BackgroundIn vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively.ResultsThe study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD.ConclusionsUntargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.

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A strategy, detailed methodology description and software are given with which the mass accuracy of U-HPLC-Orbitrap data (resolving power 50,000 FWHM) can be enhanced by an order of magnitude to sub-ppm levels. After mass accuracy enhancement all 211 reference masses have mass errors within 0.5 ppm; only 14 of these are outside the 0.2 ppm error margin. Further demonstration of mass accuracy enhancement is shown on a pre-concentrated urine sample in which evidence for 89 (342 ions) potential hydroxylated and glucuronated DHEA-metabolites is found. Although most DHEA metabolites have low-intensity mass signals, only 11 out of 342 are outside the ±1 ppm error envelop; 272 mass signals have errors below 0.5 ppm (142 below 0.2 ppm). The methodology consists of: (a) a multiple internal lock correction (here ten masses; no identity of internal lock masses is required) to avoid suppression problems of a single internal lock mass as well as to increase lock precision, (b) a multiple external mass correction (here 211 masses) to correct for calibration errors, (c) intensity dependant mass correction, (d) file averaging. The strategy is supported by ultra-fast file searching of baseline corrected, noise-reduced metAlign output. The output and efficiency of ultra-fast searching is essential in obtaining the required information to visualize the distribution of mass errors and isotope ratio deviations as a function of mass and intensity.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-010-0230-y) contains supplementary material, which is available to authorized users.

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The chemotherapeutic compound, cisplatin causes various kinds of DNA lesions but also triggers other pertubations, such as ER and oxidative stress. We and others have shown that treatment of pluripotent stem cells with cisplatin causes a plethora of transcriptional and post-translational alterations that, to a major extent, point to DNA damage response (DDR) signaling. The orchestrated DDR signaling network is important to arrest the cell cycle and repair the lesions or, in case of damage beyond repair, eliminate affected cells. Failure to properly balance the various aspects of the DDR in stem cells contributes to ageing and cancer. Here, we performed metabolic profiling by mass spectrometry of embryonic stem (ES) cells treated for different time periods with cisplatin. We then integrated metabolomics with transcriptomics analyses and connected cisplatin-regulated metabolites with regulated metabolic enzymes to identify enriched metabolic pathways. These included nucleotide metabolism, urea cycle and arginine and proline metabolism. Silencing of identified proline metabolic and catabolic enzymes indicated that altered proline metabolism serves as an adaptive, rather than a toxic response. A group of enriched metabolic pathways clustered around the metabolite S-adenosylmethionine, which is a hub for methylation and transsulfuration reactions and polyamine metabolism. Enzymes and metabolites with pro- or anti-oxidant functions were also enriched but enhanced levels of reactive oxygen species were not measured in cisplatin-treated ES cells. Lastly, a number of the differentially regulated metabolic enzymes were identified as target genes of the transcription factor p53, pointing to p53-mediated alterations in metabolism in response to genotoxic stress. Altogether, our findings reveal interconnecting metabolic pathways that are responsive to cisplatin and may serve as signaling modules in the DDR in pluripotent stem cells.

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Lipid oxidation causes food degradation and the formation of toxic compounds. Therefore, the addition to foods of compounds able to avoid, delay or minimize this degradative process is a commonly used strategy. Nevertheless, neither the identity of most of the formed compounds in this complex process nor the way in which their formation is affected by the strategy used are well known. In this context, the effect the temperature increase and the enrichment level in alpha-tocopherol on the evolution of the walnut oil oxidation, as a model of an oil rich in polyunsaturated omega-6 acyl groups, submitted to storage conditions, are tackled by 1H NMR. The study has allowed knowing the degradation kinetic of both the oil acyl groups and alpha-tocopherol, the identification of a very high number of oxylipins and the kinetic of their formation. The temperature increase accelerates the formation of all oxylipins, favouring the formation of hydroperoxy conjugated E,E-dienes and related derivatives versus that of the Z,E-isomers. The enrichment in alpha-tocopherol accelerates the formation of hydroperoxy conjugated Z,E-dienes and related derivatives, and delays in relation to the formation of the former that of the E,E-isomers and related derivatives, hindering, to a certain extent, the formation of the latter in line with the enrichment level.

Sunflower oil samples, both unenriched and enriched with four different concentrations of hydroxytyrosol acetate, were subjected to accelerated storage at 70 °C until a very advanced oxidation stage and the process was monitored by 1H NMR spectroscopy. The aim of the study is to know the effect that the presence of this antioxidant has on the oxidation process of sunflower oil under the aforementioned conditions, as well as on the formation and evolution of the concentration of a significant number of oxylipins. The oxidation process was studied globally by monitoring, during storage time, the degradation of both the linoleic acyl group of sunflower oil, which is the main component of sunflower oil, and the added hydroxytyrosol acetate. Simultaneously, the identification of up to twenty-six different types of oxylipins formed in the oxidation process and the monitoring of the evolution of their concentration over the storage time were carried out. In this way, essential information about the effect that hydroxytyrosol acetate provokes on the oxidation of this oil rich in omega-6 polyunsaturated acyl groups, has been obtained. It has also been shown that the enrichment of sunflower oil with this antioxidant under the conditions tested does not prevent the oxidation process but slows it down, affecting the entire oxidation process.