Epigenome-wide association study of incident type 2 diabetes: a meta-analysis of five prospective European cohorts.

AIMS/HYPOTHESIS: Type 2 diabetes is a complex metabolic disease with increasing prevalence worldwide. Improving the prediction of incident type 2 diabetes using epigenetic markers could help tailor prevention efforts to those at the highest risk. The aim of this study was to identify predictive methylation markers for incident type 2 diabetes by combining epigenome-wide association study (EWAS) results from five prospective European cohorts. METHODS: We conducted a meta-analysis of EWASs in blood collected 7-10 years prior to type 2 diabetes diagnosis. DNA methylation was measured with Illumina Infinium Methylation arrays. A total of 1250 cases and 1950 controls from five longitudinal cohorts were included: Doetinchem, ESTHER, KORA1, KORA2 and EPIC-Norfolk. Associations between DNA methylation and incident type 2 diabetes were examined using robust linear regression with adjustment for potential confounders. Inverse-variance fixed-effects meta-analysis of cohort-level individual CpG EWAS estimates was performed using METAL. The methylGSA R package was used for gene set enrichment analysis. Confirmation of genome-wide significant CpG sites was performed in a cohort of Indian Asians (LOLIPOP, UK). RESULTS: The meta-analysis identified 76 CpG sites that were differentially methylated in individuals with incident type 2 diabetes compared with control individuals (p values <1.1 × 10-7). Sixty-four out of 76 (84.2%) CpG sites were confirmed by directionally consistent effects and p values <0.05 in an independent cohort of Indian Asians. However, on adjustment for baseline BMI only four CpG sites remained genome-wide significant, and addition of the 76 CpG methylation risk score to a prediction model including established predictors of type 2 diabetes (age, sex, BMI and HbA1c) showed no improvement (AUC 0.757 vs 0.753). Gene set enrichment analysis of the full epigenome-wide results clearly showed enrichment of processes linked to insulin signalling, lipid homeostasis and inflammation. CONCLUSIONS/INTERPRETATION: By combining results from five European cohorts, and thus significantly increasing study sample size, we identified 76 CpG sites associated with incident type 2 diabetes. Replication of 64 CpGs in an independent cohort of Indian Asians suggests that the association between DNA methylation levels and incident type 2 diabetes is robust and independent of ethnicity. Our data also indicate that BMI partly explains the association between DNA methylation and incident type 2 diabetes. Further studies are required to elucidate the underlying biological mechanisms and to determine potential causal roles of the differentially methylated CpG sites in type 2 diabetes development.

Relevance of In Vitro Transcriptomics for In Vivo Mode of Action Assessment.

Recently, we reported an in vitro toxicogenomics comparison approach to categorize chemical substances according to similarities in their proposed toxicological modes of action. Use of such an approach for regulatory purposes requires, among others, insight into the extent of biological concordance between in vitro and in vivo findings. To that end, we applied the comparison approach to transcriptomics data from the Open TG-GATEs database for 137 substances with diverging modes of action and evaluated the outcomes obtained for rat primary hepatocytes and for rat liver. The results showed that a relatively small number of matches observed in vitro were also observed in vivo, whereas quite a large number of matches between substances were found to be relevant solely in vivo or in vitro. The latter could not be explained by physicochemical properties, leading to insufficient bioavailability or poor water solubility. Nevertheless, pathway analyses indicated that for relevant matches the mechanisms perturbed in vitro are consistent with those perturbed in vivo. These findings support the utility of the comparison approach as tool in mechanism-based risk assessment.

The Arabidopsis SUMO E3 ligase SIZ1 mediates the temperature dependent trade-off between plant immunity and growth.

Increased ambient temperature is inhibitory to plant immunity including auto-immunity. SNC1-dependent auto-immunity is, for example, fully suppressed at 28°C. We found that the Arabidopsis sumoylation mutant siz1 displays SNC1-dependent auto-immunity at 22°C but also at 28°C, which was EDS1 dependent at both temperatures. This siz1 auto-immune phenotype provided enhanced resistance to Pseudomonas at both temperatures. Moreover, the rosette size of siz1 recovered only weakly at 28°C, while this temperature fully rescues the growth defects of other SNC1-dependent auto-immune mutants. This thermo-insensitivity of siz1 correlated with a compromised thermosensory growth response, which was independent of the immune regulators PAD4 or SNC1. Our data reveal that this high temperature induced growth response strongly depends on COP1, while SIZ1 controls the amplitude of this growth response. This latter notion is supported by transcriptomics data, i.e. SIZ1 controls the amplitude and timing of high temperature transcriptional changes including a subset of the PIF4/BZR1 gene targets. Combined our data signify that SIZ1 suppresses an SNC1-dependent resistance response at both normal and high temperatures. At the same time, SIZ1 amplifies the dark and high temperature growth response, likely via COP1 and upstream of gene regulation by PIF4 and BRZ1.

A day and night difference in the response of the hepatic transcriptome to cyclophosphamide treatment.

Application of omics-based technologies is a widely used approach in research aiming to improve testing strategies for human health risk assessment. In most of these studies, however, temporal variations in gene expression caused by the circadian clock are a commonly neglected pitfall. In the present study, we investigated the impact of the circadian clock on the response of the hepatic transcriptome after exposure of mice to the chemotherapeutic agent cyclophosphamide (CP). Analysis of the data without considering clock progression revealed common responses in terms of regulated pathways between light and dark phase exposure, including DNA damage, oxidative stress, and a general immune response. The overall response, however, was stronger in mice exposed during the day. Use of time-matched controls, thereby eliminating non-CP-responsive circadian clock-controlled genes, showed that this difference in response was actually even more pronounced: CP-related responses were only identified in mice exposed during the day. Only minor differences were found in acute toxicity pathways, namely lymphocyte counts and kidney weights, indicating that gene expression is subject to time of day effects. This study is the first to highlight the impact of the circadian clock on the identification of toxic responses by omics approaches.

A novel toxicogenomics-based approach to categorize (non-)genotoxic carcinogens.

Alternative methods to detect non-genotoxic carcinogens are urgently needed, as this class of carcinogens goes undetected in the current testing strategy for carcinogenicity under REACH. A complicating factor is that non-genotoxic carcinogens act through several distinctive modes of action, which makes prediction of their carcinogenic property difficult. We have recently demonstrated that gene expression profiling in primary mouse hepatocytes is a useful approach to categorize non-genotoxic carcinogens according to their modes of action. In the current study, we improved the methods used for analysis and added mouse embryonic stem cells as a second in vitro test system, because of their features complementary to hepatocytes. Our approach involved an unsupervised analysis based on the 30 most significantly up- and down-regulated genes per chemical. Mouse embryonic stem cells and primary mouse hepatocytes were exposed to a selected set of chemicals and subsequently subjected to gene expression profiling. We focused on non-genotoxic carcinogens, but also included genotoxic carcinogens and non-carcinogens to test the robustness of this approach. Application of the optimized comparison approach resulted in improved categorization of non-genotoxic carcinogens. Mouse embryonic stem cells were a useful addition, especially for genotoxic substances, but also for detection of non-genotoxic carcinogens that went undetected by primary hepatocytes. The approach presented here is an important step forward to categorize chemicals, especially those that are carcinogenic.

In vivo murine hepatic microRNA and mRNA expression signatures predicting the (non-)genotoxic carcinogenic potential of chemicals.

There is a high need to improve the assessment of, especially non-genotoxic, carcinogenic features of chemicals. We therefore explored a toxicogenomics-based approach using genome-wide microRNA and mRNA expression profiles upon short-term exposure in mice. For this, wild-type mice were exposed for seven days to three different classes of chemicals, i.e., four genotoxic carcinogens (GTXC), seven non-genotoxic carcinogens (NGTXC), and five toxic non-carcinogens. Hepatic expression patterns of mRNA and microRNA transcripts were determined after exposure and used to assess the discriminative power of the in vivo transcriptome for GTXC and NGTXC. A final classifier set, discriminative for GTXC and NGTXC, was generated from the transcriptomic data using a tiered approach. This appeared to be a valid approach, since the predictive power of the final classifier set in three different classifier algorithms was very high for the original training set of chemicals. Subsequent validation in an additional set of chemicals revealed that the predictive power for GTXC remained high, in contrast to NGTXC, which appeared to be more troublesome. Our study demonstrated that the in vivo microRNA-ome has less discriminative power to correctly identify (non-)genotoxic carcinogen classes. The results generally indicate that single mRNA transcripts do have the potential to be applied in risk assessment, but that additional (genomic) strategies are necessary to correctly predict the non-genotoxic carcinogenic potential of a chemical.

Exploration of genome-wide DNA methylation profiles in night shift workers.

The past decades, studies indicated that night shift work is associated with adverse health effects, however, molecular mechanisms underlying these effects are poorly understood. A few previous studies have hypothesized a role for DNA-methylation (DNAm) in this relationship. We performed a cross-sectional epigenome-wide association study, to investigate if night shift work is associated with genome-wide DNAm changes and DNAm-based biological age acceleration, based on previously developed so-called 'epigenetic clocks.' Short term (2-6 years) and intermediate term (10-16 years) night shift workers, along with age and sex matched dayworkers (non-shift workers) were selected from the Lifelines Cohort Study. For genome-wide methylation analysis the Infinium Methylation EPIC array (Ilumina) was used. Linear regression analyses were used to detect differences in methylation at individual CpG-sites associated with night shift work. Pathway analysis was performed based on KEGG pathways and predictions of age acceleration in night shift workers were performed based on four previously developed epigenetic age calculators. Only in women, differences in methylation at individual CpG-sites were observed between night shift workers and non-shift workers. Most of these differentially methylated positions (DMPs) were observed in intermediate term night shift workers. Pathway analysis shows involvement of pathways related to circadian rhythm and cellular senescence. Increased age acceleration was observed only in short-term night shift workers (men and women). This might be indicative of adaptation to night shift work or a so-called healthy worker effect. In conclusion, these results show that DNA methylation changes are associated with night shift work, specifically in women.

Dissecting modes of action of non-genotoxic carcinogens in primary mouse hepatocytes.

Under REACH, the European Community Regulation on chemicals, the testing strategy for carcinogenicity is based on in vitro and in vivo genotoxicity assays. Given that non-genotoxic carcinogens are negative for genotoxicity and chronic bioassays are no longer regularly performed, this class of carcinogens will go undetected. Therefore, test systems detecting non-genotoxic carcinogens, or even better their modes of action, are required. Here, we investigated whether gene expression profiling in primary hepatocytes can be used to distinguish different modes of action of non-genotoxic carcinogens. For this, primary mouse hepatocytes were exposed to 16 non-genotoxic carcinogens with diverse modes of action. Upon profiling, pathway analysis was performed to obtain insight into the biological relevance of the observed changes in gene expression. Subsequently, both a supervised and an unsupervised comparison approach were applied to recognize the modes of action at the transcriptomic level. These analyses resulted in the detection of three of eight compound classes, that is, peroxisome proliferators, metalloids and skin tumor promotors. In conclusion, gene expression profiles in primary hepatocytes, at least in rodent hepatocytes, appear to be useful to detect some, certainly not all, modes of action of non-genotoxic carcinogens.

DNA methylation trajectories and accelerated epigenetic aging in incident type 2 diabetes.

DNA methylation (DNAm) patterns across the genome changes during aging and development of complex diseases including type 2 diabetes (T2D). Our study aimed to estimate DNAm trajectories of CpG sites associated with T2D, epigenetic age (DNAmAge), and age acceleration based on four epigenetic clocks (GrimAge, Hannum, Horvath, phenoAge) in the period 10 years prior to and up to T2D onset. In this nested case-control study within Doetinchem Cohort Study, we included 132 incident T2D cases and 132 age- and sex-matched controls. DNAm was measured in blood using the Illumina Infinium Methylation EPIC array. From 107 CpG sites associated with T2D, 10 CpG sites (9%) showed different slopes of DNAm trajectories over time (p < 0.05) and an additional 8 CpG sites (8%) showed significant differences in DNAm levels (at least 1%, p-value per time point < 0.05) at all three time points with nearly parallel trajectories between incident T2D cases and controls. In controls, age acceleration levels were negative (slower epigenetic aging), while in incident T2D cases, levels were positive, suggesting accelerated aging in the case group. We showed that DNAm levels at specific CpG sites, up to 10 years before T2D onset, are different between incident T2D cases and healthy controls and distinct patterns of clinical traits over time may have an impact on those DNAm profiles. Up to 10 years before T2D diagnosis, cases manifested accelerated epigenetic aging. Markers of biological aging including age acceleration estimates based on Horvath need further investigation to assess their utility for predicting age-related diseases including T2D.

Neuronal differentiation pathways and compound-induced developmental neurotoxicity in the human neural progenitor cell test (hNPT) revealed by RNA-seq.

There is an increased awareness that the use of animals for compound-induced developmental neurotoxicity (DNT) testing has limitations. Animal-free innovations, especially the ones based on human stem cell-based models are pivotal in studying DNT since they can mimic processes relevant to human brain development. Here we present the human neural progenitor test (hNPT), a 10-day protocol in which neural progenitor cells differentiate into a neuron-astrocyte co-culture. The study aimed to characterise differentiation over time and to find neurodevelopmental processes sensitive to compound exposure using transcriptomics. 3992 genes regulated in unexposed control cultures (p ≤ 0.001, log2FC ≥ 1) showed Gene Ontology (GO-) term enrichment for neuronal and glial differentiation, neurite extension, synaptogenesis, and synaptic transmission. Exposure to known or suspected DNT compounds (acrylamide, chlorpyrifos, fluoxetine, methyl mercury, or valproic acid) at concentrations resulting in 95% cell viability each regulated unique combinations of GO-terms relating to neural progenitor proliferation, neuronal and glial differentiation, axon development, synaptogenesis, synaptic transmission, and apoptosis. Investigation of the GO-terms 'neuron apoptotic process' and 'axon development' revealed common genes that were responsive across compounds, and might be used as biomarkers for DNT. The GO-term 'synaptic signalling', on the contrary, whilst also responsive to all compounds tested, showed little overlap in gene expression regulation patterns between the conditions. This GO-term may articulate compound-specific effects that may be relevant for revealing differences in mechanism of toxicity. Given its focus on neural progenitor cell to mature multilineage neuronal cell maturation and its detailed molecular readout based on gene expression analysis, hNPT might have added value as a tool for neurodevelopmental toxicity testing in vitro. Further assessment of DNT-specific biomarkers that represent these processes needs further studies.

Benchtop NMR spectroscopy in the analysis of substandard and falsified medicines as well as illegal drugs.

Substandard and falsified medical products may cause harm to patients and fail to treat the diseases or conditions for which they were intended. It is therefore required to have analytical methods available to assess medical product quality. Benchtop NMR spectroscopy provides a generic, inherently quantitative, analytical method capable of separating specific signals from those of a matrix. We have developed an analytical method for the analysis of active ingredients in pharmaceutical products and illegal drugs, based on benchtop NMR spectroscopy. Within its resolution limits, benchtop NMR spectroscopy is useful in determining the identity of the active ingredients in products containing acetaminophen, aspirin, caffeine, diclofenac, ibuprofen, naproxen, sildenafil, tadalafil and sibutramine, cocaine, and gamma hydroxybutyric acid, with a limit of detection of about 1 mg/mL. Furthermore, the content of the active ingredient can be determined with an error of 10%. Additionally, a chemometrics approach is shown to be useful to classify spectra in order to identify the active substances present in the sample, reducing the need for expert interpretation of the spectra acquired.

Differential effects of fluoxetine and venlafaxine in the neural embryonic stem cell test (ESTn) revealed by a cell lineage map.

There is a need for in vitro tests for the evaluation of chemicals and pharmaceuticals that may cause developmental neurotoxicity (DNT) in humans. The neural embryonic stem cell test (ESTn) is such an in vitro test that mimics early neural differentiation. The aim of this study was to define the biological domain of ESTn based on the expression of selective markers for certain cell types, and to investigate the effects of two antidepressants, fluoxetine (FLX) and venlafaxine (VNX), on neural differentiation. A cell lineage map was made to track neural differentiation and the effects of FLX and VNX in ESTn. Whole transcriptome analysis revealed differentiation from an embryonic stem cell population to a mixed culture of neural progenitors, neurons and neural crest cells 7 days into differentiation. Maturing neurons, astrocytes and oligodendrocytes were present after 13 days. Exposure to FLX or VNX led to different expression patterns between compounds at both time points. On day 7, both compounds upregulated most of the stem cell- and immature neuron markers, but had distinct effects on neural subtype markers. FLX downregulated glycinergic markers and upregulated cholinergic markers, while VNX had the opposite effect. On day 13, FLX and VNX affected their specific therapeutic targets, represented by mainly serotonergic markers by FLX- and dopaminergic and noradrenergic markers in VNX-exposed cultures, as well as oligodendrocyte and glycinergic neuron markers. This proof of concept study shows the added value of assessing DNT in ESTn through a cell lineage map and gives mechanistic insight in the potential neurodevelopmental effects of FLX and VNX. More compounds should be tested to further evaluate the use of the cell lineage map.

Protein and calorie restriction may improve outcomes in living kidney donors and kidney transplant recipients.

Previously, we and others showed that dietary restriction protects against renal ischemia-reperfusion injury in animals. However, clinical translation of preoperative diets is scarce, and in the setting of kidney transplantation these data are lacking. In this pilot study, we investigated the effects of five days of a preoperative protein and caloric dietary restriction (PCR) diet in living kidney donors on the perioperative effects in donors, recipients and transplanted kidneys. Thirty-five kidney donors were randomized into either the PCR, 30% calorie and 80% protein reduction, or control group without restrictions. Adherence to the diet and kidney function in donors and their kidney recipients were analyzed. Perioperative kidney biopsies were taken in a selected group of transplanted kidneys for gene expression analysis. All donors adhered to the diet. From postoperative day 2 up until month 1, kidney function of donors was significantly better in the PCR-group. PCR-donor kidney recipients showed significantly improved kidney function and lower incidence of slow graft function and acute rejection. PCR inhibited cellular immune response pathways and activated stress-resistance signaling. These observations are the first to show that preoperative dietary restriction induces postoperative recovery benefits in humans and may be beneficial in clinical settings involving ischemia-reperfusion injury.

Distinguishing mode of action of compounds inducing craniofacial malformations in zebrafish embryos to support dose-response modeling in combined exposures.

Knowledge on mode-of-action (MOA) is required to understand toxicological effects of compounds, notably in the context of risk assessment of mixtures. Such information is generally scarce, and often complicated by the existence of multiple MOAs per compound. Here, MOAs related to developmental craniofacial malformations were derived from literature, and assembled in a MOA network. A selection of gene expression markers was based on these MOAs. Next, these markers were verified by qPCR in zebrafish embryos, after exposure to reference compounds. These were: triazoles for inhibition of retinoic acid (RA) metabolism, AM580 and CD3254 for selective activation of respectively RA-receptor (RAR) and retinoid-X-receptor (RXR), dithiocarbamates for inhibition of lysyl oxidase, TCDD for activation of the aryl-hydrocarbon-receptor (AhR), VPA for inhibition of histone deacetylase (HDAC), and PFOS for activation of peroxisome proliferator-activated receptor-alpha (PPARα). Next, marker gene profiles for these reference compounds were used to map the profiles of test compounds to known MOAs. In this way, 2,4-dinitrophenol matched with the TCDD and RAR profiles, boric acid with RAR, endosulfan with PFOS, fenpropimorph with dithiocarbamates, PCB126 with AhR, and RA with triazoles and RAR profiles. Prochloraz showed no match. Activities of these compounds in ToxCast assays, and in silico analysis of binding affinity to the respective targets showed limited concordance with the marker gene expression profiles, but still confirmed the complex MOA profiles of reference and test compounds. Ultimately, this approach could be used to support modeling of mixture effects based on upfront knowledge of (dis)similarity of MOAs.

The effects of bariatric surgery on clinical profile, DNA methylation, and ageing in severely obese patients.

BACKGROUND: Severe obesity is a growing, worldwide burden and conventional therapies including radical change of diet and/or increased physical activity have limited results. Bariatric surgery has been proposed as an alternative therapy showing promising results. It leads to substantial weight loss and improvement of comorbidities such as type 2 diabetes. Increased adiposity is associated with changes in epigenetic profile, including DNA methylation. We investigated the effect of bariatric surgery on clinical profile, DNA methylation, and biological age estimated using Horvath's epigenetic clock. RESULTS: To determine the impact of bariatric surgery and subsequent weight loss on clinical traits, a cohort of 40 severely obese individuals (BMI = 30-73 kg/m2) was examined at the time of surgery and at three follow-up visits, i.e., 3, 6, and 12 months after surgery. The majority of the individuals were women (65%) and the mean age at surgery was 45.1 ± 8.1 years. We observed a significant decrease over time in BMI, fasting glucose, HbA1c, HOMA-IR, insulin, total cholesterol, triglycerides, LDL and free fatty acids levels, and a significant small increase in HDL levels (all p values < 0.05). Epigenome-wide association analysis revealed 4857 differentially methylated CpG sites 12 months after surgery (at Bonferroni-corrected p value < 1.09 × 10-7). Including BMI change in the model decreased the number of significantly differentially methylated CpG sites by 51%. Gene set enrichment analysis identified overrepresentation of multiple processes including regulation of transcription, RNA metabolic, and biosynthetic processes in the cell. Bariatric surgery in severely obese patients resulted in a decrease in both biological age and epigenetic age acceleration (EAA) (mean = - 0.92, p value = 0.039). CONCLUSIONS: Our study shows that bariatric surgery leads to substantial BMI decrease and improvement of clinical outcomes observed 12 months after surgery. These changes explained part of the association between bariatric surgery and DNA methylation. We also observed a small, but significant improvement of biological age. These epigenetic changes may be modifiable by environmental lifestyle factors and could be used as potential biomarkers for obesity and in the future for obesity related comorbidities.

Effects of Light-at-Night on the Rat Liver - A Role for the Autonomic Nervous System.

Exposure to light at night (LAN) has been associated with serious pathologies, including obesity, diabetes and cancer. Recently we showed that 2 h of LAN impaired glucose tolerance in rats. Several studies have suggested that the autonomic nervous system (ANS) plays an important role in communicating these acute effects of LAN to the periphery. Here, we investigated the acute effects of LAN on the liver transcriptome of male Wistar rats. Expression levels of individual genes were not markedly affected by LAN, nevertheless pathway analysis revealed clustered changes in a number of endocrine pathways. Subsequently, we used selective hepatic denervations [sympathetic (Sx), parasympathetic (Px), total (Tx, i.e., Sx plus Px), sham] to investigate the involvement of the ANS in the effects observed. Surgical removal of the sympathetic or parasympathetic hepatic branches of the ANS resulted in many, but small changes in the liver transcriptome, including a pathway involved with circadian clock regulation, but it clearly separated the four denervation groups. On the other hand, analysis of the liver metabolome was not able to separate the denervation groups, and only 6 out of 78 metabolites were significantly up- or downregulated after denervations. Finally, removal of the sympathetic and parasympathetic hepatic nerves combined with LAN exposure clearly modulated the effects of LAN on the liver transcriptome, but left most endocrine pathways unaffected. Conclusion: One-hour light-at-night acutely affects the liver transcriptome. Part of this effect is mediated via the nervous innervation, as a hepatectomy modulated and reduced the effect of LAN on liver transcripts.

Time-restricted feeding improves adaptation to chronically alternating light-dark cycles.

Disturbance of the circadian clock has been associated with increased risk of cardio-metabolic disorders. Previous studies showed that optimal timing of food intake can improve metabolic health. We hypothesized that time-restricted feeding could be a strategy to minimize long term adverse metabolic health effects of shift work and jetlag. In this study, we exposed female FVB mice to weekly alternating light-dark cycles (i.e. 12 h shifts) combined with ad libitum feeding, dark phase feeding or feeding at a fixed clock time, in the original dark phase. In contrast to our expectations, long-term disturbance of the circadian clock had only modest effects on metabolic parameters. Mice fed at a fixed time showed a delayed adaptation compared to ad libitum fed animals, in terms of the similarity in 24 h rhythm of core body temperature, in weeks when food was only available in the light phase. This was accompanied by increased plasma triglyceride levels and decreased energy expenditure, indicating a less favorable metabolic state. On the other hand, dark phase feeding accelerated adaptation of core body temperature and activity rhythms, however, did not improve the metabolic state of animals compared to ad libitum feeding. Taken together, restricting food intake to the active dark phase enhanced adaptation to shifts in the light-dark schedule, without significantly affecting metabolic parameters.

Application of the comparison approach to open TG-GATEs: A useful toxicogenomics tool for detecting modes of action in chemical risk assessment.

Mode of action information is one of the key components for chemical risk assessment as mechanistic insight leads to better understanding of potential adverse health effects of a chemical. This insight greatly facilitates assessment of human relevance and enhances the use of non-animal methods for risk assessment, as it ultimately enables extrapolation from initiating events to adverse effects. Recently, we reported an in vitro toxicogenomics comparison approach to categorize (non-)genotoxic carcinogens according to similarities in their proposed modes of action. The present study aimed to make this comparison approach generally applicable, allowing comparison of outcomes across different studies. The resulting further developed comparison approach was evaluated through application to toxicogenomics data on 18 liver toxicants in human and rat primary hepatocytes from the Open TG-GATEs database. The results showed sensible matches between compounds with (partial) overlap in mode of action, whilst matches for compounds with different modes of action were absent. Comparison of the results across species revealed pronounced and relevant differences between primary rat and human hepatocytes, underpinning that information on mode of action enhances assessment of human relevance. Thus, we demonstrate that the comparison approach now is generally applicable, facilitating its use as tool in mechanism-based risk assessment.

Mother-Specific Signature in the Maternal Transcriptome Composition of Mature, Unfertilized Zebrafish Eggs.

Maternal mRNA present in mature oocytes plays an important role in the proper development of the early embryo. As the composition of the maternal transcriptome in general has been studied with pooled mature eggs, potential differences between individual eggs are unknown. Here we present a transcriptome study on individual zebrafish eggs from clutches of five mothers in which we focus on the differences in maternal mRNA abundance per gene between and within clutches. To minimize technical interference, we used mature, unfertilized eggs from siblings. About half of the number of analyzed genes was found to be expressed as maternal RNA. The expressed and non-expressed genes showed that maternal mRNA accumulation is a non-random process, as it is related to specific biological pathways and processes relevant in early embryogenesis. Moreover, it turned out that overall the composition of the maternal transcriptome is tightly regulated as about half of the expressed genes display a less than twofold expression range between the observed minimum and maximum expression values of a gene in the experiment. Even more, the maximum gene-expression difference within clutches is for 88% of the expressed genes lower than twofold. This means that expression differences observed in maternally expressed genes are primarily caused by differences between mothers, with only limited variability between eggs from the same mother. This was underlined by the fact that 99% of the expressed genes were found to be differentially expressed between any of the mothers in an ANOVA test. Furthermore, linking chromosome location, transcription factor binding sites, and miRNA target sites of the genes in clusters of distinct and unique mother-specific gene-expression, suggest biological relevance of the mother-specific signatures in the maternal transcriptome composition. Altogether, the maternal transcriptome composition of mature zebrafish oocytes seems to be tightly regulated with a distinct mother-specific signature.

Confounding Factors in the Transcriptome Analysis of an In-Vivo Exposure Experiment.

CONFOUNDING FACTORS: In transcriptomics experimentation, confounding factors frequently exist alongside the intended experimental factors and can severely influence the outcome of a transcriptome analysis. Confounding factors are regularly discussed in methodological literature, but their actual, practical impact on the outcome and interpretation of transcriptomics experiments is, to our knowledge, not documented. For instance, in-vivo experimental factors; like Individual, Sample-Composition and Time-of-Day are potentially formidable confounding factors. To study these confounding factors, we designed an extensive in-vivo transcriptome experiment (n = 264) with UVR exposure of murine skin containing six consecutive samples from each individual mouse (n = 64). ANALYSIS APPROACH: Evaluation of the confounding factors: Sample-Composition, Time-of-Day, Handling-Stress, and Individual-Mouse resulted in the identification of many genes that were affected by them. These genes sometimes showed over 30-fold expression differences. The most prominent confounding factor was Sample-Composition caused by mouse-dependent skin composition differences, sampling variation and/or influx/efflux of mobile cells. Although we can only evaluate these effects for known cell type specifically expressed genes in our complex heterogeneous samples, it is clear that the observed variations also affect the cumulative expression levels of many other non-cell-type-specific genes. ANOVA: ANOVA analysis can only attempt to neutralize the effects of the well-defined confounding factors, such as Individual-Mouse, on the experimental factors UV-Dose and Recovery-Time. Also, by definition, ANOVA only yields reproducible gene-expression differences, but we found that these differences were very small compared to the fold changes induced by the confounding factors, questioning the biological relevance of these ANOVA-detected differences. Furthermore, it turned out that many of the differentially expressed genes found by ANOVA were also present in the gene clusters associated with the confounding factors. CONCLUSION: Hence our overall conclusion is that confounding factors have a major impact on the outcome of in-vivo transcriptomics experiments. Thus the set-up, analysis, and interpretation of such experiments should be approached with the utmost prudence.