A novel method to derive a human safety limit for PFOA by gene expression profiling and modelling.

Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant that can accumulate in the human body due to its long half-life. This substance has been associated with liver, pancreatic, testicular and breast cancers, liver steatosis and endocrine disruption. PFOA is a member of a large group of substances also known as "forever chemicals" and the vast majority of substances of this group lack toxicological data that would enable their effective risk assessment in terms of human health hazards. This study aimed to derive a health-based guidance value for PFOA intake (ng/kg BW/day) from in vitro transcriptomics data. To this end, we developed an in silico workflow comprising five components: (i) sourcing in vitro hepatic transcriptomics concentration-response data; (ii) deriving molecular points of departure using BMDExpress3 and performing pathway analysis using gene set enrichment analysis (GSEA) to identify the most sensitive molecular pathways to PFOA exposure; (iii) estimating freely-dissolved PFOA concentrations in vitro using a mass balance model; (iv) estimating in vivo doses by reverse dosimetry using a PBK model for PFOA as part of a quantitative in vitro to in vivo extrapolation (QIVIVE) algorithm; and (v) calculating a tolerable daily intake (TDI) for PFOA. Fourteen percent of interrogated genes exhibited in vitro concentration-response relationships. GSEA pathway enrichment analysis revealed that "fatty acid metabolism" was the most sensitive pathway to PFOA exposure. In vitro free PFOA concentrations were calculated to be 2.9% of the nominal applied concentrations, and these free concentrations were input into the QIVIVE workflow. Exposure doses for a virtual population of 3,000 individuals were estimated, from which a TDI of 0.15 ng/kg BW/day for PFOA was calculated using the benchmark dose modelling software, PROAST. This TDI is comparable to previously published values of 1.16, 0.69, and 0.86 ng/kg BW/day by the European Food Safety Authority. In conclusion, this study demonstrates the combined utility of an "omics"-derived molecular point of departure and in silico QIVIVE workflow for setting health-based guidance values in anticipation of the acceptance of in vitro concentration-response molecular measurements in chemical risk assessment.

Environmentally Relevant Iron Oxide Nanoparticles Produce Limited Acute Pulmonary Effects in Rats at Realistic Exposure Levels.

Iron is typically the dominant metal in the ultrafine fraction of airborne particulate matter. Various studies have investigated the toxicity of inhaled nano-sized iron oxide particles (FeOxNPs) but their results have been contradictory, with some indicating no or minor effects and others finding effects including oxidative stress and inflammation. Most studies, however, did not use materials reflecting the characteristics of FeOxNPs present in the environment. We, therefore, analysed the potential toxicity of FeOxNPs of different forms (Fe3O4, α-Fe2O3 and γ-Fe2O3) reflecting the characteristics of high iron content nano-sized particles sampled from the environment, both individually and in a mixture (FeOx-mix). A preliminary in vitro study indicated Fe3O4 and FeOx-mix were more cytotoxic than either form of Fe2O3 in human bronchial epithelial cells (BEAS-2B). Follow-up in vitro (0.003, 0.03, 0.3 µg/mL, 24 h) and in vivo (Sprague-Dawley rats, nose-only exposure, 50 µg/m3 and 500 µg/m3, 3 h/d × 3 d) studies therefore focused on these materials. Experiments in vitro explored responses at the molecular level via multi-omics analyses at concentrations below those at which significant cytotoxicity was evident to avoid detection of responses secondary to toxicity. Inhalation experiments used aerosol concentrations chosen to produce similar levels of particle deposition on the airway surface as were delivered in vitro. These were markedly higher than environmental concentrations. No clinical signs of toxicity were seen nor effects on BALF cell counts or LDH levels. There were also no significant changes in transcriptomic or metabolomic responses in lung or BEAS-2B cells to suggest adverse effects.

Mass Spectrometry Based Metabolomics Comparison of Liver Grafts from Donors after Circulatory Death (DCD) and Donors after Brain Death (DBD) Used in Human Orthotopic Liver Transplantation.

Use of marginal liver grafts, especially those from donors after circulatory death (DCD), has been considered as a solution to organ shortage. Inferior outcomes have been attributed to donor warm ischaemic damage in these DCD organs. Here we sought to profile the metabolic mechanisms underpinning donor warm ischaemia. Non-targeted Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry metabolomics was applied to biopsies of liver grafts from donors after brain death (DBD; n = 27) and DCD (n = 10), both during static cold storage (T1) as well as post-reperfusion (T2). Furthermore 6 biopsies from DBD donors prior to the organ donation (T0) were also profiled. Considering DBD and DCD together, significant metabolic differences were discovered between T1 and T2 (688 peaks) that were primarily related to amino acid metabolism, meanwhile T0 biopsies grouped together with T2, denoting the distinctively different metabolic activity of the perfused state. Major metabolic differences were discovered between DCD and DBD during cold-phase (T1) primarily related to glucose, tryptophan and kynurenine metabolism, and in the post-reperfusion phase (T2) related to amino acid and glutathione metabolism. We propose tryptophan/kynurenine and S-adenosylmethionine as possible biomarkers for the previously established higher graft failure of DCD livers, and conclude that the associated pathways should be targeted in more exhaustive and quantitative investigations.

Protein Corona Modulates Uptake and Toxicity of Nanoceria via Clathrin-Mediated Endocytosis.

Particles present in diesel exhaust have been proposed as a significant contributor to the development of acute and chronic lung diseases, including respiratory infection and allergic asthma. Nanoceria (CeO2 nanoparticles) are used to increase fuel efficiency in internal combustion engines, are present in exhaust fumes, and could affect cells of the airway. Components from the environment such as biologically derived proteins, carbohydrates, and lipids can form a dynamic layer, commonly referred to as the "protein corona" which alters cellular nanoparticle interactions and internalization. Using confocal reflectance microscopy, we quantified nanoceria uptake by lung-derived cells in the presence and absence of a serum-derived protein corona. Employing mass spectrometry, we identified components of the protein corona, and demonstrated that the interaction between transferrin in the protein corona and the transferrin receptor is involved in mediating the cellular entry of nanoceria via clathrin-mediated endocytosis. Furthermore, under these conditions nanoceria does not affect cell growth, viability, or metabolism, even at high concentration. Alternatively, despite the antioxidant capacity of nanoceria, in serum-free conditions these nanoparticles induce plasma membrane disruption and cause changes in cellular metabolism. Thus, our results identify a specific receptor-mediated mechanism for nanoceria entry, and provide significant insight into the potential for nanoparticle-dependent toxicity.

Predicting chronic copper and nickel reproductive toxicity to Daphnia pulex-pulicaria from whole-animal metabolic profiles.

The emergence of omics approaches in environmental research has enhanced our understanding of the mechanisms underlying toxicity; however, extrapolation from molecular effects to whole-organism and population level outcomes remains a considerable challenge. Using environmentally relevant, sublethal, concentrations of two metals (Cu and Ni), both singly and in binary mixtures, we integrated data from traditional chronic, partial life-cycle toxicity testing and metabolomics to generate a statistical model that was predictive of reproductive impairment in a Daphnia pulex-pulicaria hybrid that was isolated from an historically metal-stressed lake. Furthermore, we determined that the metabolic profiles of organisms exposed in a separate acute assay were also predictive of impaired reproduction following metal exposure. Thus we were able to directly associate molecular profiles to a key population response - reproduction, a key step towards improving environmental risk assessment and management.

A complete workflow for high-resolution spectral-stitching nanoelectrospray direct-infusion mass-spectrometry-based metabolomics and lipidomics.

Metabolomic and lipidomic studies measure and discover metabolic and lipid profiles in biological samples, enabling a better understanding of the metabolism of specific biological phenotypes. Accurate biological interpretations require high analytical reproducibility and sensitivity, and standardized and transparent data processing. Here we describe a complete workflow for nanoelectrospray ionization (nESI) direct-infusion mass spectrometry (DIMS) metabolomics and lipidomics. After metabolite and lipid extraction from tissues and biofluids, samples are directly infused into a high-resolution mass spectrometer (e.g., Orbitrap) using a chip-based nESI sample delivery system. nESI functions to minimize ionization suppression or enhancement effects as compared with standard electrospray ionization (ESI). Our analytical technique-named spectral stitching-measures data as several overlapping mass-to-charge (m/z) windows that are subsequently 'stitched' together, creating a complete mass spectrum. This considerably increases the dynamic range and detection sensitivity-about a fivefold increase in peak detection-as compared with the collection of DIMS data as a single wide mass-to-charge (m/z ratio) window. Data processing, statistical analysis and metabolite annotation are executed as a workflow within the user-friendly, transparent and freely available Galaxy platform (galaxyproject.org). Generated data have high mass accuracy that enables molecular formulae peak annotations. The workflow is compatible with any sample-extraction method; in this protocol, the examples are extracted using a biphasic method, with methanol, chloroform and water as the solvents. The complete workflow is reproducible, rapid and automated, which enables cost-effective analysis of >10,000 samples per year, making it ideal for high-throughput metabolomics and lipidomics screening-e.g., for clinical phenotyping, drug screening and toxicity testing.

Gene expression and metabolic responses of HepG2/C3A cells exposed to flame retardants and dust extracts at concentrations relevant to indoor environmental exposures.

Humans are routinely exposed to mixtures of flame retardants (FRs) from multiple sources including indoor dust. As a model to explore the potential effects of FR exposure from indoor dust on human health, the molecular responses of human hepatoma cells (HepG2/C3A cells) to a defined mixture of FRs and to a dust extract were investigated using multiple non-targeted omics approaches. A solvent extract of an indoor dust standard reference material SRM2585 was used as the surrogate dust sample, while a mixture of four FRs (TCEP, TCIPP, TDCIPP and HBCD) was used to mimic the FR mixture in the indoor dust. Cytotoxicity tests indicated there were no significant changes to cell viability or cell integrity after a 24- or 72-h exposure of HepG2/C3A cells to the FR mixture or to the dust extract. However, transcriptomics revealed changes in gene expression associated with the metabolism of xenobiotics (e.g. CYP1A1, CYP1A2, CYP2B6) in the dust extract group but not in the FR mixture group after a 72-h exposure. Few metabolic or lipidomic changes were detected in response to either the FR mixture or to the dust extract group. Given that the dust extract contained components that elicited a biological response, in contrast to the lack of response induced by the FR mixture, our findings suggest that the most likely causes of the molecular responses to indoor dust exposure lie in components other than the four FRs investigated, e.g. caused by PAHs or PCBs.

Defensive and adverse energy-related molecular responses precede tris (1, 3-dichloro-2-propyl) phosphate cytotoxicity.

To understand the potentially adverse effects of human exposure to tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP) and explore the underlying molecular mechanisms, combined transcriptomic and metabolomic approaches were employed to investigate the molecular responses of two human cell lines exposed to different concentrations of TDCIPP. Comparative analyses of transcriptional and metabolic profiles of HepG2/C3A and A549 cells were performed after exposure to 1, 10 and 100 µM TDCIPP for 24 and 72 h. Stress responses (e.g. xenobiotic metabolism and ABC transporter pathways) were observed at the transcriptional level after 24-h exposure to a sub-cytotoxic concentration (10 µM). Transcription of an energy metabolism-related pathway (oxidative phosphorylation) was down-regulated more severely at 100 µM TDCIPP exposure, accompanied by the suppression of pathways relevant to cell proliferation (e.g. cell cycle and DNA replication), while no significant cytotoxic effects were observed. Functional metabolic changes were observed after 72 h in HepG2/C3A cells exposed to 100 µM TDCIPP that corresponded to changes detected at the transcriptional level after 24 h. Taken together, defensive responses to chemical exposure and energy-related changes both precede the cytotoxic effects of TDCIPP in HepG2/C3A cells.

Transcriptomic and metabolomic approaches to investigate the molecular responses of human cell lines exposed to the flame retardant hexabromocyclododecane (HBCD).

The potential for human exposure to the brominated flame retardant, hexabromocyclododecane (HBCD) has given rise to health concerns, yet there is relatively limited knowledge about its possible toxic effects and the underlying molecular mechanisms that may mediate any impacts on health. In this study, unbiased transcriptomic and metabolomic approaches were employed to investigate the potential molecular changes that could lead to the toxicity of HBCD under concentrations relevant to human exposure conditions using in vitro models. A concentration-dependent cytotoxic effect of HBCD to A549 and HepG2/C3A cells was observed based on MTT assays or CCK-8 assays with EC50 values of 27.4 µM and 63.0 µM, respectively. Microarray-based transcriptomics and mass spectrometry-based metabolomics revealed few molecular changes in A549 cells or HepG2/C3A cells following a 24-hour exposure to several sub-lethal concentrations (2 to 4000 nM) of HBCD. Quantification of the level of HBCD in the HepG2/C3A exposed cells suggested that the flame retardant was present at concentrations several orders of magnitude higher than those reported to occur in human tissues. We conclude that at the concentrations known to be achievable following exposure in humans, HBCD exhibits no detectable acute toxicity in A549 cells, representative of the lung, or in HepG2/C3A cells, that are hepatocytes with some xenobiotic metabolic capacity.

Drug Redeployment to Kill Leukemia and Lymphoma Cells by Disrupting SCD1-Mediated Synthesis of Monounsaturated Fatty Acids.

The redeployed drug combination of bezafibrate and medroxyprogesterone acetate (designated BaP) has potent in vivo anticancer activity in acute myelogenous leukemia (AML) and endemic Burkitt lymphoma (eBL) patients; however, its mechanism-of-action is unclear. Given that elevated fatty acid biosynthesis is a hallmark of many cancers and that these drugs can affect lipid metabolism, we hypothesized that BaP exerts anticancer effects by disrupting lipogenesis. We applied mass spectrometry-based lipidomics and gene and protein expression measurements of key lipogenic enzymes [acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and stearoyl CoA desaturase 1 (SCD1)] to AML and eBL cell lines treated with BaP. BaP treatment decreased fatty acid and phospholipid biosynthesis from (13)C D-glucose. The proportion of phospholipid species with saturated and monounsaturated acyl chains was also decreased after treatment, whereas those with polyunsaturated chains increased. BaP decreased SCD1 protein levels in each cell line (0.46- to 0.62-fold; P < 0.023) and decreased FASN protein levels across all cell lines (0.87-fold decrease; P = 1.7 × 10(-4)). Changes to ACC1 protein levels were mostly insignificant. Supplementation with the SCD1 enzymatic product, oleate, rescued AML and e-BL cells from BaP cell killing and decreased levels of BaP-induced reactive oxygen species, whereas supplementation with the SCD1 substrate (and FASN product), palmitate, did not rescue cells. In conclusion, these data suggest that the critical anticancer actions of BaP are decreases in SCD1 levels and monounsaturated fatty acid synthesis. To our knowledge, this is the first time that clinically available antileukemic and antilymphoma drugs targeting SCD1 have been reported.

High-resolution mass spectrometry provides novel insights into products of human metabolism of organophosphate and brominated flame retardants.

The high resolution, accurate mass, and fast scanning features of the Orbitrap(TM) mass spectrometer, combined with the separation power of ultrahigh-performance liquid chromatography were applied for the first time to study the metabolic profiles of several organic flame retardants (FRs) present in indoor dust. To mimic real-life exposure, in vitro cultured HepG2 human hepatocyte cell lines were exposed simultaneously to various FRs in an indoor dust extract for 24 h. Target parent FRs, hexabromocyclododecanes (α-, ß-, and γ-HBCDs), tris-2-chloroethyl phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCIPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), were separated in a single run for the first time using alternating positive and negative heated ESI source. Further metabolite separation and identification was achieved using full scan (70,000 full width at half maximum (FWHM)), accurate mass (up to 1 ppm) spectrometry. Structural confirmation was performed via all ion fragmentation (AIF) spectra using the optional higher collisional dissociation (HCD) cell and MS/MS analysis. First insights into human metabolism of HBCDs revealed several hydroxylated and debrominated phase I metabolites, in addition to conjugated phase II glucuronides. Furthermore, various hydroxylated, oxidized, and conjugated metabolites of chlorinated phosphorous FRs were identified, leading to the suggestion of α-oxidation as a significant metabolic pathway for these compounds.

The effect of perhexiline on myocardial protection during coronary artery surgery: a two-centre, randomized, double-blind, placebo-controlled trial.

OBJECTIVES: Perhexiline is thought to modulate metabolism by inhibiting mitochondrial carnitine palmitoyltransferase-1, reducing fatty acid uptake and increasing carbohydrate utilization. This study assessed whether preoperative perhexiline improves markers of myocardial protection in patients undergoing coronary artery bypass graft surgery and analysed its effect on the myocardial metabolome. METHODS: In a prospective, randomized, double-blind, placebo-controlled trial, patients at two centres were randomized to receive either oral perhexiline or placebo for at least 5 days prior to surgery. The primary outcome was a low cardiac output episode in the first 6 h. All pre-specified analyses were conducted according to the intention-to-treat principle with a statistical power of 90% to detect a relative risk of 0.5 and a conventional one-sided α-value of 0.025. A subset of pre-ischaemic left ventricular biopsies was analysed using mass spectrometry-based metabolomics. RESULTS: Over a 3-year period, 286 patients were randomized, received the intervention and were included in the analysis. The incidence rate of a low cardiac output episode in the perhexiline arm was 36.7% (51/139) vs 34.7% (51/147) in the control arm [odds ratio (OR) 0.92, 95% confidence interval (CI) 0.56-1.50, P = 0.74]. Perhexiline was associated with a reduction in the cardiac index at 6 h [difference in means 0.19, 95% CI 0.07-0.31, P = 0.001] and an increase in inotropic support in the first 12 h (OR 0.55, 95% CI 0.34-0.89, P = 0.015). There were no significant differences in myocardial injury with troponin-T or electrocardiogram, reoperation, renal dysfunction or length of stay. No difference in the preischaemic left ventricular metabolism was identified between groups on metabolomics analysis. CONCLUSIONS: Preoperative perhexiline does not improve myocardial protection in patients undergoing coronary surgery and in fact reduced perioperative cardiac output, increasing the need for inotropic support. Perhexiline has no significant effect on the mass spectrometry-visible polar myocardial metabolome in vivo in humans, supporting the suggestion that it acts via a pathway that is independent of myocardial carnitine palmitoyltransferase inhibition and may explain the lack of clinical benefit observed following surgery. CLINICALTRIALSGOV ID: NCT00845364.

Exo-metabolome of Pseudovibrio sp. FO-BEG1 analyzed by ultra-high resolution mass spectrometry and the effect of phosphate limitation.

Oceanic dissolved organic matter (DOM) is an assemblage of reduced carbon compounds, which results from biotic and abiotic processes. The biotic processes consist in either release or uptake of specific molecules by marine organisms. Heterotrophic bacteria have been mostly considered to influence the DOM composition by preferential uptake of certain compounds. However, they also secrete a variety of molecules depending on physiological state, environmental and growth conditions, but so far the full set of compounds secreted by these bacteria has never been investigated. In this study, we analyzed the exo-metabolome, metabolites secreted into the environment, of the heterotrophic marine bacterium Pseudovibrio sp. FO-BEG1 via ultra-high resolution mass spectrometry, comparing phosphate limited with phosphate surplus growth conditions. Bacteria belonging to the Pseudovibrio genus have been isolated worldwide, mainly from marine invertebrates and were described as metabolically versatile Alphaproteobacteria. We show that the exo-metabolome is unexpectedly large and diverse, consisting of hundreds of compounds that differ by their molecular formulae. It is characterized by a dynamic recycling of molecules, and it is drastically affected by the physiological state of the strain. Moreover, we show that phosphate limitation greatly influences both the amount and the composition of the secreted molecules. By assigning the detected masses to general chemical categories, we observed that under phosphate surplus conditions the secreted molecules were mainly peptides and highly unsaturated compounds. In contrast, under phosphate limitation the composition of the exo-metabolome changed during bacterial growth, showing an increase in highly unsaturated, phenolic, and polyphenolic compounds. Finally, we annotated the detected masses using multiple metabolite databases. These analyses suggested the presence of several masses analogue to masses of known bioactive compounds. However, the annotation was successful only for a minor part of the detected molecules, underlining the current gap in knowledge concerning the biosynthetic ability of marine heterotrophic bacteria.

Disruption of DNA methylation via S-adenosylhomocysteine is a key process in high incidence liver carcinogenesis in fish.

Interactions between epigenome and the environment in biology and in disease are of fundamental importance. The incidence of hepatocellular adenomas in flatfish exceeds 20% in some environments forming a unique opportunity to study environmental tumorigenesis of general relevance to cancer in humans. We report the novel finding of marked DNA methylation and metabolite concentration changes in histopathologically normal tissue distal to tumors in fish liver. A multi-"omics" discovery approach led to targeted and quantitative gene transcription analyses and metabolite analyses of hepatocellular adenomas and histologically normal liver tissue in the same fish. We discovered a remarkable and consistent global DNA hypomethylation, modification of DNA methylation and gene transcription, and disruption of one-carbon metabolism in distal tissue compared to livers of non-tumor-bearing fish. The mechanism of this disruption is linked not to depletion of S-adenosylmethionine, as is often a feature of mammalian tumors, but to a decrease in choline and elevated S-adenosylhomocysteine, a potent inhibitor of DNA methyltransferase. This novel feature of normal-appearing tissue of tumor-bearing fish helps to understand the unprecedentedly high incidence of tumors in fish sampled from the field and adds weight to the controversial epigenetic progenitor model of tumorigenesis. With further studies, the modifications may offer opportunities as biomarkers of exposure to environmental factors influencing disease.

Proton NMR-based metabolite analyses of archived serial paired serum and urine samples from myeloma patients at different stages of disease activity identifies acetylcarnitine as a novel marker of active disease.

BACKGROUND: Biomarker identification is becoming increasingly important for the development of personalized or stratified therapies. Metabolomics yields biomarkers indicative of phenotype that can be used to characterize transitions between health and disease, disease progression and therapeutic responses. The desire to reproducibly detect ever greater numbers of metabolites at ever diminishing levels has naturally nurtured advances in best practice for sample procurement, storage and analysis. Reciprocally, since many of the available extensive clinical archives were established prior to the metabolomics era and were not processed in such an 'ideal' fashion, considerable scepticism has arisen as to their value for metabolomic analysis. Here we have challenged that paradigm. METHODS: We performed proton nuclear magnetic resonance spectroscopy-based metabolomics on blood serum and urine samples from 32 patients representative of a total cohort of 1970 multiple myeloma patients entered into the United Kingdom Medical Research Council Myeloma IX trial. FINDINGS: Using serial paired blood and urine samples we detected metabolite profiles that associated with diagnosis, post-treatment remission and disease progression. These studies identified carnitine and acetylcarnitine as novel potential biomarkers of active disease both at diagnosis and relapse and as a mediator of disease associated pathologies. CONCLUSIONS: These findings show that samples conventionally processed and archived can provide useful metabolomic information that has important implications for understanding the biology of myeloma, discovering new therapies and identifying biomarkers potentially useful in deciding the choice and application of therapy.

Towards a system level understanding of non-model organisms sampled from the environment: a network biology approach.

The acquisition and analysis of datasets including multi-level omics and physiology from non-model species, sampled from field populations, is a formidable challenge, which so far has prevented the application of systems biology approaches. If successful, these could contribute enormously to improving our understanding of how populations of living organisms adapt to environmental stressors relating to, for example, pollution and climate. Here we describe the first application of a network inference approach integrating transcriptional, metabolic and phenotypic information representative of wild populations of the European flounder fish, sampled at seven estuarine locations in northern Europe with different degrees and profiles of chemical contaminants. We identified network modules, whose activity was predictive of environmental exposure and represented a link between molecular and morphometric indices. These sub-networks represented both known and candidate novel adverse outcome pathways representative of several aspects of human liver pathophysiology such as liver hyperplasia, fibrosis, and hepatocellular carcinoma. At the molecular level these pathways were linked to TNF alpha, TGF beta, PDGF, AGT and VEGF signalling. More generally, this pioneering study has important implications as it can be applied to model molecular mechanisms of compensatory adaptation to a wide range of scenarios in wild populations.

Metabolomics of microliter hemolymph samples enables an improved understanding of the combined metabolic and transcriptional responses of Daphnia magna to cadmium.

Omic technologies offer unprecedented opportunities to better understand mode(s)-of-toxicity and downstream secondary effects by providing a holistic view of the molecular changes underlying physiological disruption. Crustacean hemolymph represents a largely untapped biochemical resource for such toxicity studies. We sought to characterize changes in the hemolymph metabolome and whole-body transcriptome to reveal early processes leading to chronic toxicity in the indicator species, Daphnia magna, after 24-h sublethal cadmium exposure (18 µg/L, corresponding to 1/10 LC(50)). We first confirmed that metabolites can be detected and identified in small volumes (∼3-6 µL) of D. magna hemolymph using Fourier transform ion cyclotron resonance mass spectrometry and NMR spectroscopy. Subsequently, mass spectrometry based metabolomics of hemolymph identified disruption to two major classes of metabolites: amino acids and fatty acids. These findings were compared to differentially expressed genes identified by a D. magna 44k oligonucleotide microarray, which included decreased levels of digestive enzymes and increased expression of cuticle proteins and oxidative stress response genes. The combination of metabolic and transcriptional changes revealed through KEGG pathway analysis and gene ontology, respectively, enabled a more complete understanding of how cadmium disrupts nutrient uptake and metabolism, ultimately resulting in decreased energy reserves and chronic toxicity.

Hypoxia triggers major metabolic changes in AML cells without altering indomethacin-induced TCA cycle deregulation.

Our previous studies have shown that the nonsteroidal anti-inflammatory drug indomethacin exhibits antileukemic activity in vitro and can inhibit the aldo-keto reductase AKR1C3, which we identified as a novel target in acute myeloid leukemia. However, the antileukemic actions of indomethacin are likely to be complex and extend beyond inhibition of either AKR1C3 or cycloxygenases. To further understand the antileukemic activity of indomethacin we have used untargeted nuclear magnetic resonance-based metabolic analysis to characterize the responses of KG1a and K562 cell lines in both normal culture conditions and in hypoxia, which better represents the tumor environment in vivo. Hypoxia induced dramatic metabolic changes in untreated KG1a and K562, including adaptation of both phospholipid and glycolytic metabolism. Despite these changes, both cell lines sustained relatively unaltered mitochondrial respiration. The administration of indomethacin induced similar metabolic responses regardless of the oxygen level in the environment. Notable exceptions included metabolites associated with de novo fatty acid synthesis and choline phospholipid metabolism. Collectively, these results suggest that leukemia cells have the inherent ability to tolerate changes in oxygen tension while maintaining an unaltered mitochondrial respiration. However, the administration of indomethacin significantly increased oxidative stress in both KG1a and K562, inducing mitochondrial dysfunction, regardless of the oxygenation conditions. These findings emphasize the particular pertinence of the tricarboxylic acid cycle to the survival of cancer cells and may explain why some antileukemic drugs have been discovered and developed successfully despite the use of culture conditions that do not reflect the hypoxic environment of cancer cells in vivo.

Metabolic responses produced by crude versus dispersed oil in Chinook salmon pre-smolts via NMR-based metabolomics.

Crude oil spills from tankers remain a serious threat along coastal California. Resource managers require information on the acute toxicity of treated and untreated oil, and their sublethal effects on wildlife. This investigation compared the toxic actions of the water-accommodated fraction (WAF) and the chemically-enhanced WAF (CEWAF; Corexit 9500) of Prudhoe Bay crude oil in pre-smolt Chinook salmon (Oncorhynchus tshawytscha) via nuclear magnetic resonance (NMR)-based metabolomics. Metabolite profiles from muscle samples, after 96h exposures, were measured using 1D (1)H NMR and compared via principal component analysis. It was determined that both WAF and CEWAF produced similar profiles in which amino acids, lactate and ATP comprised the highest intensity signals. Overall, metabolic substrates and growth measurements did not show residual effects of short-term exposure on long-term development. In conclusion, the 96h LC(50)s indicate dispersant application significantly decreased hydrocarbon potency and identified metabolites may be bio-indicators of hydrocarbon stress from hydrocarbon exposure.

Combined bezafibrate and medroxyprogesterone acetate: potential novel therapy for acute myeloid leukaemia.

BACKGROUND: The majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis. PRINCIPAL FINDINGS: Here we demonstrate the potent anti-leukaemic activity of the combination of the lipid-regulating drug bezafibrate (BEZ) and the sex hormone medroxyprogesterone acetate (MPA) against AML cell lines and primary AML cells. The combined activity of BEZ and MPA (B/M) converged upon the increased synthesis and reduced metabolism of prostaglandin D(2) (PGD(2)) resulting in elevated levels of the downstream highly bioactive, anti-neoplastic prostaglandin 15-deoxy Delta(12,14) PGJ(2) (15d-PGJ(2)). BEZ increased PGD(2) synthesis via the generation of reactive oxygen species (ROS) and activation of the lipid peroxidation pathway. MPA directed prostaglandin synthesis towards 15d-PGJ(2) by inhibiting the PGD(2) 11beta -ketoreductase activity of the aldo-keto reductase AKR1C3, which metabolises PGD(2) to 9alpha11beta-PGF(2alpha). B/M treatment resulted in growth arrest, apoptosis and cell differentiation in both AML cell lines and primary AML cells and these actions were recapitulated by treatment with 15d-PGJ(2). Importantly, the actions of B/M had little effect on the survival of normal adult myeloid progenitors. SIGNIFICANCE: Collectively our data demonstrate that B/M treatment of AML cells elevated ROS and delivered the anti-neoplastic actions of 15d-PGJ(2). These observations provide the mechanistic rationale for the redeployment of B/M in elderly and relapsed AML.