colchicine and bleomycin, clearly induced micronuclei. Reproducible results were difficult to obtain for some substances, which tended to be those acting at specific stages of the cell cycle. Cytosine arabinoside, diethylstilboestrol and 5-fluorouracil were classified as equivocal. Urethane and thiabendazole were classified as negative. The two presumed non-genotoxic compounds, mannitol and clofibrate, did not induce micronuclei. Repeat testing, exposing cells at both an early and late time after mitogenic stimulation, was needed to detect substances classified as equivocal. These results show the importance of achieving sufficient inhibition of nuclear division to avoid the possibility of missing an effect. The evaluation of micronuclei in mononucleated as well as binucleated cells was particularly useful to detect aneugens. There were no false positive results using lymphocytes, indicating a high specificity. It is concluded that the clastogenic or aneugenic potential in vitro of the substances tested was correctly identified in this study, but that refining the protocol to take into account factors such as the stages of the cell cycle exposed to the compound, or the duration of recovery would be likely to improve the sensitivity of detection using lymphocytes. [Copyright &y& Elsevier]

Worldwide, lung cancer is the most prevalent and lethal malignant disease. In addition to avoidance of the most predominant risk factor, i.e., tobacco use, consumption of high amounts of vegetables and fruits could be an effective means of preventing lung cancer. However, the molecular mechanisms underlying lung cancer risk reduction by vegetables are not clear. In the present study, the effect of vegetables on gene expression changes in the lungs of female C57Bl/6 mice was investigated using cDNA microarray technology. The mice were fed 1 of 8 diets for 2 wk: a control diet containing no vegetables (diet 1); a diet containing a vegetable mixture at 100 (diet 2, 10% dose), 200 (diet 3, 20% dose), or 400 (diet 4, 40% dose) g/kg; or a diet containing cauliflower at 70 (diet 5, 7% dose); carrots at 73 (diet 6, 7.3% dose); peas at 226 (diet 7, 22.6% dose); or onions at 31 (diet 8, 3.1% dose) g/kg. The vegetable mixture consisted of these 4 individual vegetables. After the mice were killed, the lungs were removed and total RNA was isolated from the lungs for expression analysis of 602 genes involved in pathways of (anti)-carcinogenesis. The results of this study suggest that individual vegetables have a higher potential of modulating genes (5 from the 8 modulated genes) in favor of lung cancer risk prevention, in comparison with the vegetable mixture (2 from the 7 modulated genes); the other gene modulations are expected to enhance lung cancer risk. The pathways involved were miscellaneous and included cell growth, apoptosis, biotransformation, and immune response. Furthermore, carrots were able to modulate most gene expressions, and most of these effects occurred in processes that favored lung cancer risk prevention. The current study provides more insight into the genetic mechanisms by which vegetables, in particular carrots, can prevent lung cancer risk. [ABSTRACT FROM AUTHOR]

There is abundant epidemiological evidence that vegetable consumption decreases colorectal cancer (CRC) risk. However, the molecular targets in the genome are mostly unknown. The present study investigated the effects of vegetable consumption on gene expression in the colon mucosa of female C57BI/6 mice using cDNA microarray technology. Mice were fed one of 8 diets: a control diet containing no vegetables (diet 1); a diet containing 100 g/kg (diet 2, 10% dose), 200 g/kg (diet 3, 20% dose), or 400 g/kg (diet 4, 40% dose) of a vegetable mixture; or a diet containing 70 g/kg of cauliflower (diet 5, 7% dose), 73 g/kg of carrots (diet 6, 7.3% dose), 226 g/kg of peas (diet 7, 22.6% dose); or 31 g/kg of onions (diet 8, 3.1 % dose). The vegetable mixture used in diets 2 to 4 consisted of the 4 individual vegetables used in diets 5 to 8: cauliflower (30% wet wt), carrots (30% wet wt), peas (30% wet wt), and onions (10% wet wt). To assess gene expression changes, colonic mucosal cells were collected after the mice were killed. Total ANA was isolated and microarray technology was used to measure the expression levels of 602 genes simultaneously. For 39 genes, significant dose-dependent effects were found, although in general the relations were not linear. For 15 genes, the altered expression could indeed explain reduced cancer risk at various stages of CRC development. Eleven genes were modulated by the vegetable mixture as well as by one or more of the individual vegetables. For 7 of the genes, the modulation by the mixture was due to the effect of a particular vegetable. These genes are of particular interest because they were consistently affected and could be involved in the prevention of CRC by vegetable consumption. [ABSTRACT FROM AUTHOR]

Benzo(a)pyrene (BaP) is a ubiquitous carcinogen resulting from incomplete combustion of organic compounds and also present at high levels in cigarette smoke. A wide range of biological effects has been attributed to BaP and its genotoxic metabolite BPDE, but the contribution to BaP toxicity of intermediary metabolites generated along the detoxification path remains unknown. Here, we report for the first time how 3-OH-BaP, 9,10-diol and BPDE, three major BaP metabolites, temporally relate to BaP-induced transcriptomic alterations in HepG2 cells. Since BaP is also known to induce AhR activation, we additionally evaluated TCDD to source the expression of non-genotoxic AhR-mediated patterns. 9,10-Diol was shown to activate several transcription factor networks related to BaP metabolism (AhR), oxidative stress (Nrf2) and cell proliferation (HIF-1α, AP-1) in particular at early time points, while BPDE influenced expression of genes involved in cell energetics, DNA repair and apoptotic pathways. Also, in order to grasp the role of BaP and its metabolites in chemical hepatocarcinogenesis, we compared expression patterns from BaP(-metabolites) and TCDD to a signature set of approximately nine thousand gene expressions derived from hepatocellular carcinoma (HCC) patients. While transcriptome modulation by TCDD appeared not significantly related to HCC, BaP and BPDE were shown to deregulate metastatic markers via non-genotoxic and genotoxic mechanisms and activate inflammatory pathways (NF-κβ signaling, cytokine-cytokine receptor interaction). BaP also showed strong repression of genes involved in cholesterol and fatty acid biosynthesis. Altogether, this study provides new insights into BaP-induced toxicity and sheds new light onto its mechanism of action as a hepatocarcinogen.

Acetaminophen (APAP) is a readily available over-the-counter drug and is one of the most commonly used analgesics/antipyretics worldwide. Large interindividual variation in susceptibility toward APAP-induced liver failure has been reported. However, the exact underlying factors causing this variability in susceptibility are still largely unknown. The aim of this study was to better understand this variability in response to APAP by evaluating interindividual differences in gene expression changes and APAP metabolite formation in primary human hepatocytes (PHH) from several donors (n = 5) exposed in vitro to a non-toxic to toxic APAP dose range. To evaluate interindividual variation, gene expression data/levels of metabolites were plotted against APAP dose/donor. The correlation in APAP dose response between donors was calculated by comparing data points from one donor to the data points of all other donors using a Pearson-based correlation analysis. From that, a correlation score/donor for each gene/metabolite was defined, representing the similarity of the omics response to APAP in PHH of a particular donor to all other donors. The top 1 % highest variable genes were selected for further evaluation using gene set overrepresentation analysis. The biological processes in which the genes with high interindividual variation in expression were involved include liver regeneration, inflammatory responses, mitochondrial stress responses, hepatocarcinogenesis, cell cycle, and drug efficacy. Additionally, the interindividual variation in the expression of these genes could be associated with the variability in expression levels of hydroxyl/methoxy-APAP and C8H13O5N-APAP-glucuronide. The before-mentioned metabolites or their derivatives have also been reported in blood of humans exposed to therapeutic APAP doses. Possibly these findings can contribute to elucidating the causative factors of interindividual susceptibility toward APAP.

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. Performing pathway analyses of significant mRNAs and metabolites separately and integrated, resulted in more relevant pathways for the latter. Integrated analyses showed pathways involved in cell cycle and cellular metabolism to be significantly changed. Moreover, pathways involved in protein processing of the endoplasmic reticulum, bile acid biosynthesis and cholesterol metabolism were significantly affected. Our findings indicate that an integrated approach combining metabonomics and transcriptomics data derived from representative in vitro models, with bioinformatics can improve our understanding of the mechanisms of action underlying drug-induced hepatotoxicity. Furthermore, we showed that integrating multiple omics and thereby analyzing genes, microRNAs and metabolites of the opposed model for drug-induced cholestasis can give valuable information about mechanisms of drug-induced cholestasis in vitro and therefore could be used in toxicity screening of new drug candidates at an early stage of drug discovery., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Large differences in toxicity responses occur within the human population. In this study we evaluate whether interindividual variation in baseline enzyme activity (EA)/gene expression (GE) levels in liver predispose for the variation in toxicity responses by assessing dose-response relationships for several prototypical hepatotoxicants. Baseline levels of cytochrome-P450 (CYP) GE/EA were measured in precision-cut human liver slices. Slices (n=4-5/compound) were exposed to a dose-range of acetaminophen, aflatoxin B1, benzo(α) pyrene or 2-nitrofluorene. Interindividual variation in induced genotoxicity (COMET-assay and CDKN1A/p21 GE) and cytotoxicity (lactate dehydrogenase-leakage), combined with NQO1- and GSTM1-induced GE-responses for oxidative stress and GE-responses of several CYPs was evaluated. The benchmark dose-approach was applied as a tool to model exposure responses on an individual level. Variation in baseline CYP levels, both GE and EA, can explain variation in compound exposure-responses on an individual level. Network analyses enable the definition of key parameters influencing interindividual variation after compound exposure. For 2-nitrofluorene, this analysis suggests involvement of CYP1B1 in the metabolism of this compound, which represents a novel finding. In this study, GSTM1 which is known to be highly polymorphic within the human population, but so far could not be linked to toxicity in acetaminophen-poisoned patients, is suggested to cause interindividual variability in acetaminophen-metabolism, dependent on the individual's gene expression-responses of CYP-enzymes. This study demonstrates that using interindividual variation within network modelling provides a source for the definition of essential and even new parameters involved in compound-related metabolism. This information might enable ways to make more quantitative estimates of human risks., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Current testing models for predicting drug-induced liver injury are inadequate, as they basically under-report human health risks. We present here an approach towards developing pathways based on hepatotoxicity-associated gene groups derived from two types of publicly accessible hepatotoxicity databases, in order to develop drug-induced liver injury biomarker profiles. One human liver 'omics-based and four text-mining-based databases were explored for hepatotoxicity-associated gene lists. Over-representation analysis of these gene lists with a hepatotoxicant-exposed primary human hepatocytes data set showed that human liver 'omics gene lists performed better than text-mining gene lists and the results of the latter differed strongly between databases. However, both types of databases contained gene lists demonstrating biomarker potential. Visualizing those in pathway format may aid in interpreting the biomolecular background. We conclude that exploiting existing and openly accessible databases in a dedicated manner seems promising in providing venues for translational research in toxicology and biomarker development.

The mycotoxin Ochratoxin A (OTA) is a potent renal carcinogen in male rats. Transcriptomic studies on OTA (4 in vitro, 6 in vivo, 2 in vitro/in vivo) have been reviewed. The aim of 6 of them was mainly mechanistic whereas the rest had mostly predictive (1) or evaluation (5) purposes. An overall tendency towards gene expression downregulation was observed, probably as a result of protein synthesis inhibition. DNA damage response genes were not deregulated in most of the studies. Genes involved in acute renal injury, cell survival and cell proliferation were upregulated in several in vivo studies. Apoptosis genes were deregulated in vitro but less affected in vivo; activation of several MAPKs has been observed. Many genes related to oxidative stress or involved in cell-to-cell interaction pathways (Wnt) or cytoskeleton structure appeared to be deregulated either in vitro or in vivo. Regucalcin was highly downregulated in vivo and other calcium homeostasis genes were significantly deregulated in vitro. Genes related to OTA transport (OATs) and metabolism (CYPs) appeared downregulated in vivo. Overall, the mechanism of action of OTA remains unclear, however transcriptomic data have contributed to new mechanistic hypothesis generation and to in vitro-in vivo comparison., (Copyright © 2013. Published by Elsevier Ltd.)

Flanders, Belgium, is one of the most densely populated areas in Europe. The Flemish Environment and Health Survey (2002-2006) aimed at determining exposure to pollutants of neonates, adolescents, and older adults and to assess associated biological and health effects. This study investigated genome wide gene expression changes associated with a range of environmental pollutants, including cadmium, lead, PCBs, dioxin, hexachlorobenzene, p,p'-DDE, benzene, and PAHs. Gene expression levels were measured in peripheral blood cells of 20 adults with relatively high and 20 adults with relatively low combined internal exposure levels, all non-smokers aged 50-65. Pearson correlation was used to analyze associations between pollutants and gene expression levels, separately for both genders. Pollutant- and gender-specific correlation analysis results were obtained. For organochlorine pollutants, analysis within genders revealed that genes were predominantly regulated in opposite directions in males and females. Significantly modulated pathways were found to be associated with each of the exposure biomarkers measured. Pathways and/or genes related to estrogen and STAT5 signaling were correlated to organochlorine exposures in both genders. Our work demonstrates that gene expression in peripheral blood is influenced by environmental pollutants. In particular, gender-specific changes are associated with organochlorine pollutants, including gender-specific modulation of endocrine related pathways and genes. These pathways and genes have previously been linked to endocrine disruption related disorders, which in turn have been associated with organochlorine exposure. Based on our results, we recommend that males and females be considered separately when analyzing gene expression changes associated with exposures that may include chemicals with endocrine disrupting properties., (Copyright © 2013 Wiley Periodicals, Inc.)

Drug-induced hepatotoxicity is a leading cause of attrition for candidate pharmaceuticals in development. New preclinical screening methods are crucial to predict drug toxicity prior to human studies. Of all in vitro hepatotoxicity models, primary human hepatocytes are considered as 'the gold standard.' However, their use is hindered by limited availability and inter-individual variation. These barriers may be overcome by using primary mouse hepatocytes. We used differential in gel electrophoresis (DIGE) to study large-scale protein expression of primary mouse hepatocytes. These hepatocytes were exposed to three well-defined hepatotoxicants: acetaminophen, amiodarone, and cyclosporin A. Each hepatotoxicant induces a different hepatotoxic phenotype. Based on the DIGE results, the mRNA expression levels of deregulated proteins from cyclosporin A-treated cells were also analyzed. We were able to distinguish cyclosporin A from controls, as well as acetaminophen and amiodarone-treated samples. Cyclosporin A induced endoplasmic reticulum (ER) stress and altered the ER-Golgi transport. Moreover, liver carboxylesterase and bile salt sulfotransferase were differentially expressed. These proteins were associated with a protective adaptive response against cyclosporin A-induced cholestasis. The results of this study are comparable with effects in HepG2 cells. Therefore, we suggest both models can be used to analyze the cholestatic properties of cyclosporin A. Furthermore, this study showed a conserved response between primary mouse hepatocytes and HepG2 cells. These findings collectively lend support for use of omics strategies in preclinical toxicology, and might inform future efforts to better link preclinical and clinical research in rational drug development.

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. Based on EnsEMBL genes, we demonstrate that RNA-seq detects ca 20% more genes than microarray-based technology but almost threefold more significantly differentially expressed genes. Functional enrichment analyses show that RNA-seq yields more insight into the biology and mechanisms related to the toxic effects caused by BaP, i.e., two- to fivefold more affected pathways and biological processes. Additionally, we demonstrate that RNA-seq allows detecting alternative isoform expression in many genes, including regulators of cell death and DNA repair such as TP53, BCL2 and XPA, which are relevant for genotoxic responses. Moreover, potentially novel isoforms were found, such as fragments of known transcripts, transcripts with additional exons, intron retention or exon-skipping events. The biological function(s) of these isoforms remain for the time being unknown. Finally, we demonstrate that RNA-seq enables the investigation of allele-specific gene expression, although no changes could be observed. Our results provide evidence that RNA-seq is a powerful tool for toxicology, which, compared with microarrays, is capable of generating novel and valuable information at the transcriptome level for characterizing deleterious effects caused by chemicals.

Potassium bromate (KBrO(3)) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO(3) in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO(3) for 6, 24 and 72 h. Pathway analysis identified "glutathione metabolism", "Nrf2-mediated oxidative stress" and "tight junction (TJ) signalling" as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO(3) exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N-Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.

The concept of mechanistic toxicogenomics implies that compound-induced changes in gene expression profiles provide valuable information about their mode of action. A growing number of research groups have presented evidence that whole-genome gene expression profiling techniques might be used as tools for in vivo and in vitro generation of gene signatures and elucidation of molecular mechanisms after exposure to toxic compounds. An important issue to be investigated is the in vivo relevance of in vitro-obtained data. In the current study, we compare the gene expression profiles generated in vitro, after exposing conventional and epigenetically stabilized primary rat hepatocytes to well-known genotoxic hepatocarcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 2-nitrofluorene) with those derived in vivo after oral exposure of rats to these compounds. Similar statistical tools were applied on both sets of data. The major molecular pathways affected in the in vivo setting were DNA damage, detoxification and cell survival response, as previously described. In the conventional hepatocyte cultures, two of the three genotoxicants showed quite similar responses as in vivo with respect to these pathways. The third compound (2-nitrofluorene) revealed in vitro response which was not observed in vivo. In the epigenetically stabilized hepatocytes, in contrast to what was expected, the responses were less relevant for the in vivo situation. This study highlights the importance of in vitro/in vivo comparison of data that are generated using in vitro models and shows that conventional primary rat hepatocyte cultures represent an appropriate in vitro model to retrieve mechanistic information on the exposure to genotoxicants.

At present, substantial efforts are focused on the development of in vitro assays coupled with "omics" technologies for the identification of carcinogenic substances as an alternative to the classical 2-year rodent carcinogenicity bioassay. A prerequisite for the eventual regulatory acceptance of such assays, however, is the in vivo relevance of the observed in vitro findings. In the current study, hepatocarcinogen-induced gene expression profiles generated after the exposure of conventional cultures of primary rat hepatocytes to three non-genotoxic carcinogens (methapyrilene hydrochloride, piperonyl butoxide, and Wy-14643), three genotoxic carcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-nitrofluorene), and two non-carcinogens (nifedipine and clonidine) are compared with previously obtained in vivo data after oral administration for up to 14 days of the same hepatocarcinogens to rats. In addition to the comparison of deregulated genes and functions per compound between in vivo and in vitro models, the major discriminating cellular pathways found in vivo in livers of exposed rats were examined for deregulation in vitro. Further, in vivo-derived gene signatures for the identification of genotoxic versus non-genotoxic carcinogens are used to classify in vitro-tested hepatocarcinogens and non-carcinogens. In the primary hepatocyte cultures, two out of the three tested genotoxic carcinogens mimicked the in vivo-relevant DNA damage response and were correctly assessed. Exposure to the non-genotoxic hepatocarcinogens, however, triggered a relatively weak response in the in vitro system, with no clear similarities to in vivo. This study contributes to the further optimization of toxicogenomics predictive tools when applied in in vitro settings.

The safety assessment for pharmaceuticals includes in vivo repeated dose toxicity tests in laboratory animals. These in vivo studies often generate false negative results and unexpected toxicity. The appearance of this unexpected toxicity is one of the major reasons for the drawback of a drug from the market. The liver is often a target organ in toxicology since it is responsible for the metabolism and elimination of chemical compounds. Therefore, there is need for new screening methods which classify hepatotoxic compounds earlier in development. This will lead to safer drugs and a more efficient drug discovery process. Furthermore, these new screening methods are preferably in vitro test systems, aiming at reducing the use of laboratory animals. In this review the possibilities of proteomics and its promising results for improving current predictive and mechanistic toxicological studies are described. Biomarkers or protein panels for hepatotoxic mechanisms, which reflect the in vivo situation, need to be identified to allow a better toxicity screening. Therefore, in vivo studies and in vitro cell models are discussed and evaluated with regard to the protein expression of their metabolic enzymes, their similarities with liver, their use for analyzing toxicological mechanisms and hepatotoxicity screening. Studies in which proteomics are combined with other omics-technologies are also presented. The results from these integrated data analyses can be used for the development of improved panels of biomarkers for toxicity screening., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

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. Temporal profiles for functional gene sets demonstrate both early and late effects in up- and downregulation of relevant gene sets involved in cell cycle, apoptosis, DNA repair, and metabolism of amino acids and lipids. Many significant transcription regulation networks appeared to be around TF that are proto-oncogenes or tumor suppressor genes. The time series analysis tool Short Time-series Expression Miner (STEM) was used to identify time-dependent correlation of pathways, gene sets, TF networks, and biological parameters. Most correlations are with DNA adduct levels, which is an early response, and less with the later responses on G1 and S phase cells. The majority of the modulated genes in the Reactome pathways can be regulated by several of these TF, e.g., 73% by nuclear factor-kappa B and 34-42% by c-MYC, SRF, AP1, and E2F1. All these TF can also regulate one or more of the others. Our data indicate that a complex network of a few TF is responsible for the majority of the transcriptional changes induced by BaP. This network hardly changes over time, despite that the transcriptional profiles clearly alter, suggesting that also other regulatory mechanisms are involved.

Reactive oxygen species-induced oxidative stress in the colon is involved in inflammatory bowel diseases and suggested to be associated with colorectal cancer risk. However, our insight in molecular responses to different oxygen radicals is still fragmentary. Therefore, we studied global gene expression by an extensive time series (0.08, 0.25, 0.5, 1, 2, 4, 8, 16, or 24 h) analyses in human colon cancer (caco-2) cells after exposure to H(2)O(2) or the superoxide anion donor menadione. Differences in gene expression were investigated by hybridization on two-color microarrays against nonexposed time-matched control cells. Next to gene expression, correlations with related phenotypic markers (8-oxodG levels and cell cycle arrest) were investigated. Gene expression analysis resulted in 1404 differentially expressed genes upon H(2)O(2) challenge and 979 genes after menadione treatment. Further analysis of gene expression data revealed how these oxidant responses can be discriminated. Time-dependent coregulated genes immediately showed a pulse-like response to H(2)O(2), while the menadione-induced expression is not restored over 24 h. Pathway analyses demonstrated that H(2)O(2) immediately influences pathways involved in the immune function, while menadione constantly regulated cell cycle-related pathways Altogether, this study offers a novel and detailed insight in the similarities and differences of the time-dependent oxidative stress responses induced by the oxidants H(2)O(2) and menadione and show that these can be discriminated regarding their modulation of particular colon carcinogenesis-related mechanisms.

Metabolites of the human carcinogen 4-aminobiphenyl (4-ABP) form hemoglobin (Hb) adducts, which represent a useful biomarker for exposure. However, not every individual responds to a similar degree to 4-ABP exposure, and variations in 4-ABP-Hb adduct formation might be explained by genetic polymorphisms in genes coding for enzymes involved in 4-ABP metabolism. 4-ABP-Hb adducts were measured in blood samples from 57 smoking and 10 non-smoking volunteers. An association was found between cigarette smoking and 4-ABP-Hb adduct levels in smokers (R(2) = 0.5, P < 0.001). Subsequently, subjects were genotyped for 12 polymorphisms in seven genes involved in biotransformation reactions. From this selection of polymorphisms, a significant impact was found for the CYP1B1 Leu(432)Val polymorphism (P = 0.021), which has been reported to lead to a decrease in enzyme activity. Indeed higher levels of 4-ABP-Hb adducts were observed in homo- and heterozygous carriers of the CYP1B1 (432)Leu as compared to the double CYP1B1 (432)Val genotype. A significant interaction between these CYP1B1 genotypes and the level of exposure was found (P = 0.003). Noteworthy, a saturation effect was observed for 4-ABP-Hb adduct formation at high smoking doses limited to carriers of the CYP1B1 (432)Leu allele. No effect of polymorphisms in other genes were found. This is the first study in humans suggesting a crucial role of the CYP1B1 enzyme in 4-ABP metabolism, indicating a protective effect of the CYP1B1 Leu(432)Val polymorphism against the formation of 4-ABP-Hb adduct levels, depending on the smoking dose.

Microarray technology is being used increasingly to study gene expression of biological systems on a large scale. Both interlaboratory and interplatform differences are known to contribute to variability in microarray data. In this study we have investigated data from different platforms and laboratories on the transcriptomic profile of HepG2 cells exposed to benzo(a)pyrene (BaP). RNA samples generated in two different laboratories were analyzed using both Agilent oligonucleotide microarrays and Cancer Research UK (CR-UK) cDNA microarrays. Comparability of the expression profiles was assessed at various levels including correlation and overlap between the data, clustering of the data and affected biological processes. Overlap and correlation occurred, but it was not possible to deduce whether choice of platform or interlaboratory differences contributed more to the data variation. Principal component analysis (PCA) and hierarchical clustering of the expression profiles indicated that the data were most clearly defined by duration of exposure to BaP, suggesting that laboratory and platform variability does not mask the biological effects. Real-time quantitative PCR was used to validate the two array platforms and indicated that false negatives, rather than false positives, are obtained with both systems. All together these results suggest that data from similar biological experiments analyzed on different microarray platforms can be combined to give a more complete transcriptomic profile. Each platform gives a slight variation in the BaP-gene expression response and, although it cannot be stated which is more correct, combining the two data sets is more informative than considering them individually.

The frequent use of rodent hepatic in vitro systems in pharmacological and toxicological investigations challenges extrapolation of in vitro results to the situation in vivo and interspecies extrapolation from rodents to humans. The toxicogenomics approach may aid in evaluating relevance of these model systems for human risk assessment by direct comparison of toxicant-induced gene expression profiles and infers mechanisms between several systems. In the present study, acetaminophen (APAP) was used as a model compound to compare gene expression responses between rat and human using in vitro cellular models, hepatocytes, and between rat in vitro and in vivo. Comparison at the level of modulated biochemical pathways and biological processes rather than at that of individual genes appears preferable as it increases the overlap between various systems. Pathway analysis by T-profiler revealed similar biochemical pathways and biological processes repressed in rat and human hepatocytes in vitro, as well as in rat liver in vitro and in vivo. Repressed pathways comprised energy-consuming biochemical pathways, mitochondrial function, and oxidoreductase activity. The present study is the first that used a toxicogenomics-based parallelogram approach, extrapolating in vitro to in vivo and interspecies, to reveal relevant mechanisms indicative of APAP-induced liver toxicity in humans in vivo.

Although exposure to polycyclic aromatic hydrocarbons (PAHs) occurs mostly through mixtures, hazard and risk assessment are mostly based on the effects caused by individual compounds. The objective of the current study was to investigate whether interactions between PAHs occur, focusing on gene expression (as measured by cDNA microarrays) and DNA adduct formation. The effects of benzo[a]pyrene or dibenzo[a,h]anthracene (DB[a,h]A) alone and in binary mixtures with another PAH (DB[a,h]A, benzo[b]fluoranthene, fluoranthene or dibenzo[a,l]pyrene) were investigated using precision-cut rat liver slices. All compounds significantly modulated the expression of several genes, but overlap between genes affected by the mixture and by the individual compounds was relatively small. All mixtures showed an antagonistic response on total gene expression profiles. Moreover, at the level of individual genes, mostly antagonism was evident, with additivity and synergism observed for only a few genes. As far as DNA adduct formation is concerned, the binary mixtures generally caused antagonism. The effects in liver slices suggest a lower carcinogenic potency of PAH mixtures than estimated based on additivity of individual compounds.