White-Nose Syndrome Disrupts the Splenic Lipidome of Little Brown Bats (Myotis lucifugus) at Early Disease Stages.

White-nose syndrome (WNS)-positive little brown bats (Myotis lucifugus) may exhibit immune responses including increased cytokine and pro-inflammatory mediator gene levels. Bioactive lipid mediators (oxylipins) formed by enzymatic oxidation of polyunsaturated fatty acids can contribute to these immune responses, but have not been investigated in WNS pathophysiology. Nonenzymatic conversion of polyunsaturated fatty acids can also occur due to reactive oxygen species, however, these enantiomeric isomers will lack the same signaling properties. In this study, we performed a series of targeted lipidomic approaches on laboratory Pseudogymnoascus destructans-inoculated bats to assess changes in their splenic lipidome, including the formation of lipid mediators at early stages of WNS. Hepatic lipids previously identified were also resolved to a higher structural detail. We compared WNS-susceptible M. lucifugus to a WNS-resistant species, the big brown bat (Eptesicus fuscus). Altered splenic lipid levels were only observed in M. lucifugus. Differences in splenic free fatty acids included both omega-3 and omega-6 compounds. Increased levels of an enantiomeric monohydroxy DHA mixture were found, suggesting nonenzymatic formation. Changes in previously identified hepatic lipids were confined to omega-3 constituents. Together, these results suggest that increased oxidative stress, but not an inflammatory response, is occurring in bats at early stages of WNS that precedes fat depletion. These data have been submitted to metabolomics workbench and assigned a study number ST002304.

Low dose radiation upregulates Ras/p38 and NADPH oxidase in mouse colon two months after exposure.

BACKGROUND: Exposure to ionizing is known to cause persistent cellular oxidative stress and NADPH oxidase (Nox) is a major source of cellular oxidant production. Chronic oxidative stress is associated with a myriad of human diseases including gastrointestinal cancer. However, the roles of NADPH oxidase in relation of long-term oxidative stress in colonic epithelial cells after radiation exposure are yet to be clearly established. METHODS AND RESULTS: Mice were exposed either to sham or to 0.5 Gy γ radiation, and NADPH oxidase, oxidative stress, and related signaling pathways were assessed in colon samples 60 days after exposure. Radiation exposure led to increased expression of colon-specific NADPH oxidase isoform, Nox1, as well as upregulation of its modifiers such as Noxa1 and Noxo1 at the mRNA and protein level. Co-immunoprecipitation experiments showed enhanced binding of Rac1, an activator of NADPH oxidase, to Nox1. Increased 4-hydroxynonenal, 8-oxo-dG, and γH2AX along with higher protein carbonylation levels suggest increased oxidative stress after radiation exposure. Immunoblot analysis demonstrates upregulation of Ras/p38 pathway, and Gata6 and Hif1α after irradiation. Increased staining of ß-catenin, cyclinD1, and Ki67 after radiation was also observed. CONCLUSIONS: In summary, data show that exposure to a low dose of radiation was associated with upregulation of NADPH oxidase and its modifiers along with increased Ras/p38/Gata6 signaling in colon. When considered along with oxidative damage and proliferative markers, our observations suggest that the NADPH oxidase pathway could be playing a critical role in propagating long-term oxidative stress after radiation with implications for colon carcinogenesis.

GADD45 in Stress Signaling, Cell Cycle Control, and Apoptosis.

GADD45 is a gene family consisting of GADD45A, GADD45B, and GADD45G that is often induced by DNA damage and other stress signals associated with growth arrest and apoptosis. Many of these roles are carried out via signaling mediated by p38 mitogen-activated protein kinases (MAPKs). The GADD45 proteins can contribute to p38 activation either by activation of upstream kinase(s) or by direct interaction, as well as suppression of p38 activity in certain cases. In vivo, there are important tissue and cell type specific differences in the roles for GADD45 in MAPK signaling. In addition to being p53-regulated, GADD45A has also been found to contribute to p53 activation via p38. Like other stress and signaling proteins, GADD45 proteins show complex regulation and numerous effectors. More recently, aberrant GADD45 expression has been found in several human cancers, but the mechanisms behind these findings largely remain to be understood.

Predominant contribution of the dose received from constituent heavy-ions in the induction of gastrointestinal tumorigenesis after simulated space radiation exposure.

Gastrointestinal (GI) cancer risk among astronauts after encountering galactic cosmic radiation (GCR) is predicted to exceed safe permissible limits in long duration deep-space missions. Current predictions are based on relative biological effectiveness (RBE) values derived from in-vivo studies using single-ion beams, while GCR is essentially a mixed radiation field composed of protons (H), helium (He), and heavy ions. Therefore, a sequentially delivered proton (H) → Helium (He) → Oxygen (O) → Silicon (Si) beam was designed to simulate simplified-mixed-field GCR (Smf-GCR), and Apc1638N/+ mice were total-body irradiated to sham or γ (157Cs) or Smf-GCR followed by assessment of GI-tumorigenesis at 150 days post-exposure. Further, GI-tumor data from equivalent doses of heavy-ions (i.e., 0.05 Gy of O and Si) in 0.5 Gy of Smf-GCR were compared to understand the contributions of heavy-ions in GI-tumorigenesis. The Smf-GCR-induced tumor and carcinoma count were significantly greater than γ-rays, and male preponderance for GI-tumorigenesis was consistent with our earlier findings. Comparison of tumor data from Smf-GCR and equivalent doses of heavy ions revealed an association between higher GI-tumorigenesis where dose received from heavy-ions contributed to > 95% of the total GI-tumorigenic effect observed after Smf-GCR. This study provides the first experimental evidence that cancer risk after GCR exposure could largely depend on doses received from constituent heavy-ions.

Biofluid Metabolomics of Mice Exposed to External Low-Dose Rate Radiation in a Novel Irradiation System, the Variable Dose-Rate External 137Cs Irradiator.

An important component of ionizing radiation (IR) exposure after a radiological incident may include low-dose rate (LDR) exposures either externally or internally, such as from 137Cs deposition. In this study, a novel irradiation system, VAriable Dose-rate External 137Cs irradiatoR (VADER), was used to expose male and female mice to a variable LDR irradiation over a 30 d time span to simulate fall-out-type exposures in addition to biofluid collection from a reference dose rate (0.8 Gy/min). Radiation markers were identified by untargeted metabolomics and random forests. Mice exposed to LDR exposures were successfully identified from control groups based on their urine and serum metabolite profiles. In addition to metabolites commonly perturbed after IR exposure, we identified and validated a novel metabolite (hexosamine-valine-isoleucine-OH) that increased up to 150-fold after LDR and 80-fold after conventional exposures in urine. A multiplex panel consisting of hexosamine-valine-isoleucine-OH with other urinary metabolites (N6,N6,N6-trimethyllysine, carnitine, 1-methylnicotinamide, and α-ketoglutaric acid) achieved robust classification performance using receiver operating characteristic curve analysis, irrespective of the dose rate or sex. These results show that in terms of biodosimetry, dysregulated energy metabolism is associated with IR exposure for both LDR and conventional IR exposures. These mass spectrometry data have been deposited to the NIH data repository via Metabolomics Workbench with study IDs ST001790, ST001791, ST001792, ST001793, and ST001806.

Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells.

Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant's mode of action. To address the limited approaches available for identifying epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration-response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4 h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (RGL1, NEU1, GPR183) or cellular stress-response genes (ATF3, CDKN1A, GADD45A) analyzed by TaqMan qPCR assays after 4 h of exposure. Whole transcriptomes were profiled after 4 h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.

Space radiation triggers persistent stress response, increases senescent signaling, and decreases cell migration in mouse intestine.

Proliferative gastrointestinal (GI) tissue is radiation-sensitive, and heavy-ion space radiation with its high-linear energy transfer (high-LET) and higher damaging potential than low-LET γ-rays is predicted to compromise astronauts' GI function. However, much uncertainty remains in our understanding of how heavy ions affect coordinated epithelial cell migration and extrusion, which are essential for GI homeostasis. Here we show using mouse small intestine as a model and BrdU pulse labeling that cell migration along the crypt-villus axis is persistently decreased after a low dose of heavy-ion 56Fe radiation relative to control and γ-rays. Wnt/ß-catenin and its downstream EphrinB/EphB signaling are key to intestinal epithelial cell (IEC) proliferation and positioning during migration, and both are up-regulated after 56Fe radiation. Conversely, factors involved in cell polarity and adhesion and cell-extracellular matrix interactions were persistently down-regulated after 56Fe irradiation-potentially altering cytoskeletal remodeling and cell extrusion. 56Fe radiation triggered a time-dependent increase in γH2AX foci and senescent cells but without a noticeable increase in apoptosis. Some senescent cells acquired the senescence-associated secretory phenotype, and this was accompanied by increased IEC proliferation, implying a role for progrowth inflammatory factors. Collectively, this study demonstrates a unique phenomenon of heavy-ion radiation-induced persistently delayed IEC migration involving chronic sublethal genotoxic and oncogenic stress-induced altered cytoskeletal dynamics, which were seen even a year later. When considered along with changes in barrier function and nutrient absorption factors as well as increased intestinal tumorigenesis, our in vivo data raise a serious concern for long-duration deep-space manned missions.

Effects of Low Dose Space Radiation Exposures on the Splenic Metabolome.

Future space missions will include a return to the Moon and long duration deep space roundtrip missions to Mars. Leaving the protection that Low Earth Orbit provides will unavoidably expose astronauts to higher cumulative doses of space radiation, in addition to other stressors, e.g., microgravity. Immune regulation is known to be impacted by both radiation and spaceflight and it remains to be seen whether prolonged effects that will be encountered in deep space can have an adverse impact on health. In this study, we investigated the effects in the overall metabolism of three different low dose radiation exposures (γ-rays, 16O, and 56Fe) in spleens from male C57BL/6 mice at 1, 2, and 4 months after exposure. Forty metabolites were identified with significant enrichment in purine metabolism, tricarboxylic acid cycle, fatty acids, acylcarnitines, and amino acids. Early perturbations were more prominent in the γ irradiated samples, while later responses shifted towards more prominent responses in groups with high energy particle irradiations. Regression analysis showed a positive correlation of the abundance of identified fatty acids with time and a negative association with γ-rays, while the degradation pathway of purines was positively associated with time. Taken together, there is a strong suggestion of mitochondrial implication and the possibility of long-term effects on DNA repair and nucleotide pools following radiation exposure.

Arsenite and cadmium promote the development of mammary tumors.

Previous studies demonstrate that the heavy metal cadmium and the metalloid arsenite activate estrogen receptor-alpha in breast cancer cells by forming a high-affinity complex with the ligand-binding domain of the receptor and that environmentally relevant doses of cadmium have estrogen-like activity in vivo. The present study showed that in estrogen-receptor positive cells, arsenite and cadmium increased the global expression of estrogen-responsive genes and that an environmentally relevant dose of arsenite also had estrogen-like activity in vivo. Similar to estrogens, exposure of ovariectomized animals to arsenite induced the expression of the progesterone receptor, GREB1, and c-fos in the mammary gland and the expression of complement C3, c-fos, and cyclin D1 in the uterus and the increase was blocked by the antiestrogen ICI-182,780. When virgin female animals were fed a diet, that mimics exposure to either arsenite or cadmium, and challenged with the chemical carcinogen dimethylbenzanthracene, there was an increase in the incidence of mammary tumors and a decrease in the time to tumor onset, but no difference in the total number of tumors, tumor multiplicity, or total tumor volume. Together with published results, these data showed that environmentally relevant amounts of arsenite and cadmium had estrogen-like activity in vivo and promoted mammary tumorigenesis.

Development and validation of a high-throughput transcriptomic biomarker to address 21st century genetic toxicology needs.

Interpretation of positive genotoxicity findings using the current in vitro testing battery is a major challenge to industry and regulatory agencies. These tests, especially mammalian cell assays, have high sensitivity but suffer from low specificity, leading to high rates of irrelevant positive findings (i.e., positive results in vitro that are not relevant to human cancer hazard). We developed an in vitro transcriptomic biomarker-based approach that provides biological relevance to positive genotoxicity assay data, particularly for in vitro chromosome damage assays, and propose its application for assessing the relevance of the in vitro positive results to carcinogenic hazard. The transcriptomic biomarker TGx-DDI (previously known as TGx-28.65) readily distinguishes DNA damage-inducing (DDI) agents from non-DDI agents. In this study, we demonstrated the ability of the biomarker to classify 45 test agents across a broad set of chemical classes as DDI or non-DDI. Furthermore, we assessed the biomarker's utility in derisking known irrelevant positive agents and evaluated its performance across analytical platforms. We correctly classified 90% (9 of 10) of chemicals with irrelevant positive findings in in vitro chromosome damage assays as negative. We developed a standardized experimental and analytical protocol for our transcriptomics biomarker, as well as an enhanced application of TGx-DDI for high-throughput cell-based genotoxicity testing using nCounter technology. This biomarker can be integrated in genetic hazard assessment as a follow-up to positive chromosome damage findings. In addition, we propose how it might be used in chemical screening and assessment. This approach offers an opportunity to significantly improve risk assessment and reduce cost.

Liquid Chromatography-Mass Spectrometry-Based Metabolomics of Nonhuman Primates after 4 Gy Total Body Radiation Exposure: Global Effects and Targeted Panels.

Rapid assessment of radiation signatures in noninvasive biofluids may aid in assigning proper medical treatments for acute radiation syndrome (ARS) and delegating limited resources after a nuclear disaster. Metabolomic platforms allow for rapid screening of biofluid signatures and show promise in differentiating radiation quality and time postexposure. Here, we use global metabolomics to differentiate temporal effects (1-60 d) found in nonhuman primate (NHP) urine and serum small molecule signatures after a 4 Gy total body irradiation. Random Forests analysis differentially classifies biofluid signatures according to days post 4 Gy exposure. Eight compounds involved in protein metabolism, fatty acid ß oxidation, DNA base deamination, and general energy metabolism were identified in each urine and serum sample and validated through tandem MS. The greatest perturbations were seen at 1 d in urine and 1-21 d in serum. Furthermore, we developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method to quantify a six compound panel (hypoxanthine, carnitine, acetylcarnitine, proline, taurine, and citrulline) identified in a previous training cohort at 7 d after a 4 Gy exposure. The highest sensitivity and specificity for classifying exposure at 7 d after a 4 Gy exposure included carnitine and acetylcarnitine in urine and taurine, carnitine, and hypoxanthine in serum. Receiver operator characteristic (ROC) curve analysis using combined compounds show excellent sensitivity and specificity in urine (area under the curve [AUC] = 0.99) and serum (AUC = 0.95). These results highlight the utility of MS platforms to differentiate time postexposure and acquire reliable quantitative biomarker panels for classifying exposed individuals.

Fabric Phase Sorptive Extraction-A Metabolomic Preprocessing Approach for Ionizing Radiation Exposure Assessment.

The modern application of mass spectrometry-based metabolomics to the field of radiation assessment and biodosimetry has allowed for the development of prompt biomarker screenings for radiation exposure. Our previous work on radiation assessment, in easily accessible biofluids (such as urine, blood, saliva), has revealed unique metabolic perturbations in response to radiation quality, dose, and dose rate. Nevertheless, the employment of swift injury assessment in the case of a radiological disaster still remains a challenge as current sample processing can be time consuming and cause sample degradation. To address these concerns, we report a metabolomics workflow using a mass spectrometry-compatible fabric phase sorptive extraction (FPSE) technique. FPSE employs a matrix coated with sol-gel poly(caprolactone-b-dimethylsiloxane-b-caprolactone) that binds both polar and nonpolar metabolites in whole blood, eliminating serum processing steps. We confirm that the FPSE preparation technique combined with liquid chromatography-mass spectrometry can distinguish radiation exposure markers such as taurine, carnitine, arachidonic acid, α-linolenic acid, and oleic acid found 24 h after 8 Gy irradiation. We also note the effect of different membrane fibers on both metabolite extraction efficiency and the temporal stabilization of metabolites in whole blood at room temperature. These findings suggest that the FPSE approach could work in future technology to triage irradiated individuals accurately, via biomarker screening, by providing a novel method to stabilize biofluids between collection and sample analysis.

Impact of inflammatory signaling on radiation biodosimetry: mouse model of inflammatory bowel disease.

BACKGROUND: Ionizing Radiation (IR) is a known pro-inflammatory agent and in the process of development of biomarkers for radiation biodosimetry, a chronic inflammatory disease condition could act as a confounding factor. Hence, it is important to develop radiation signatures that can distinguish between IR-induced inflammatory responses and pre-existing disease. In this study, we compared the gene expression response of a genetically modified mouse model of inflammatory bowel disease (Il10-/-) with that of a normal wild-type mouse to potentially develop transcriptomics-based biodosimetry markers that can predict radiation exposure in individuals regardless of pre-existing inflammatory condition. RESULTS: Wild-type (WT) and Il10-/- mice were exposed to whole body irradiation of 7 Gy X-rays. Gene expression responses were studied using high throughput whole genome microarrays in peripheral blood 24 h post-irradiation. Analysis resulted in identification of 1962 and 1844 genes differentially expressed (p < 0.001, FDR < 10%) after radiation exposure in Il10-/- and WT mice respectively. A set of 155 genes was also identified as differentially expressed between WT and Il10-/- mice at the baseline pre-irradiation level. Gene ontology analysis revealed that the 155 baseline differentially expressed genes were mainly involved in inflammatory response, glutathione metabolism and collagen deposition. Analysis of radiation responsive genes revealed that innate immune response and p53 signaling processes were strongly associated with up-regulated genes, whereas B-cell development process was found to be significant amongst downregulated genes in the two genotypes. However, specific immune response pathways like MHC based antigen presentation, interferon signaling and hepatic fibrosis were associated with radiation responsive genes in Il10-/- mice but not WT mice. Further analysis using the IPA prediction tool revealed significant differences in the predicted activation status of T-cell mediated signaling as well as regulators of inflammation between WT and Il10-/- after irradiation. CONCLUSIONS: Using a mouse model we established that an inflammatory disease condition could affect the expression of many radiation responsive genes. Nevertheless, we identified a panel of genes that, regardless of disease condition, could predict radiation exposure. Our results highlight the need for consideration of pre-existing conditions in the population in the process of development of reliable biodosimetry markers.

Gas Chromatography/Mass Spectrometry Metabolomics of Urine and Serum from Nonhuman Primates Exposed to Ionizing Radiation: Impacts on the Tricarboxylic Acid Cycle and Protein Metabolism.

Ionizing radiation (IR) directly damages cells and tissues or indirectly damages them through reactive free radicals that may lead to longer term adverse sequelae such as cancers, persistent inflammation, or possible death. Potential exposures include nuclear reactor accidents, improper disposal of equipment containing radioactive materials or medical errors, and terrorist attacks. Metabolomics (comprehensive analysis of compounds <1 kDa) by mass spectrometry (MS) has been proposed as a tool for high-throughput biodosimetry and rapid assessment of exposed dose and triage needed. While multiple studies have been dedicated to radiation biomarker discovery, many have utilized liquid chromatography (LC) MS platforms that may not detect particular compounds (e.g., small carboxylic acids or isomers) that complementary analytical tools, such as gas chromatography (GC) time-of-flight (TOF) MS, are ideal for. The current study uses global GC-TOF-MS metabolomics to complement previous LC-MS analyses on nonhuman primate biofluids (urine and serum) 7 days after exposure to 2, 4, 6, 7, and 10 Gy IR. Multivariate data analysis was used to visualize differences between control and IR exposed groups. Univariate analysis was used to determine a combined 26 biomarkers in urine and serum that significantly changed after exposure to IR. We found several metabolites involved in tricarboxylic acid cycle function, amino acid metabolism, and host microbiota that were not previously detected by global and targeted LC-MS studies.

A Serum Small Molecule Biosignature of Radiation Exposure from Total Body Irradiated Patients.

The potential for radiological accidents and nuclear terrorism has increased the need for the development of new rapid biodosimetry methods. In addition, in a clinical setting the issue of an individual's radiosensitivity should be taken into consideration during radiotherapy. We utilized metabolomics and lipidomics to investigate changes of metabolites in serum samples following exposure to total body ionizing radiation in humans. Serum was collected prior to irradiation, at 3-8 h after a single dose of 1.25-2 Gy, and at 24 h with a total delivered dose of 2-3.75 Gy. Metabolomics revealed perturbations in glycerophosphocholine, phenylalanine, ubiquinone Q2, and oxalic acid. Alterations were observed in circulating levels of lipids from monoacylglycerol, triacylglycerol, phosphatidylcholine, and phosphatidylglycerol lipid classes. Polyunsaturated fatty acids were some of the most dysregulated lipids, with increased levels linked to proinflammatory processes. A targeted metabolomics approach for eicosanoids was also employed. The results showed a rapid response for proinflammatory eicosanoids, with a dampening of the signal at the later time point. Sex differences were observed in the markers from the untargeted approach but not the targeted method. The ability to identify and quantify small molecules in blood can therefore be utilized to monitor radiation exposure in human populations.

Regulation of Cytochrome P450 2B10 (CYP2B10) Expression in Liver by Peroxisome Proliferator-activated Receptor-ß/δ Modulation of SP1 Promoter Occupancy.

Alcoholic liver disease is a pathological condition caused by overconsumption of alcohol. Because of the high morbidity and mortality associated with this disease, there remains a need to elucidate the molecular mechanisms underlying its etiology and to develop new treatments. Because peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) modulates ethanol-induced hepatic effects, the present study examined alterations in gene expression that may contribute to this disease. Chronic ethanol treatment causes increased hepatic CYP2B10 expression inPparß/δ+/+ mice but not in Pparß/δ-/- mice. Nuclear and cytosolic localization of the constitutive androstane receptor (CAR), a transcription factor known to regulate Cyp2b10 expression, was not different between genotypes. PPARγ co-activator 1α, a co-activator of both CAR and PPARß/δ, was up-regulated in Pparß/δ+/+ liver following ethanol exposure, but not in Pparß/δ-/- liver. Functional mapping of the Cyp2b10 promoter and ChIP assays revealed that PPARß/δ-dependent modulation of SP1 promoter occupancy up-regulated Cyp2b10 expression in response to ethanol. These results suggest that PPARß/δ regulates Cyp2b10 expression indirectly by modulating SP1 and PPARγ co-activator 1α expression and/or activity independent of CAR activity. Ligand activation of PPARß/δ attenuates ethanol-induced Cyp2b10 expression in Pparß/δ+/+ liver but not in Pparß/δ-/- liver. Strikingly, Cyp2b10 suppression by ligand activation of PPARß/δ following ethanol treatment occurred in hepatocytes and was mediated by paracrine signaling from Kupffer cells. Combined, results from the present study demonstrate a novel regulatory role of PPARß/δ in modulating CYP2B10 that may contribute to the etiology of alcoholic liver disease.

Wip1 directly dephosphorylates NLK and increases Wnt activity during germ cell development.

Mice null for wild-type p53-induced phosphatase 1 (WIP1) display defects in testis development and spermatogenesis, resulting in reduced fertility. However, the molecular mechanism underlying these abnormalities in the testis remains uncharacterized. We report that the phosphatase activity of WIP1 increases Wnt activity through Nemo-like kinase (NLK). WIP1 directly interacted with NLK, which is highly homologous to p38 MAPK, a WIP1 substrate, and dephosphorylated its activation site. The WIP1-mediated inhibition of NLK activity markedly decreased the phosphorylation of lymphoid enhancer-binding factor 1 (LEF1), enhancing its interaction with ß-catenin. Additionally, WIP1 depletion impaired germ cell development, as evidenced by the expression of Oct4 and the germ cell-specific markers Ddx4, Nanos3 and Dnd1 during the development of germ cells from Oct4-GFP transgenic (OG2) mouse embryonic stem cells (mESCs). The expression of WIP1, whose level was significantly lower after the differentiation of germ cells from mESCs, occurred in parallel with the expression of germ cell development markers and SRY-box 17 (Sox17), a downstream target of Wnt. These results indicate that WIP1 is essential for germ cell development, which is known to require Wnt activity.

Information-dependent enrichment analysis reveals time-dependent transcriptional regulation of the estrogen pathway of toxicity.

The twenty-first century vision for toxicology involves a transition away from high-dose animal studies to in vitro and computational models (NRC in Toxicity testing in the 21st century: a vision and a strategy, The National Academies Press, Washington, DC, 2007). This transition requires mapping pathways of toxicity by understanding how in vitro systems respond to chemical perturbation. Uncovering transcription factors/signaling networks responsible for gene expression patterns is essential for defining pathways of toxicity, and ultimately, for determining the chemical modes of action through which a toxicant acts. Traditionally, transcription factor identification is achieved via chromatin immunoprecipitation studies and summarized by calculating which transcription factors are statistically associated with up- and downregulated genes. These lists are commonly determined via statistical or fold-change cutoffs, a procedure that is sensitive to statistical power and may not be as useful for determining transcription factor associations. To move away from an arbitrary statistical or fold-change-based cutoff, we developed, in the context of the Mapping the Human Toxome project, an enrichment paradigm called information-dependent enrichment analysis (IDEA) to guide identification of the transcription factor network. We used a test case of activation in MCF-7 cells by 17ß estradiol (E2). Using this new approach, we established a time course for transcriptional and functional responses to E2. ERα and ERß were associated with short-term transcriptional changes in response to E2. Sustained exposure led to recruitment of additional transcription factors and alteration of cell cycle machinery. TFAP2C and SOX2 were the transcription factors most highly correlated with dose. E2F7, E2F1, and Foxm1, which are involved in cell proliferation, were enriched only at 24 h. IDEA should be useful for identifying candidate pathways of toxicity. IDEA outperforms gene set enrichment analysis (GSEA) and provides similar results to weighted gene correlation network analysis, a platform that helps to identify genes not annotated to pathways.

Quantitative Metabolomic Analysis of Urinary Citrulline and Calcitroic Acid in Mice after Exposure to Various Types of Ionizing Radiation.

With the safety of existing nuclear power plants being brought into question after the Fukushima disaster and the increased level of concern over terrorism-sponsored use of improvised nuclear devices, it is more crucial to develop well-defined radiation injury markers in easily accessible biofluids to help emergency-responders with injury assessment during patient triage. Here, we focused on utilizing ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify and quantitate the unique changes in the urinary excretion of two metabolite markers, calcitroic acid and citrulline, in mice induced by different forms of irradiation; external γ irradiation at a low dose rate (LDR) of 3.0 mGy/min and a high dose rate (HDR) of 1.1 Gy/min, and internal exposure to Cesium-137 ((137)Cs) and Strontium-90 ((90)Sr). The multiple reaction monitoring analysis showed that, while exposure to (137)Cs and (90)Sr induced a statistically significant and persistent decrease, similar doses of external γ beam at the HDR had the opposite effect, and the LDR had no effect on the urinary levels of these two metabolites. This suggests that the source of exposure and the dose rate strongly modulate the in vivo metabolomic injury responses, which may have utility in clinical biodosimetry assays for the assessment of exposure in an affected population. This study complements our previous investigations into the metabolomic profile of urine from mice internally exposed to (90)Sr and (137)Cs and to external γ beam radiation.

Metabolomic and lipidomic analysis of serum from mice exposed to an internal emitter, cesium-137, using a shotgun LC-MS(E) approach.

In this study ultra performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry in the MS(E) mode was used for rapid and comprehensive analysis of metabolites in the serum of mice exposed to internal exposure by Cesium-137 ((137)Cs). The effects of exposure to (137)Cs were studied at several time points after injection of (137)CsCl in mice. Over 1800 spectral features were detected in the serum of mice in positive and negative electrospray ionization modes combined. Detailed statistical analysis revealed that several metabolites associated with amino acid metabolism, fatty acid metabolism, and the TCA cycle were significantly perturbed in the serum of (137)Cs-exposed mice compared with that of control mice. While metabolites associated with the TCA cycle and glycolysis increased in their serum abundances, fatty acids such as linoleic acid and palmitic acid were detected at lower levels in serum after (137)Cs exposure. Furthermore, phosphatidylcholines (PCs) were among the most perturbed ions in the serum of (137)Cs-exposed mice. This is the first study on the effects of exposure by an internal emitter in serum using a UPLC-MS(E) approach. The results have put forth a panel of metabolites, which may serve as potential serum markers to (137)Cs exposure.