Pathways for ligand activated nuclear receptors to unravel the genomic responses induced by hepatotoxicants.

The liver is a vital organ in vertebrates that can be subject to disease, among others due to exposure to toxic xenobiotic compounds. A group of transcription factors named ligand activated nuclear receptors (LANR) influence and regulate important liver functions, and can be activated by many xenobiotic compounds, which thereby can cause hepatotoxicity. Systematic analysis of the gene pathways regulated by LANR using modern 'omics technologies is important for investigating modes-of-action of hepatotoxicants. So far, these pathways are not publicly available in a format that allows these studies. We used PathVisio to build liver-specific LANR pathways, both for rats and humans. Since many LANR pathways are linked to each other, we also merged them into a meta-pathway. The pathways are in a GPML-format that enables pathway statistics and visualisations, and will be made available to the public through WikiPathways. We demonstrate the performance of these novel pathways in evaluating transcriptomic studies from the Japanese toxicogenomics project database (Open TG-GATEs). We show that the new pathways can be used to accurately analyse and visualize the effects of prototypical hepatotoxicants in important liver processes, and thus to evaluate the possible mode-of-actions of hepatotoxic xenobiotic compounds by assessing which LANRs are possible targets.

Baseline and genotoxic compound induced gene expression profiles in HepG2 and HepaRG compared to primary human hepatocytes.

Efforts are put into developing toxicogenomics-based toxicity testing methods using in vitro human cell models for improving human risk assessment/replacing animal models. Human in vitro liver models include HepG2, HepaRG and primary human hepatocytes (PHH). Studies on comparability/applicability of these cell types mainly focus on assessing baseline biotransformation capacities/cytochrome P450-inducibility, but compound-induced gene expression profiles are at least as important. Therefore, we compared baseline and aflatoxin B1- and benzo(α)pyrene-induced gene expression profiles in HepG2, HepaRG and PHH (11-13 donors). At baseline, all liver models differ from each other with respect to whole genome gene expression levels. PHH show profound inter-individual differences, and are most similar to HepaRG. After compound exposure, induced gene expression profiles are more similar between cell models, especially for benzo(α)pyrene. Pathways involved in compound metabolism are induced in all 3 models, while others are more pronounced in a specific cell model. Examples are transcriptomic modifications of carbohydrate-related genes (HepaRG) and of receptor-related genes (PHH) after benzo(α)pyrene exposure, and of cell cycle-related genes (HepG2) after aflatoxin B1 exposure. PHH gene expression responses are the most heterogeneous. In conclusion, at base line level PHH are more similar to HepaRG than to HepG2, but for toxicogenomics applications both cell lines perform equally well in comparison to PHH.

Toxicogenomic profiles in relation to maternal immunotoxic exposure and immune functionality in newborns.

A crucial period for the development of the immune system occurs in utero. This results in a high fetal vulnerability to immunotoxic exposure, and indeed, immunotoxic effects have been reported, demonstrating negative effects on immune-related health outcomes and immune functionality. Within the NewGeneris cohort BraMat, a subcohort of the Norwegian Mother and Child Cohort Study (MoBa), immunotoxicity was demonstrated for polychlorinated biphenyls and dioxins, showing associations between estimated maternal intake levels and reduced measles vaccination responses in the offspring at the age of 3. The present study aimed to investigate this link at the transcriptomic level within the same BraMat cohort. To this end, whole-genome gene expression in cord blood was investigated and found to be associated with maternal Food Frequency Questionnaires-derived exposure estimates and with vaccination responses in children at 3 years of age. Because the literature reports gender specificity in the innate, humoral, and cell-mediated responses to viral vaccines, separate analysis for males and females was conducted. Separate gene sets for male and female neonates were identified, comprising genes significantly correlating with both 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and polychlorinated biphenyls (PCB) exposure and with measles vaccination response. Noteworthy, genes correlating negatively with exposure in general show positive correlations with antibody levels and vice versa. For both sexes, these included immune-related genes, suggesting immunosuppressive effects of maternal exposure to TCDD and PCB at the transcriptomic level in neonates in relation to measles vaccination response 3 years later.

A transcriptomics-based in vitro assay for predicting chemical genotoxicity in vivo.

The lack of accurate in vitro assays for predicting in vivo toxicity of chemicals together with new legislations demanding replacement and reduction of animal testing has triggered the development of alternative methods. This study aimed at developing a transcriptomics-based in vitro prediction assay for in vivo genotoxicity. Transcriptomics changes induced in the human liver cell line HepG2 by 34 compounds after treatment for 12, 24, and 48 h were used for the selection of gene-sets that are capable of discriminating between in vivo genotoxins (GTX) and in vivo nongenotoxins (NGTX). By combining transcriptomics with publicly available results for these chemicals from standard in vitro genotoxicity studies, we developed several prediction models. These models were validated by using an additional set of 28 chemicals. The best prediction was achieved after stratification of chemicals according to results from the Ames bacterial gene mutation assay prior to transcriptomics evaluation after 24h of treatment. A total of 33 genes were selected for discriminating GTX from NGTX for Ames-positive chemicals and 22 for Ames-negative chemicals. Overall, this method resulted in 89% accuracy and 91% specificity, thereby clearly outperforming the standard in vitro test battery. Transcription factor network analysis revealed HNF3a, HNF4a, HNF6, androgen receptor, and SP1 as main factors regulating the expression of classifiers for Ames-positive chemicals. Thus, the classical bacterial gene mutation assay in combination with in vitro transcriptomics in HepG2 is proposed as an upgraded in vitro approach for predicting in vivo genotoxicity of chemicals holding a great promise for reducing animal experimentations on genotoxicity.

Transcriptomic fingerprints in human peripheral blood mononuclear cells indicative of genotoxic and non-genotoxic carcinogenic exposure.

For evaluating genotoxic exposure in human populations a number of biomarkers has been successfully applied over the last 30 years to determine early biological effects due to exposure to carcinogens. Despite their success, these early biological effect markers provide limited mechanistic insight, and do not allow detection of exposure to non-genotoxic carcinogens. Gene expression profiling forms a promising tool for the development of new biomarkers in blood cells to overcome these limitations. The aim of our research was to identify novel genomics-based candidate markers for genotoxic and non-genotoxic carcinogen exposure in human peripheral blood cells (PBMC). Whole genome gene expression changes were investigated following 20 h of in vitro exposure to a high and low concentration of eight genotoxic and three non-genotoxic carcinogenic compounds using whole genome microarrays. Per condition, PBMC of five independent donors were exposed, all in the presence of human liver S9. Sets of genes, as well as biological pathways indicative of genotoxic exposure and of non-genotoxic carcinogenic exposure were identified. Furthermore, networks were built using the genotoxic and non-genotoxic gene sets, showing the majority of the genes to be interlinked and revealing distinctive transcription factors for both classes. The identification of these potential candidate marker genes might contribute to the development of genomic based biomarkers of carcinogen exposure.

Comparison of phenotypic and transcriptomic effects of false-positive genotoxins, true genotoxins and non-genotoxins using HepG2 cells.

The conventional in vitro assays for genotoxicity assessment of chemicals are characterised by a high false-positive rate, thus failing to correctly predict their in vivo genotoxic effects. This study aimed to identify the cellular mechanisms induced by the false-positive genotoxins quercetin, 8-Hydroxyquinoline and 17-beta oestradiol in comparison to true genotoxins and non-genotoxins, by combining in vitro phenotypic parameters with transcriptomics data from HepG2 cells. The effects of these compounds on the phosphorylation of H2AX, cell cycle distribution and whole genome gene expression following treatment for 12, 24 and 48 h were compared with the effects of true genotoxins [benzo[a]pyrene and aflatoxin B1] and non-genotoxins (2,3,7,8-tetrachlorodibenzodioxin, cyclosporin A and ampicillin C). Quercetin induced similar phenotypic effects as true genotoxins and to some extent similar gene expression alterations. Different gene expression changes were also observed, including the up-regulation of DNA repair-related genes. 8-Hydroxyquinoline and 17-beta oestradiol showed no similarities to the true genotoxins at both the phenotypic and the transcriptomic level. In a classification approach, classifiers were selected to discriminate between genotoxins and non-genotoxins. Subsequent analysis for the false-positive compounds showed quercetin to be predicted as genotoxic and 8-hydroxyquinoline and 17-beta oestradiol as non-genotoxic. Our results support that transcriptomics analysis of compound effects in HepG2 leads to similar results with phenotypic analysis and provides additional mechanistic information. Therefore, combined evaluation of gene expression alterations and relevant functional end points using HepG2 cells may contribute to the better understanding of modes-of-action of chemicals and the correct evaluation of their genotoxic properties.

In vitro cytokine release from human peripheral blood mononuclear cells in the assessment of the immunotoxic potential of chemicals.

Alternative methods to the use of animals in testing of chemicals are needed. We investigated if the immunotoxic potential of 12 dietary toxicants could be predicted from effects on cytokine release from human peripheral blood mononuclear cells (PBMC) after in vitro exposure. Nine cytokines were selected to reflect different types of immune responses. The toxicants were classified as immunotoxic or non-immunotoxic substances according to the published in vivo data. Isolated human PBMC were exposed for 20 h to three concentrations of each of the 12 substances in the presence of human liver S9 fraction. After further incubation of PBMC in fresh medium containing the mitogen phytohemagglutinin (PHA, 10 µg/ml) for 48 h, release of the nine selected cytokines into the supernatant as well as cell proliferation were measured by Luminex technology™ and the BrdU incorporation assay, respectively. All 12 substances investigated affected the release of one or more cytokines, and each of the substances showed different cytokine release patterns. Within the limitations of the study design, the present study suggests that the effect of the substances on mitogen-induced cytokine release from PBMC cannot predict their immunotoxic potential, but may be useful in mechanistic studies.

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

Well-established in vitro methods for testing the genotoxic potency of chemicals--such as the Ames/Salmonella test, the mouse lymphoma assay, the micronucleus test and the chromosomal aberration test--show a high false-positive rate for predicting in vivo genotoxicity and carcinogenicity. Thus, there is a need for more reliable in vitro assays. We investigated whether gene expression profiling in metabolically competent primary mouse hepatocytes is capable of discriminating true genotoxic (GTX) compounds from false-positive genotoxic (FP-GTX) compounds. Sandwich-cultured primary hepatocytes from male C57Bl6 mice were treated for 24 and 48 h with five true GTX and five FP-GTX compounds. Whole genome gene expression modifications were analysed by means of Affymetrix mouse genome 430 2.0 microarrays. Filtered genes were used for hierarchical clustering and class prediction methods. Classifiers were generated by prediction analysis of microarray using a leave-one-compound-out method and selecting the genes that were common to the 10 training sets. For the training compounds, all but one were correctly classified. Validation of the classification model with five new compounds resulted in a 100% correct classification at 24 h and 80% at 48 h. The generated classifiers were mostly involved in metabolic and biosynthetic processes, immune responses and apoptosis. Applying genes whose expression change correlates with γH2AX foci, a measure for DNA damage, did not improve the classification. The present study shows that gene expression profiling in primary mouse hepatocytes is capable of discriminating between true GTX and FP-GTX compounds.

A pro-inflammatory genotype predisposes to Barrett's esophagus.

INTRODUCTION: Severity of mucosal inflammation is shown to be associated with Barrett's esophagus (BE) development in animals. It has therefore been postulated that a strong pro-inflammatory host response predisposes to BE. AIM: To determine the impact of cytokine gene polymorphisms on the development of BE. METHODS: The multiplex SNaPshot method was used to determine interleukin (IL)-12B (A+1188C), IL-10 (C-592A, C-819T, A-1082G), IL-8 (A-251T), IL-6 (G-174C) and IL-2 (G-330T) gene polymorphisms in 255 patients with BE and 247 patients with reflux esophagitis (RE). RESULTS: The presence of the IL-12B C-allele, which is associated with increased IL-12p70 expression, was more frequently observed in BE than in RE patients [odds ratio (OR) 1.8; 95% confidence interval (CI) 1.2-2.7; P = 0.007). The risk of BE was increased in patients in whom the IL-12B C-allele coincided with a hiatal hernia (OR 2.9; 95% CI 1.32-6.58; P = 0.008). The IL-10(-1082) GG genotype, which is associated with higher IL-10 levels, was also associated with a decreased risk of BE when it was associated with the IL-12B C-allele, indicating IL-10-dependent down-regulation of IL-12p70 expression. A combination of the IL-12B AA genotype and the IL-10 AA or AG genotypes was associated with RE (OR 1.4; 95% CI 1.05-1.85; P = 0.011). CONCLUSION: A genetic profile predisposing to a strong pro-inflammatory host response, mediated by IL-12p70 and partially dependent on IL-10, is associated with BE. This risk further increases when this genotype coincides with a hiatal hernia, suggesting that exposure to gastroesophageal reflux in the presence of a pro-inflammatory genetic background is a driving force in the development of BE.

A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling.

A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte-based in vitro system was developed with special focus on metabolic competence. Therefore, a mixture of CYP450 inducers, phenobarbital, dexamethasone and beta-naphthoflavone, was added to culture medium of sandwich-cultured rat hepatocytes. The resulting modified model was evaluated by comparing its genome-wide expression profiles with liver and a standard model without the inducer mixture. Metabolic capacity for CYP450 enzymes showed that the modified model resembled more closely the in vivo situation. Gene expression results revealed large differences between in vivo and both in vitro models. The slight differences between the two sandwich models were predominantly represented by gene expression changes in CYP450s. Importantly, in the modified model, expression ratios of the phase I and the majority of phase II genes more closely resembled liver in vivo. The CYP450 enzyme activities corresponded with gene expression data. In conclusion, for toxicological applications using sandwich-cultured hepatocytes, the modified model may be preferred.

Genome-wide differential gene expression in children exposed to air pollution in the Czech Republic.

The Teplice area in the Czech Republic is a mining district where elevated levels of air pollution including airborne carcinogens, have been demonstrated, especially during winter time. This environmental exposure can impact human health; in particular children may be more vulnerable. To study the impact of air pollution in children at the transcriptional level, peripheral blood cells were subjected to whole genome response analysis, in order to identify significantly modulated biological pathways and processes as a result of exposure. Using genome-wide oligonucleotide microarrays, we investigated differential gene expression in children from the Teplice area (n=23) and compared them with children from the rural control area of Prachatice (n=24). In an additional approach, individual gene expressions were correlated with individual peripheral blood lymphocyte micronuclei frequencies, in order to evaluate the linkage of individual gene expressions with an established biomarker of effect that is representative for increased genotoxic risk. Children from the Teplice area showed a significantly higher average micronuclei frequency than Prachatice children (p=0.023). For considerable numbers of genes, the expression differed significantly between the children from the two areas. Amongst these genes, considerable numbers of genes were observed to correlate significantly with the frequencies of micronuclei. The main biological process that appeared significantly affected overall was nucleosome assembly. This suggests an effect of air pollution on the primary structural unit of the condensed DNA. In addition, several other pathways were modulated. Based on the results of this study, we suggest that transcriptomic analysis represents a promising biomarker for environmental carcinogenesis.

Comparison of supervised clustering methods to discriminate genotoxic from non-genotoxic carcinogens by gene expression profiling.

Prediction of the toxic properties of chemicals based on modulation of gene expression profiles in exposed cells or animals is one of the major applications of toxicogenomics. Previously, we demonstrated that by Pearson correlation analysis of gene expression profiles from treated HepG2 cells it is possible to correctly discriminate and predict genotoxic from non-genotoxic carcinogens. Since to date many different supervised clustering methods for discrimination and prediction tests are available, we investigated whether application of the methods provided by the Whitehead Institute and Stanford University improved our initial prediction. Four different supervised clustering methods were applied for this comparison, namely Pearson correlation analysis (Pearson), nearest shrunken centroids analysis (NSC), K-nearest neighbour analysis (KNN) and Weighted voting (WV). For each supervised clustering method, three different approaches were followed: (1) using all the data points for all treatments, (2) exclusion of the samples with marginally affected gene expression profiles and (3) filtering out the gene expression signals that were hardly altered. On the complete data set, NSC, KNN and WV outperformed the Pearson test, but on the reduced data sets no clear difference was observed. Exclusion of samples with marginally affected profiles improved the prediction by all methods. For the various prediction models, gene sets of different compositions were selected; in these 27 genes appeared three times or more. These 27 genes are involved in many different biological processes and molecular functions, such as apoptosis, cell cycle control, regulation of transcription, and transporter activity, many of them related to the carcinogenic process. One gene, BAX, was selected in all 10 models, while ZFP36 was selected in 9, and AHR, MT1E and TTR in 8. Summarising, this study demonstrates that several supervised clustering methods can be used to discriminate certain genotoxic from non-genotoxic carcinogens by gene expression profiling in vitro in HepG2 cells. None of the methods clearly outperforms the others.

Discrimination of genotoxic from non-genotoxic carcinogens by gene expression profiling.

Two general mechanisms are implicated in chemical carcinogenesis. The first involves direct damage to DNA, referred to as genotoxic (GTX), to which the cell responds by repair of the damages, arrest of the cell cycle or induction of apoptosis. The second is non-DNA damaging, non-genotoxic (NGTX), in which a wide variety of cellular processes may be involved. Therefore, it can be hypothesized that modulation of the underlying gene expression patterns is profoundly distinct between GTX and NGTX carcinogens, and thus that expression profiling is applicable for classification of chemical carcinogens as GTX or NGTX. We investigated this hypothesis by analysing modulation of gene expression profiles induced by 20 chemical carcinogens in HepG2 cells with application of cDNA microarrays that contain 597 toxicologically relevant genes. In total, 22 treatments were included, divided in two sets. The training set consisted of 16 treatments (nine genotoxins and seven non-genotoxins) and the validation set of six treatments (three and three). Class discrimination models based on Pearson correlation analyses for the 20 most discriminating genes were developed with data from the training set, where after the models were tested with all data. Using all data, the correctness for classification of the carcinogens from the training set was clearly better than that for the validation set, namely 81 and 33%, respectively. Exclusion of the treatments that had only marginal effects on the expression profiles, improved the discrimination for the training and validation sets to 92 and 100% correctness, respectively. Exclusion of the gene expression signals that were hardly altered also improved classification, namely to 94 and 80%. Therefore, our study proves the principle that gene expression profiling can discriminate carcinogens with major differences in their mode of actions, namely genotoxins versus non-genotoxins.

Lack of clastogenic effects in cultured human lymphocytes treated with hydroquinone.

Hydroquinone (HQ) occurs in the environment as a result of manmade processes as well as in natural products from plants and animals. The compound has been reported to produce chromosomal effects in some in vivo and in vitro animal models. However, its potential to produce similar effects in human lymphocytes is less clear. To obtain more information on the clastogenic potential of HQ in human cells, its ability to induce structural chromosomal aberrations in human lymphocytes in vitro has been examined, both in the absence and presence of exogenous metabolic activation. Moreover, the effect of HQ pre-incubation on peroxide induced clastogenicity was studied, because HQ has putative chemopreventive activity as well. It was found that HQ was cytotoxic, but did not induce chromosomal aberrations in human lymphocytes cultured in vitro. Additionally, it was observed that pre-incubation of lymphocytes with HQ resulted in a concentration dependent reduction of the H2O2 induced chromosomal aberrations (P=0.069). However, this effect was present at 12 mM H2O2 only, because of high cytotoxicity at higher dosages.

The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo.

There is increasing evidence that chemicals/test substances cannot only have adverse effects, but that there are many substances that can (also) have a beneficial effect on health. As this journal regularly publishes papers in this area and has every intention in continuing to do so in the near future, it has become essential that studies reported in this journal reflect an adequate level of scientific scrutiny. Therefore a set of essential characteristics of studies has been defined. These basic requirements are default properties rather than non-negotiables: deviations are possible and useful, provided they can be justified on scientific grounds. The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo concern the following areas: (1) Hypothesis-driven study design; (2) The nature of the test substance; (3) Valid and invalid test systems; (4) The selection of dose levels and gender; (5) Reversal of the effects induced by oxidants, carcinogens and mutagens; (6) Route of administration; (7) Number and validity of test variables; (8) Repeatability and reproducibility; (9) Statistics; and (10) Quality Assurance.

Monitoring for DNA damage of humans occupationally exposed to methyl bromide.

BACKGROUND: Methyl bromide (MeBr) is a methylating agent, weak mutagen and possible animal carcinogen. A molecular epidemiological study to examine human exposure to, and consequent DNA damage by MeBr was conducted in an area where this agent is used extensively for soil sterilisation in greenhouses. MATERIALS AND METHODS: During the first part of the study, blood samples were collected from 21 persons within 24 hours after use of MeBr for greenhouse sterilisation, as well as from 19 non-exposed subjects. Personal air sampling was also carried out, indicating mean air concentrations for different subjects in the range 11-78 mg/m3. In the second part of the study, an attempt was made to examine professional applicators of MeBr who suffered particularly high exposures (mean exposures, based on personal monitoring 23-165 mg/m3). The levels of N7-methylguanine and O6-methylguanine, two DNA adducts known to be induced by MeBr, were assessed in blood leukocyte DNA. RESULTS: Concerning the first part, two subjects (one exposed and one control) were found to be positive for N7-methylguanine, while none of the blood samples analysed had detectable levels of O6-methylguanine. Among 6 such persons examined during the second part, 2 were found positive for N7-methylguanine while none was positive for O6-methylguanine. CONCLUSION: Within the detection power of this limited study, no significant evidence of induction of DNA damage in blood leukocyte DNA by MeBr was found.