Toxicogenomics directory of chemically exposed human hepatocytes.

A long-term goal of numerous research projects is to identify biomarkers for in vitro systems predicting toxicity in vivo. Often, transcriptomics data are used to identify candidates for further evaluation. However, a systematic directory summarizing key features of chemically influenced genes in human hepatocytes is not yet available. To bridge this gap, we used the Open TG-GATES database with Affymetrix files of cultivated human hepatocytes incubated with chemicals, further sets of gene array data with hepatocytes from human donors generated in this study, and publicly available genome-wide datasets of human liver tissue from patients with non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular cancer (HCC). After a curation procedure, expression data of 143 chemicals were included into a comprehensive biostatistical analysis. The results are summarized in the publicly available toxicotranscriptomics directory ( http://wiki.toxbank.net/toxicogenomics-map/ ) which provides information for all genes whether they are up- or downregulated by chemicals and, if yes, by which compounds. The directory also informs about the following key features of chemically influenced genes: (1) Stereotypical stress response. When chemicals induce strong expression alterations, this usually includes a complex but highly reproducible pattern named 'stereotypical response.' On the other hand, more specific expression responses exist that are induced only by individual compounds or small numbers of compounds. The directory differentiates if the gene is part of the stereotypical stress response or if it represents a more specific reaction. (2) Liver disease-associated genes. Approximately 20 % of the genes influenced by chemicals are up- or downregulated, also in liver disease. Liver disease genes deregulated in cirrhosis, HCC, and NASH that overlap with genes of the aforementioned stereotypical chemical stress response include CYP3A7, normally expressed in fetal liver; the phase II metabolizing enzyme SULT1C2; ALDH8A1, known to generate the ligand of RXR, one of the master regulators of gene expression in the liver; and several genes involved in normal liver functions: CPS1, PCK1, SLC2A2, CYP8B1, CYP4A11, ABCA8, and ADH4. (3) Unstable baseline genes. The process of isolating and the cultivation of hepatocytes was sufficient to induce some stress leading to alterations in the expression of genes, the so-called unstable baseline genes. (4) Biological function. Although more than 2,000 genes are transcriptionally influenced by chemicals, they can be assigned to a relatively small group of biological functions, including energy and lipid metabolism, inflammation and immune response, protein modification, endogenous and xenobiotic metabolism, cytoskeletal organization, stress response, and DNA repair. In conclusion, the introduced toxicotranscriptomics directory offers a basis for a rationale choice of candidate genes for biomarker evaluation studies and represents an easy to use source of background information on chemically influenced genes.

Unraveling toxicological mechanisms and predicting toxicity classes with gene dysregulation networks.

The use of genes for distinguishing classes of toxicity has become well established. In this paper we combine the reconstruction of a gene dysregulation network (GDN) with a classifier to assign unseen compounds to their appropriate class. Gene pairs in the GDN are dysregulated in the sense that they are linked by a common expression pattern in one class and differ in this pattern in another class. The classifier gives a quantitative measure on this difference by its prediction accuracy. As an in-depth example, gene pairs were selected that were dysregulated between skin cells treated with either sensitizers or irritants. Pairs with known and novel markers were found such as HMOX1 and ZFAND2A, ATF3 and PPP1R15A, OXSR1 and HSPA1B, ZFP36 and MAFF. The resulting GDN proved biologically valid as it was well-connected and enriched in known interactions, processes and common regulatory motifs for pairs. Classification accuracy was improved when compared with conventional classifiers. As the dysregulated patterns for heat shock responding genes proved to be distinct from those of other stress genes, we were able to formulate the hypothesis that heat shock genes play a specific role in sensitization, apart from other stress genes. In conclusion, our combined approach creates added value for classification-based toxicogenomics by obtaining novel, well-distinguishing and biologically interesting measures, suitable for the formulation of hypotheses on functional relationships between genes and their relevance for toxicity class differences.

'Omics analysis of low dose acetaminophen intake demonstrates novel response pathways in humans.

Acetaminophen is the primary cause of acute liver toxicity in Europe/USA, which led the FDA to reconsider recommendations concerning safe acetaminophen dosage/use. Unfortunately, the current tests for liver toxicity are no ideal predictive markers for liver injury, i.e. they only measure acetaminophen exposure after profound liver toxicity has already occurred. Furthermore, these tests do not provide mechanistic information. Here, 'omics techniques (global analysis of metabolomic/gene-expression responses) may provide additional insight. To better understand acetaminophen-induced responses at low doses, we evaluated the effects of (sub-)therapeutic acetaminophen doses on metabolite formation and global gene-expression changes (including, for the first time, full-genome human miRNA expression changes) in blood/urine samples from healthy human volunteers. Many known and several new acetaminophen-metabolites were detected, in particular in relation to hepatotoxicity-linked, oxidative metabolism of acetaminophen. Transcriptomic changes indicated immune-modulating effects (2g dose) and oxidative stress responses (4g dose). For the first time, effects of acetaminophen on full-genome human miRNA expression have been considered and confirmed the findings on mRNA level. 'Omics techniques outperformed clinical chemistry tests and revealed novel response pathways to acetaminophen in humans. Although no definitive conclusion about potential immunotoxic effects of acetaminophen can be drawn from this study, there are clear indications that the immune system is triggered even after intake of low doses of acetaminophen. Also, oxidative stress-related gene responses, similar to those seen after high dose acetaminophen exposure, suggest the occurrence of possible pre-toxic effects of therapeutic acetaminophen doses. Possibly, these effects are related to dose-dependent increases in levels of hepatotoxicity-related metabolites.

A dictionary to identify small molecules and drugs in free text.

MOTIVATION: From the scientific community, a lot of effort has been spent on the correct identification of gene and protein names in text, while less effort has been spent on the correct identification of chemical names. Dictionary-based term identification has the power to recognize the diverse representation of chemical information in the literature and map the chemicals to their database identifiers. RESULTS: We developed a dictionary for the identification of small molecules and drugs in text, combining information from UMLS, MeSH, ChEBI, DrugBank, KEGG, HMDB and ChemIDplus. Rule-based term filtering, manual check of highly frequent terms and disambiguation rules were applied. We tested the combined dictionary and the dictionaries derived from the individual resources on an annotated corpus, and conclude the following: (i) each of the different processing steps increase precision with a minor loss of recall; (ii) the overall performance of the combined dictionary is acceptable (precision 0.67, recall 0.40 (0.80 for trivial names); (iii) the combined dictionary performed better than the dictionary in the chemical recognizer OSCAR3; (iv) the performance of a dictionary based on ChemIDplus alone is comparable to the performance of the combined dictionary. AVAILABILITY: The combined dictionary is freely available as an XML file in Simple Knowledge Organization System format on the web site http://www.biosemantics.org/chemlist.

The contact allergen dinitrochlorobenzene (DNCB) and respiratory allergy in the Th2-prone Brown Norway rat.

All LMW respiratory allergens known to date can also induce skin allergy in test animals. The question here was if in turn skin allergens can induce allergy in the respiratory tract. Respiratory allergy was tested in Th2-prone Brown Norway (BN) rats by dermal sensitization with the contact allergen dinitrochlorobenzene (DNCB; 1%, day 0; 0.5%, day 7) and a head/nose-only inhalation challenge of 27mg/m3 of DNCB (15 min, day 21), using a protocol that successfully identified chemical respiratory allergens. Skin allergy to DNCB was examined in BN rats and Th1-prone Wistar rats in a local lymph node assay followed by a topical patch challenge of 0.1% DNCB. Sensitization of BN rats via the skin induced DNCB-specific IgG in serum, but not in all animals, and an increased number of CD4+ cells in the lung parenchyma. Subsequent inhalation challenge with DNCB did not provoke apneas or allergic inflammation (signs of respiratory allergy) in the BN rats. However, microarray analysis of mRNA isolated from the lung revealed upregulation of the genes for Ccl2 (MCP-1), Ccl4 (MIP-1beta), Ccl7 and Ccl17. Skin challenge induced considerably less skin irritation and allergic dermatitis in the BN rat than in the Wistar rat. In conclusion, the Th2-prone BN rat appeared less sensitive to DNCB than the Wistar rat; nevertheless, DNCB induced allergic inflammation in the skin of BN rats but even a relatively high challenge concentration did not induce allergy in the respiratory tract, although genes associated with allergy were upregulated in lung tissue.

Pathway and single gene analyses of inhibited Caco-2 differentiation by ascorbate-stabilized quercetin suggest enhancement of cellular processes associated with development of colon cancer.

The aim was to investigate mechanisms contributing to quercetin's previously described effects on cell-proliferation and -differentiation, which contradicted its proposed anticarcinogenic potency. In a 10-day experiment, 40 microM quercetin stabilized by 1 mM ascorbate reduced Caco-2 differentiation up to 50% (p < 0.001). Caco-2 RNA from days 5 and 10, hybridized on HG-U133A2.0 Affymetrix GeneChips(R), showed 1,743 affected genes on both days (p < 0.01). All 14 Caco-2 differentiation-associated genes showed decreased expression (p < 0.01), including intestinal alkaline phosphatase, that was confirmed technically (qRT-PCR) and functionally (enzyme-activity). The 1,743 genes contributed to 27 pathways (p < 0.05) categorized under six gene ontology (GO) processes, including apoptosis and cell-cycle. Genes within these GO-processes showed fold changes that suggest increased cell-survival and -proliferation. Furthermore, quercetin down-regulated expression of genes involved in tumor-suppression and phase II metabolism, and up-regulated oncogenes. Gene expression changes mediated by ascorbate-stabilized quercetin were concordant with those occurring in human colorectal carcinogenesis ( approximately 80-90%), but were opposite to those previously described for Caco-2 cells exposed to quercetin without ascorbate ( approximately 75-90%). In conclusion, gene expression among Caco-2 cells exposed to ascorbate-stabilized quercetin showed mechanisms contrary to what is expected for a cancer-preventive agent. Whether this unexpected in vitro effect is relevant in vivo, remains to be elucidated.

Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2.

The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001

Systems toxicology: applications of toxicogenomics, transcriptomics, proteomics and metabolomics in toxicology.

Toxicogenomics can facilitate the identification and characterization of toxicity, as illustrated in this review. Toxicogenomics, the application of the functional genomics technologies (transcriptomics, proteomics and metabolomics) in toxicology enables the study of adverse effects of xenobiotic substances in relation to structure and activity of the genome. The advantages and limitations of the different technologies are evaluated, and the prospects for integration of the technologies into a systems biology or systems toxicology approach are discussed. Applications of toxicogenomics in various laboratories around the world show that the crucial steps and sequence of events at the molecular level can be studied to provide detailed insights into mechanisms of toxic action. Toxicogenomics allowed for more sensitive and earlier detection of adverse effects in (animal) toxicity studies. Furthermore, the effects of exposure to mixtures could be studied in more detail. This review argues that in the (near) future, human health risk assessment will truly benefit from toxicogenomics (systems toxicology).

Integrated assessment by multiple gene expression analysis of quercetin bioactivity on anticancer-related mechanisms in colon cancer cells in vitro.

BACKGROUND: Many different mechanisms are involved in nutrient-related prevention of colon cancer. In this study, a comprehensive assessment of the spectrum of possible biological actions of the bioactive compound quercetin is made using multiple gene expression analysis. Quercetin is a flavonoid that can inhibit proliferation of tumor cells and reduce the number of aberrant crypt foci, although increase of number of colon tumors was also reported. AIM OF THE STUDY: In order to elucidate possible mechanisms involved in its mode of action the effect of quercetin on expression of 4000 human genes in Caco-2 cells was studied and related to functional effects. METHODS: Caco-2 cells were exposed to 5 or 50 microM quercetin for 48 hours, differential expression of 4000 human genes was studied using microarrays and related to functional effects. Differentially expressed genes were categorized in seven functional groups: cell cycle and differentiation, apoptosis, tumor suppressor genes and oncogenes, cell adhesion and cell-cell interaction, transcription, signal transduction and energy metabolism. Also, cell proliferation and cell cycle distribution were measured. RESULTS: Quercetin (5 microM) downregulated expression of cell cycle genes (for example CDC6, CDK4 and cyclin D1), downregulated cell proliferation and induced cell cycle arrest in Caco-2 cells. After exposure to 50 microM quercetin cell proliferation decreased to 51.3% of control, and further decrease of the percentage of cells in the G1 phase coincided with an increase of the percentage of cells in the sub-G1 phase. Quercetin upregulated expression of several tumor suppressor genes. In addition, genes involved in signal transduction pathways like beta catenin/TCF signalling and MAPK signal transduction were influenced by quercetin. CONCLUSIONS: This study shows that large-scale gene expression analysis in combination with functional assays yields a considerable amount of information on (anti-)carcinogenic potential of food components like quercetin.

Toxicogenomic analysis of gene expression changes in rat liver after a 28-day oral benzene exposure.

Benzene is an industrial chemical, component of automobile exhaust and cigarette smoke. After hepatic bioactivation benzene induces bone marrow, blood and hepatic toxicity. Using a toxicogenomics approach this study analysed the effects of benzene at three dose levels on gene expression in the liver after 28 daily doses. NMR based metabolomics was used to assess benzene exposure by identification of characteristic benzene metabolite profiles in urine. The 28-day oral exposure to 200 and 800 mg/kg/day but not 10 mg/kg/day benzene-induced hematotoxicity in male Fisher rats. Additionally these upper dose levels slightly reduced body weight and increased relative liver weights. Changes in hepatic gene expression were identified with oligonucleotide microarrays at all dose levels including the 10 mg/kg/day dose level where no toxicity was detected by other methods. The benzene-induced gene expression changes were related to pathways of biotransformation, glutathione synthesis, fatty acid and cholesterol metabolism and others. Some of the effects on gene expression observed here have previously been observed after induction of acute hepatic necrosis with bromobenzene and acetaminophen. In conclusion, changes in hepatic gene expression were found after treatment with benzene both at the toxic and non-toxic doses. The results from this study show that toxicogenomics identified hepatic effects of benzene exposure possibly related to toxicity. The findings aid to interpret the relevance of hepatic gene expression changes in response to exposure to xenobiotics. In addition, the results have the potential to inform on the mechanisms of response to benzene exposure.

Toxicogenomics of bromobenzene hepatotoxicity: a combined transcriptomics and proteomics approach.

Toxicogenomics is a novel approach integrating the expression analysis of thousands of genes (transcriptomics) or proteins (proteomics) with classical methods in toxicology. Effects at the molecular level are related to pathophysiological changes of the organisms, enabling detailed comparison of mechanisms and early detection and prediction of toxicity. This report addresses the value of the combined use of transcriptomics and proteomics technologies in toxicology. Acute hepatotoxicity was induced in rats by bromobenzene administration resulting in depleted glutathione levels and reduced average body weights, 24hr after dosage. These physiological symptoms coincided with many changes of hepatic mRNA and protein content. Gene induction confirmed involvement of glutathione-S-transferase isozymes and epoxide hydrolase in bromobenzene metabolism and identified many genes possibly relevant in bromobenzene toxicity. Observed glutathione depletion coincided with induction of the key enzyme in glutathione biosynthesis, gamma-glutamylcysteine synthetase. Oxidative stress was apparent from strong upregulation of heme oxygenase, peroxiredoxin 1 and other genes. Bromobenzene-induced protein degradation was suggested from two-dimensional gel electrophoresis, upregulated mRNA levels for proteasome subunits and lysosomal cathepsin L, whereas also genes were upregulated with a role in protein synthesis. Both protein and gene expression profiles from treated rats were clearly distinct from controls as shown by principal component analysis, and several proteins found to significantly change upon bromobenzene treatment were identified by mass spectrometry. A modest overlap in results from proteomics and transcriptomics was found. This work indicates that transcriptomics and proteomics technologies are complementary to each other and provide new possibilities in molecular toxicology.

Bromobenzene-induced hepatotoxicity at the transcriptome level.

Rats were exposed to three levels of bromobenzene, sampled at 6, 24, and 48 h, and liver gene expression profiles were determined to identify dose and time-related changes. Expression of many genes changed transiently, and dependent on the dose. Few changes were identified after 6 h, but many genes were differentially expressed after 24 h, while after 48 h, only the high dose elicited large effects. Differentially expressed genes were involved in drug metabolism (upregulated GSTs, mEH, NQO1, Mrps, downregulated CYPs, sulfotransferases), oxidative stress (induced HO-1, peroxiredoxin, ferritin), GSH depletion (induced GCS-l, GSTA, GSTM) the acute phase response, and in processes like cholesterol, fatty acid and protein metabolism, and intracellular signaling. Trancriptional regulation via the electrophile and sterol response elements seemed to mediate part of the response to bromobenzene. Recovery of the liver was suggested in response to BB by the altered expression of genes involved in protein synthesis and cytoskeleton rearrangement. Furthermore, after 48 h, rats in the mid dose group showed no toxicity, and gene expression patterns resembled the normal situation. For certain genes (e.g., CYP4A, metallothioneins), intraday variation in expression levels was found, regardless of the treatment. Selected cDNA microarray measurements were confirmed using the specific and sensitive branched DNA signal amplification assay.

Effect of thiabendazole on some rat hepatic xenobiotic metabolising enzymes.

The effect of thiabendazole (TB) on some rat hepatic xenobiotic metabolising enzymes has been investigated. Male Sprague-Dawley rats were fed control diet or diets containing 102-5188 ppm TB for 28 days. As a positive control for induction of hepatic xenobiotic metabolism, rats were also fed diets containing 1457 and 10,155 ppm butylated hydroxytoluene (BHT). Treatment with TB and BHT resulted in dose-dependent increases in relative liver weight. TB was found to be a mixed inducer of cytochrome P450 (CYP) forms in the CYP1A and CYP2B subfamilies. The administration of high doses of TB resulted in the induction of 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-depentylase activities, CYP1A1, CYP1A2, CYP2B1 and CYP2B1/2 mRNA levels and CYP1A2 and CYP2B1/2 apoprotein levels. In contrast, BHT was a CYP2B form inducer, increasing 7-pentoxyresorufin O-depentylase activity, CYP2B1 and CYP2B1/2 mRNA levels and CYP2B1/2 apoprotein levels. Both TB and BHT induced GSH S-transferase activities towards a range of substrates. In addition, TB and BHT markedly induced GSTP1 mRNA levels, but had only a small effect on GSTT1 mRNA levels. In summary, these results demonstrate that TB induces both phase I and II xenobiotic metabolising enzymes in rat liver.

Toxicology of chemical mixtures: a challenging quest along empirical sciences.

This paper describes the "quest" of our institute trying to assess the toxicology of chemical mixtures. In this overview, we will discuss some critical developments in hazard identification and risk assessment of chemical mixtures during these past 15 years. We will stand still at empirical and mechanistic modeling. "Empirical" means that only information on doses or concentrations and effects is available in addition to an often empirically selected quantitative dose-response relationship. Empirical models have played a dominant role in the last decade to identify health and safety characteristics of chemical mixtures. Many of these models are based on the work of pioneers in mixture toxicology who defined three basic types of action for combinations of chemicals: simple similar action, simple dissimilar action and interaction. Nowadays, empirical models are mainly based on response-surface analysis and make use of advanced statistical designs. However, possible interactions between components in a mixture can also be given in terms of mechanistic models. In terms of "mechanistic" (or biological) understanding, interactions between compounds may occur in the kinetic phase (processes of uptake, distribution, metabolism and excretion) or in the dynamic phase (effects of chemicals on the receptor, cellular target or organ). A biological phenomenon such as competitive agonism as described for mixtures of drugs (biotransformation enzymes) or sensory irritants (nerve receptors) can accurately predict the effect of any of these mixtures. Thus, far mechanistic and empirical analyses of interactions are usually unrelated. It is one of the future challenges for mixtures research to combine information from both approaches. Also, our current biology-based models have their limitations, since they cannot integrate every relevant biological mechanism. In this respect, mechanistic modeling of mixtures may benefit from the developments coming from the arena of molecular biology (toxicogenomics) which offers an in-depth analysis of several involved enzymatic pathways in parallel through the use of a systems biology approach. This was illustrated with mixtures of food additives known to affect the liver. Key to further maturation of mixture toxicology is collaboration of experimental toxicologists, biomathematicians, biologists, pharmacologists, model developers, molecular biologists and bioinformaticians to ensure parallel and coordinated research in this challenging area of toxicology. For this reason, the next sequel will be even more challenging and exciting to that first 15 years of empirical testing.

Next-generation text-mining mediated generation of chemical response-specific gene sets for interpretation of gene expression data.

BACKGROUND: Availability of chemical response-specific lists of genes (gene sets) for pharmacological and/or toxic effect prediction for compounds is limited. We hypothesize that more gene sets can be created by next-generation text mining (next-gen TM), and that these can be used with gene set analysis (GSA) methods for chemical treatment identification, for pharmacological mechanism elucidation, and for comparing compound toxicity profiles. METHODS: We created 30,211 chemical response-specific gene sets for human and mouse by next-gen TM, and derived 1,189 (human) and 588 (mouse) gene sets from the Comparative Toxicogenomics Database (CTD). We tested for significant differential expression (SDE) (false discovery rate -corrected p-values < 0.05) of the next-gen TM-derived gene sets and the CTD-derived gene sets in gene expression (GE) data sets of five chemicals (from experimental models). We tested for SDE of gene sets for six fibrates in a peroxisome proliferator-activated receptor alpha (PPARA) knock-out GE dataset and compared to results from the Connectivity Map. We tested for SDE of 319 next-gen TM-derived gene sets for environmental toxicants in three GE data sets of triazoles, and tested for SDE of 442 gene sets associated with embryonic structures. We compared the gene sets to triazole effects seen in the Whole Embryo Culture (WEC), and used principal component analysis (PCA) to discriminate triazoles from other chemicals. RESULTS: Next-gen TM-derived gene sets matching the chemical treatment were significantly altered in three GE data sets, and the corresponding CTD-derived gene sets were significantly altered in five GE data sets. Six next-gen TM-derived and four CTD-derived fibrate gene sets were significantly altered in the PPARA knock-out GE dataset. None of the fibrate signatures in cMap scored significant against the PPARA GE signature. 33 environmental toxicant gene sets were significantly altered in the triazole GE data sets. 21 of these toxicants had a similar toxicity pattern as the triazoles. We confirmed embryotoxic effects, and discriminated triazoles from other chemicals. CONCLUSIONS: Gene set analysis with next-gen TM-derived chemical response-specific gene sets is a scalable method for identifying similarities in gene responses to other chemicals, from which one may infer potential mode of action and/or toxic effect.

Delay and impairment in brain development and function in rat offspring after maternal exposure to methylmercury.

Maternal exposure to the neurotoxin methylmercury (MeHg) has been shown to have adverse effects on neural development of the offspring in man. Little is known about the underlying mechanisms by which MeHg affects the developing brain. To explore the neurodevelopmental defects and the underlying mechanism associated with MeHg exposure, the cerebellum and cerebrum of Wistar rat pups were analyzed by [(18)F]FDG PET functional imaging, field potential analysis, and microarray gene expression profiling. Female rat pups were exposed to MeHg via maternal diet during intrauterinal and lactational period (from gestational day 6 to postnatal day (PND)10), and their brain tissues were sampled for the analysis at weaning (PND18-21) and adulthood (PND61-70). The [(18)F]FDG PET imaging and field potential analysis suggested a delay in brain activity and impaired neural function by MeHg. Genome-wide transcriptome analysis substantiated these findings by showing (1) a delay in the onset of gene expression related to neural development, and (2) alterations in pathways related to both structural and functional aspects of nervous system development. The latter included changes in gene expression of developmental regulators, developmental phase-associated genes, small GTPase signaling molecules, and representatives of all processes required for synaptic transmission. These findings were observed at dose levels at which only marginal changes in conventional developmental toxicity endpoints were detected. Therefore, the approaches applied in this study are promising in terms of yielding increased sensitivity compared with classical developmental toxicity tests.

A toxicogenomics-based parallelogram approach to evaluate the relevance of coumarin-induced responses in primary human hepatocytes in vitro for humans in vivo.

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.

Parallelogram approach using rat-human in vitro and rat in vivo toxicogenomics predicts acetaminophen-induced hepatotoxicity in humans.

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.

Transcriptome and proteome profiling of colon mucosa from quercetin fed F344 rats point to tumor preventive mechanisms, increased mitochondrial fatty acid degradation and decreased glycolysis.

Quercetin has been shown to act as an anticarcinogen in experimental colorectal cancer (CRC). The aim of the present study was to characterize transcriptome and proteome changes occurring in the distal colon mucosa of rats supplemented with 10 g quercetin/kg diet for 11 wk. Transcriptome data analyzed with Gene Set Enrichment Analysis showed that quercetin significantly downregulated the potentially oncogenic mitogen-activated protein kinase (Mapk) pathway. In addition, quercetin enhanced expression of tumor suppressor genes, including Pten, Tp53, and Msh2, and of cell cycle inhibitors, including Mutyh. Furthermore, dietary quercetin enhanced genes involved in phase I and II metabolism, including Fmo5, Ephx1, Ephx2, and Gpx2. Quercetin increased PPARalpha target genes, and concomitantly enhanced expression of genes involved in mitochondrial fatty acid (FA) degradation. Proteomics performed in the same samples revealed 33 affected proteins, of which four glycolysis enzymes and three heat shock proteins were decreased. A proteome-transcriptome comparison showed a low correlation, but both pointed out toward altered energy metabolism. In conclusion, transcriptomics combined with proteomics showed that dietary quercetin evoked changes contrary to those found in colorectal carcinogenesis. These tumor-protective mechanisms were associated with a shift in energy production pathways, pointing at decreased cytoplasmic glycolysis and toward increased mitochondrial FA degradation.