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 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.

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.

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.

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.

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.

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.

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.

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.

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.

Valuable lessons-learned in transcriptomics experimentation.

We have collected several valuable lessons that will help improve transcriptomics experimentation. These lessons relate to experiment design, execution, and analysis. The cautions, but also the pointers, may help biologists avoid common pitfalls in transcriptomics experimentation and achieve better results with their transcriptome studies.