Validation of the Hem-Col capillary blood collection system for routine laboratory analyses.

At home collection of capillary blood using Hem-Col tubes (Labonovum) could offer a solution to patients with chronic conditions, who require frequent laboratory analyses. The collection tubes contain a conservation buffer to stabilize analytes for up to 5 days. In this validation study it was investigated whether analytes are measured accurately in Hem-Col tubes 5 days after collection. Forty-six healthy volunteers donated blood via venepuncture as well as capillary blood by finger prick using Hem-Col tubes. The analytes were measured within 2 h for the venepuncture and after 120 h for the Hem-Col method. The results of each analyte were analysed using Passing-Bablok regression analyses. The analytes that met the predefined acceptance criteria were total cholesterol, LDL-cholesterol, thyroid stimulating hormone (TSH) and glycated haemoglobin (HbA1c). HDL-cholesterol, C-reactive protein (CRP), ferritin, bilirubin total, creatinine, gGT and triglycerides met two out of three acceptance criteria. All other analytes did not meet the predefined criteria. The Hem-Col method is suitable for the measurement of total cholesterol, LDL-cholesterol, thyroid stimulating hormone (TSH) and glycated haemoglobin (HbA1c). However, due to this limited set of valid tests and practical limitations, routine application of this novel collection system in daily practice is limited.

Mitochondrial ATP Depletion Disrupts Caco-2 Monolayer Integrity and Internalizes Claudin 7.

Objective:In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Methods: Human Caco-2 cells were cultured for 10 days in culture flasks or for 14 days on transwell inserts in either glucose-containing or galactose-containing medium. Mitochondria were visualized and cellular respiration and levels of oxidative phosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletion was induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability was assessed using transepithelial electrical resistance (TEER) and fluorescein flux. Gene expression and cellular distribution of tight junction proteins were analyzed. Results: Caco-2 cells cultured in galactose-containing, but not in glucose-containing, medium showed increased mitochondrial connectivity, oxygen consumption rates and levels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP, rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability. In-depth studies with PA showed a six fold decrease in cellular ATP and revealed increased gene expression of tight junction proteins (TJP) 1 and 2, occludin, and claudin 1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearly redistributed from the cellular membrane into the cytoplasm, while the others were not (TJP1, occludin) or slightly (claudin 2, actin) affected. In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Conclusions: Well-functioning mitochondria are essential for maintaining cellular energy status and monolayer integrity of galactose grown Caco-2 cells. Energy depletion-induced Caco-2 monolayer permeability may be facilitated by changes in the distribution of claudin 7.

Effects of a wide range of dietary nicotinamide riboside (NR) concentrations on metabolic flexibility and white adipose tissue (WAT) of mice fed a mildly obesogenic diet.

SCOPE: Metabolic flexibility is the ability to switch metabolism between carbohydrate oxidation (CHO) and fatty acid oxidation (FAO) and is a biomarker for metabolic health. The effect on metabolic health of nicotinamide riboside (NR) as an exclusive source of vitamin B3 is unknown and is examined here for a wide range of NR. DESIGN AND METHODS: Nine-week-old male C57BL/6JRcc mice received a semi-purified mildly obesogenic (40 en% fat) diet containing 0.14% L-tryptophan and either 5, 15, 30, 180, or 900 mg NR per kg diet for 15 weeks. Body composition and metabolic parameters were analyzed. Metabolic flexibility was measured using indirect calorimetry. Gene expression in epididymal white adipose tissue (eWAT) was measured using qRT-PCR . RESULTS: The maximum delta respiratory exchange ratio when switching from CHO to FAO (maxΔRERCHO1→FAO ) and when switching from FAO to CHO (maxΔRERFAO→CHO2 ) were largest in 30 mg NR per kg diet (30NR). In eWAT, the gene expression of Pparγ, a master regulator of adipogenesis, and of Sod2 and Prdx3, two antioxidant genes, were significantly upregulated in 30NR compared to 5NR. CONCLUSION: 30NR is most beneficial for metabolic health, in terms of metabolic flexibility and eWAT gene expression, of mice on an obesogenic diet.

The effect of endurance exercise on intestinal integrity in well-trained healthy men.

Exercise is one of the external factors associated with impairment of intestinal integrity, possibly leading to increased permeability and altered absorption. Here, we aimed to examine to what extent endurance exercise in the glycogen-depleted state can affect intestinal permeability toward small molecules and protein-derived peptides in relation to markers of intestinal function. Eleven well-trained male volunteers (27 ± 4 years) ingested 40 g of casein protein and a lactulose/rhamnose (L/R) solution after an overnight fast in resting conditions (control) and after completing a dual - glycogen depletion and endurance - exercise protocol (first protocol execution). The entire procedure was repeated 1 week later (second protocol execution). Intestinal permeability was measured as L/R ratio in 5 h urine and 1 h plasma. Five-hour urine excretion of betacasomorphin-7 (BCM7), postprandial plasma amino acid levels, plasma fatty acid binding protein 2 (FABP-2), serum pre-haptoglobin 2 (preHP2), plasma glucagon-like peptide 2 (GLP2), serum calprotectin, and dipeptidylpeptidase-4 (DPP4) activity were studied as markers for excretion, intestinal functioning and recovery, inflammation, and BCM7 breakdown activity, respectively. BCM7 levels in urine were increased following the dual exercise protocol, in the first as well as the second protocol execution, whereas 1 h-plasma L/R ratio was increased only following the first exercise protocol execution. FABP2, preHP2, and GLP2 were not changed after exercise, whereas calprotectin increased. Plasma citrulline levels following casein ingestion (iAUC) did not increase after exercise, as opposed to resting conditions. Endurance exercise in the glycogen depleted state resulted in a clear increase of BCM7 accumulation in urine, independent of DPP4 activity and intestinal permeability. Therefore, strenuous exercise could have an effect on the amount of food-derived bioactive peptides crossing the epithelial barrier. The health consequence of increased passage needs more in depth studies.

Improved muscle function and quality after diet intervention with leucine-enriched whey and antioxidants in antioxidant deficient aged mice.

Antioxidant (AOX) deficiencies are commonly observed in older adults and oxidative stress has been suggested to contribute to sarcopenia. Here we investigate if 1) low levels of dietary antioxidants had a negative impact on parameters of muscle mass, function and quality, and 2) to study if nutritional interventions with AOX and/or leucine-enriched whey protein could improve these muscle parameters in aged mice. 18-months-old mice were fed a casein-based antioxidant-deficient (lowox) diet or a casein-based control-diet (CTRL) for 7 months. During the last 3 months, lowox-mice were subjected to either: a) continued lowox, b) supplementation with vitamin A/E, Selenium and Zinc (AOX), c) substitution of casein with leucine-enriched whey protein (PROT) or d) a combination of both AOX and PROT (TOTAL). After 7 months lowox-mice displayed lower muscle strength and more muscle fatigue compared to CTRL. Compared to lowox-mice, PROT-mice showed improved muscle power, grip strength and less muscle fatigue. AOX-mice showed improved oxidative status, less muscle fatigue, improved grip strength and mitochondrial dynamics compared to lowox-mice. The TOTAL-mice showed the combined effects of both interventions compared to lowox-mice. In conclusion, nutritional intervention with AOX and/or leucine-enriched whey protein can play a role in improving muscle health in a AOX-deficient mouse model.

Dynamic changes in energy metabolism upon embryonic stem cell differentiation support developmental toxicant identification.

Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in embryonic development, the modulation of energy metabolism in in vitro models, such as the EST, is not yet described. The present study is among the first studies that analyses whole genome expression data to specifically characterize metabolic changes upon ESC early differentiation. Our transcriptomic analyses showed activation of glycolysis, truncated activation of the tricarboxylic acid (TCA) cycle, activation of lipid synthesis, as well as activation of glutaminolysis during the early phase of ESC differentiation. Taken together, this energy metabolism profile points towards energy metabolism reprogramming in the provision of metabolites for biosynthesis of cellular constituents. Next, we defined a gene set that describes this energy metabolism profile. We showed that this gene set could be successfully applied in the EST to identify developmental toxicants known to modulate cellular biosynthesis (5-fluorouracil and methoxyacetic acid), while other developmental toxicants or the negative control did not modulate the expression of this gene set. Our description of dynamic changes in energy metabolism during early ESC differentiation, as well as specific identification of developmental toxicants modulating energy metabolism, is an important step forward in the definition of the applicability domain of the EST.

Reprogrammed metabolism of cancer cells as a potential therapeutic target.

Metabolism in cancer cells is reprogrammed. Cancer cells largely depend on glycolysis for ATP production. The metabolic alterations in cancer cells facilitate resistance to cell death as well as biosynthesis of nucleotides and lipids, building blocks for growth. The reprogrammed metabolism is increasingly seen as a target in cancer therapy. This review describes the metabolic reprogramming of cancer cells and illustrates how this is related to cell cycle and apoptosis resistance. Is also describes various scenarios for targeting cancer cell metabolism and highlights options for interventions with nutrition and bioactive food components.

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.

Complementary detection of embryotoxic properties of substances in the neural and cardiac embryonic stem cell tests.

In developmental toxicity testing, in vitro screening assays are highly needed to increase efficiency and to reduce animal use. A promising in vitro assay is the cardiac embryonic stem cell test (ESTc), in which the effect of developmental toxicants on cardiomyocyte differentiation is assessed. Recently, we developed a neural differentiation variant of the stem cell test (neural embryonic stem cell test [ESTn]). In both of these models, we have previously performed a series of transcriptomic studies to characterize gene expression changes (1) across time during normal differentiation and (2) in response to a series of developmental toxicants in the ESTn and ESTc. Here, using the cumulative of these studies, we compared gene expression profiles of ESTn and ESTc over time as well as model-specific changes induced by seven compounds, comprising six known in vivo developmental toxicants and one negative control. Time-related gene expression profiles showed similarities between the two EST systems. However, specific genes could be identified changing over time differently in each model related to the two different lineages of differentiation. Interestingly, compound-induced gene expression changes were generally model specific, especially for methylmercury and flusilazole, which were predicted better in ESTn and ESTc, respectively. Valproic acid-induced gene expression changes were most comparable out of the six developmental toxicants between the ESTn and ESTc. Direct transcriptomic comparisons between the ESTn and ESTc indicate that combined transcriptomic analyses support and complement each other. Therefore, a combined approach incorporating ESTc and ESTn may improve developmental toxicant detection over individual assays.

Dose response analysis of monophthalates in the murine embryonic stem cell test assessed by cardiomyocyte differentiation and gene expression.

The embryonic stem cell test (EST) is based on compound-induced inhibition of cardiomyocyte differentiation of pluripotent stem cells. We examined the use of transcriptomics to assess concentration-effect relationships and performed potency ranking within a chemical class. Three embryotoxic phthalate monoesters, monobutyl phthalate (MBuP), monobenzyl phthalate (MBzP) and mono-(2-ethylhexyl) phthalate (MEHP) and the non-embryotoxic monomethyl phthalate (MMP) were studied for their effects on gene expression. Effects on gene expression were observed at concentrations that did not inhibit cardiomyocyte differentiation or induce cytotoxicity. The embryotoxic phthalate monoesters altered the expression of 668 commonly expressed genes in a concentration-dependent fashion. The same potency ranking was observed for morphology and gene expression (MEHP>MBzP>MBuP>MMP). These results indicate that integrating transcriptomics provides a sensitive method to measure the dose-dependent effects of phthalate monoester exposure and enables potency ranking based on a common mode of action within a class of compounds. Transcriptomic approaches may improve the applicability of the EST, in terms of sensitivity and specificity.

Comparison of MeHg-induced toxicogenomic responses across in vivo and in vitro models used in developmental toxicology.

Toxicogenomic evaluations may improve toxicity prediction of in vitro-based developmental models, such as whole embryo culture (WEC) and embryonic stem cells (ESC), by providing a robust mechanistic marker which can be linked with responses associated with developmental toxicity in vivo. While promising in theory, toxicogenomic comparisons between in vivo and in vitro models are complex due to inherent differences in model characteristics and experimental design. Determining factors which influence these global comparisons are critical in the identification of reliable mechanistic-based markers of developmental toxicity. In this study, we compared available toxicogenomic data assessing the impact of the known teratogen, methylmercury (MeHg) across a diverse set of in vitro and in vivo models to investigate the impact of experimental variables (i.e. model, dose, time) on our comparative assessments. We evaluated common and unique aspects at both the functional (Gene Ontology) and gene level of MeHg-induced response. At the functional level, we observed stronger similarity in MeHg-response between mouse embryos exposed in utero (2 studies), ESC, and WEC as compared to liver, brain and mouse embryonic fibroblast MeHg studies. These findings were strongly correlated to the presence of a MeHg-induced developmentally related gene signature. In addition, we identified specific MeHg-induced gene expression alterations associated with developmental signaling and heart development across WEC, ESC and in vivo systems. However, the significance of overlap between studies was highly dependent on traditional experimental variables (i.e. dose, time). In summary, we identify promising examples of unique gene expression responses which show in vitro-in vivo similarities supporting the relevance of in vitro developmental models for predicting in vivo developmental toxicity.

The embryonic stem cell test combined with toxicogenomics as an alternative testing model for the assessment of developmental toxicity.

One of the most studied in vitro alternative testing methods for identification of developmental toxicity is the embryonic stem cell test (EST). Although the EST has been formally validated, the applicability domain as well as the predictability of the model needs further study to allow successful implementation of the EST as an alternative testing method in regulatory toxicity testing. Genomics technologies have already provided a proof of principle of their value in identification of toxicants such as carcinogenic compounds. Also within the EST, gene expression profiling has shown its value in the identification of developmental toxicity and in the evaluation of factors critical for risk assessment, such as dose and time responses. It is expected that the implementation of genomics into the EST will provide a more detailed end point evaluation as compared to the classical morphological scoring of differentiation cultures. Therefore, genomics may contribute to improvement of the EST, both in terms of definition of its applicability domain as well as its predictive capacity. In the present review, we present the progress that has been made with regard to the prediction of developmental toxicity using the EST combined with transcriptomics. Furthermore, we discuss the developments of additional aspects required for further optimization of the EST, including kinetics, the use of human embryonic stem cells (ESC) and computational toxicology. Finally, the current and future use of the EST model for prediction of developmental toxicity in testing strategies and in regulatory toxicity evaluations is discussed.

Gene set assembly for quantitative prediction of developmental toxicity in the embryonic stem cell test.

The embryonic stem cell test (EST) is an in vitro method for predicting developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation. We previously described how gene expression analysis in the EST can be used to describe normal ESC differentiation as well as identify compound developmental toxicity, by means of our differentiation track algorithm. In this study, we combined raw data from our three previous studies in a new integrated analysis, to identify a gene set that allows for improved prediction. By evaluating predictions of 100,000 randomly selected gene sets, we identified which genes contribute significantly to the prediction reliability. By additional cross-validation, we identified a set of 52 genes that allows for improved prediction of toxicity. The correlation between the predictions using this gene set and the magnitude of the EST endpoint was 0.85, and the gene set predicted developmental toxicity with 83% accuracy (area under the curve 89%). If compounds with ineffective data because of a too low tested concentration or too much variation between samples were excluded, even 100% accuracy could be reached based on 15 compounds. This novel gene set consists mainly of genes involved in the stem cell differentiation or other developmental processes. We expect that this set can be of use in future studies aimed at improving the EST for risk assessment, thus making a next step towards regulatory implementation of this method.

Discriminating classes of developmental toxicants using gene expression profiling in the embryonic stem cell test.

The embryonic stem cell test (EST) has been shown to be a promising in vitro method for the prediction of developmental toxicity. In our previous studies, we demonstrated that the implementation of gene expression analysis in the EST may further improve the identification of developmental toxicants. In the present study, we investigated if gene expression profiling could be used to discriminate compound classes with distinct modes of action (MoA) using the EST protocol. Gene expression data of our previous study were used and were analyzed of embryonic stem cell (ESC) differentiation cultures exposed to six compounds belonging to two classes with distinct MoA, namely phthalates and triazoles. We used three approaches to study class-characteristic gene regulation that may be useful for discrimination of compound classes. First, at the individual gene level, gene signatures characteristic for each class were identified that successfully discriminated both classes using principal component analysis. Second, at the functional level, enriched gene ontology (GO) biological processes showed their usefulness for class discrimination via hierarchical clustering. Third, two previously identified gene sets, which we designed to predict developmental toxicity, appeared successful in separating phthalate from triazole compounds. In summary, we established the possibility to discriminate between compound classes in the EST system using three different specific transcriptomics-based approaches. Differential gene expression information specific for the class of compound under study may be employed to optimize prioritization of compounds within that class for further testing.

Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures.

The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 µM) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.

Evaluation of developmental toxicant identification using gene expression profiling in embryonic stem cell differentiation cultures.

The murine embryonic stem cell test (EST) is an alternative testing method designed to assess potential developmental toxicity of compounds. The implementation of transcriptomics in the EST has been shown to reduce the culture duration and improve endpoint evaluation and is expected to result in an enhanced predictability and definition of the applicability domain. We evaluated the identification of developmental toxicity in the EST using two gene sets ("Van_Dartel_heartdiff_24h" and "EST biomarker genes") defined in our earlier studies. Nonexposed embryonic stem cells (ESC) differentiation cultures were sampled 0, 24, and 48 h after initiation of differentiation. Additionally, cultures exposed to 12 diverse well-characterized positive and negative developmental toxicants were isolated 24 h after the onset of exposure. Inhibition of ESC differentiation was evaluated in parallel by morphological scoring on culture day 10. Transcriptomics analysis was conducted using the Affymetrix Gene Chips platform. We applied principal component analysis on the basis of the two predefined gene sets to define the "differentiation track" that represents ESC differentiation. The significance of derivations in the gene expression-based differentiation track because of compound exposures were evaluated to determine developmental toxicity of tested compounds. We successfully predicted developmental toxicity using transcriptomics for 83% (10/12) and 67% (8/12) of the compounds, respectively, using the two predefined gene sets ("Van_Dartel_heartdiff_24h" and "EST biomarker genes"). Our study suggests that the application of transcriptomics may improve the applicability of the EST for the prediction of the developmental toxicity of chemicals.

Identification by gene coregulation mapping of novel genes involved in embryonic stem cell differentiation.

A combined analysis of data from a series of literature studies can lead to more reliable results than that based on a single study. A common problem in performing combined analyses of literature microarray gene expression data is that the original raw data are not always available and not always easy to combine in one analysis. We propose an approach that does not require analyzing original raw data, but instead takes literature gene sets derived from (supplementary) tables as input and uses gene co-occurrence in these sets for mapping a co-regulation network. An algorithm for this method was applied to a collection of literature-derived gene sets related to embryonic stem cell (ESC) differentiation. In the resulting network, genes involved in similar biological processes or expressed at similar time points during differentiation were found to cluster together. Using this information, we identified 43 genes not previously associated with cardiac ESC differentiation for which we were able to assign a putative novel biological function. For 6 of these genes (Apobec2, Cth, Ptges, Rrad, Zfp57, and 2410146L05Rik), literature data on mouse knockout phenotypes support their putative function. Three other genes (Rcor2, Zfp503, and Hspb3) are part of major pathways within the network and therefore likely mechanistically relevant candidate genes. We anticipate that these 43 genes can help to improve the understanding of the molecular events underlying ESC differentiation. Moreover, the approach introduced here can be more widely applied to identify possible novel gene functions in biological processes.

Proteome profiling of mouse embryonic stem cells to define markers for cell differentiation and embryotoxicity.

In search for alternative methods for developmental toxicity testing, we investigated whether embryonic stem cell (ESC) differentiation and its modulation by toxic exposure could be monitored by proteome profiling. We compared the proteomes of mouse ESC, differentiating ESC (DIF) and differentiating ESC exposed to the embryotoxicant monobutyl phthalate (MBP). Experiments were performed in duplicates for each cell culture and the proteins extracted from the cells were separated by one-dimensional SDS-PAGE. The identified proteins were quantified using a label-free quantitative method based on counting the observed peptides as an index of protein abundance. Fifty-seven proteins were upregulated in DIF relative to ESC, whereas 42 proteins were downregulated. Most of the upregulated proteins could be correlated with cardiomyocyte functionality. In contrast, the downregulated proteins were principally pluripotency markers, chaperones and ribosomal proteins. Higher expression levels of enzymes involved in DNA mismatch repair (MSH6) and in methylation reactions (MAT2A, AHCY) were also detected in the ESC, suggesting that these processes are more active in the ESC. In addition, the detection of gluthatione S-transferase alpha 4 (GSTA4) and Park7 DJ-1 protein (antioxidant) in ESC indicates that these cells have potential detoxification mechanisms. Furthermore, MBP affected the expression of 33 proteins, including MYH6, a cardiomyocyte-specific protein, which was significantly downregulated. MBP also affected the expression levels of chaperones, metabolic enzymes and chromatin modulating proteins, suggesting that MBP alters the differentiation process. Western blot analysis of MYH6 and HSPB1 confirmed the proteomic data. In addition, a favorable correlation was observed between protein expression and published changes in the expression of related genes at the transcriptomics level. Together, the results reveal potential protein markers that may be used as endpoints in an ESC based animal free alternative test for embryotoxicity though further studies are required for confirmation.

Monitoring developmental toxicity in the embryonic stem cell test using differential gene expression of differentiation-related genes.

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

Transcriptomics-based identification of developmental toxicants through their interference with cardiomyocyte differentiation of embryonic stem cells.

The embryonic stem cell test (EST) predicts developmental toxicity based on the inhibition of cardiomyocyte differentiation of embryonic stem cells (ESC). The subjective endpoint, the long culture duration together with the undefined applicability domain and related predictivity need further improvement to facilitate implementation of the EST into regulatory strategies. These aspects may be improved by studying gene expression changes in the ESC differentiation cultures and their modulation by compound exposure using transcriptomics. Here, we tested the developmental toxicants monobutyl phthalate and 6-aminonicotinamide. ESC were allowed to differentiated, and cardiomyocyte differentiation was assessed after 10 days of culture. RNA of solvent controls was collected after 0, 24, 48, 72 and 96 h of exposure, and RNA of developmental-toxicant-exposed cultures was collected after 24 and 96 h. Samples were hybridized to DNA microarrays, and 1355 genes were found differentially expressed among the unexposed experimental groups. These regulated genes were involved in differentiation-related processes, and Principal Component Analysis (PCA) based on these genes showed that the unexposed experimental groups appeared in chronological order in the PCA, which can therefore be regarded as a continuous representation of the differentiation track. The developmental-toxicant-exposed cultures appeared to deviate significantly from this differentiation track, which confirms the compound-modulating effects on the differentiation process. The incorporation of transcriptomics in the EST is expected to provide a more informative and improved endpoint in the EST as compared with morphology, allowing early detection of differentiation modulation. Furthermore, this approach may improve the definition of the applicability domain and predictivity of the EST.