Metabolomics as a tool for the study of drug-induced hepatotoxicity

Drug-induced liver injury (DILI) is a major health and economic problem and the leading cause of hepatic dysfunction, drug failure during clinical testing and post-market withdrawal of approved drugs. Pre-clinical testing should be able to detect potential hepatotoxins early in the drug development process in order to minimize health risks and financial losses. Several liver-derived in vitro models have been developed to be used in pharmacology and toxicology research to understand the mechanism of DILI and to evaluate potential hepatotoxicity of new chemical entities. Although they fail to reproduce the complexity of a whole organ, their low cost, high reproducibility, and the possibility of a human origin make them a good complement to traditional in vivo tests. Monoparametric strategies used in in vitro toxicity testing have been proved insufficient to predict human DILI. The application of the new 'omics' technologies allows the simultaneous determination of multiple parameters in a single biological sample and represents a more sensitive, comprehensive and powerful tool for the study of hepatotoxic events. Among them, metabolomics measures the downstream products of the 'omics cascade', thus representing a closer approximation to phenotype than the study of genes, transcripts or proteins. Based on the previous evidences we decided to evaluate whether metabolomics, in combination with in vitro cellular models and in vivo animal models, could be a useful tool for the disvovery of characteristic patterns associated to specific mechanisms of DILI. First, we defined a suitable framework that, thanks to a careful design of sample analysis and the incorporation of different internal standards and quality controls, allowed us to perform metabolomic analysis within a quality assurance environment. Then, uni- and multivariate statistical tools were selected in order to be able to identify mechanism-specific alterations and to develop predictive/classificatory models. Finally, we optimized a sample processing and analysis strategy that allowed the differential extraction and detection of a broad range of metabolites ranging from highly polar to highly apolar ones thus maximizing metabolome coverage. The application of the developed tools to HepG2 cells exposed to subcytotoxic concentrations of model hepatotoxins acting through different mechanisms of toxicity allowed us to identify specific metabolomic patterns associated to each of the mechanisms of interest. Moreover, the application of multivariate data analysis techniques allowed the development of predictive/classificatory models able not only to distinguish between toxic and non-toxic compounds, but also to specifically classify drugs according to their mechanism of hepatotoxicity. The proposed strategy could be of interest for the identification of early markers of hepatotoxicy and for the prediction of mechanism of hepatotoxicity of new drug entities. The usefulness of the analytical strategy was also confirmed by its application with in vivo models using both medaka (Oryzias latipes) and rat. Studies in medaka revealed common liver altered pathways with HepG2 cells, suggesting medaka as a useful model for human hepatotoxicity prediction. Toxicity studies in rats allowed us to identify common serum markers of hepatotoxicity which could be used as biomarkers in pre-clinical studies or even extrapolated to humans

Los elementos traza (Mg, Sr, Ha, Fe, Mn) en carbonatos: ambiente genético del Karst del techo de la unidad intermedia de la Cuenca neógena de Madrid.

Este trabajo trata de caracterizar geoquímicamente el karst del techo de la U. Intermedia del Mioceno en las partes centrales de la Cuenca de Madrid. Para ello se han estudiado una serie de elementos traza (Mg, Sr, Na, Fe y Mn) mediante fluorescencia de rayos X. En base a este estudio se han distinguido dos tipos de procesos: De reemplazamiento y/o recristalización, que implican la litificación y homogeneización química de un sedimento calcítico-dolomítico; y procesos de precipitación química de espeleotemas en función de su contenido en Sr, asignando un probable origen aragonítico a los de mayor contenido en Sr

Calcitization of Mg±Ca carbonate and Ca sulphate deposits in a continental Tertiary basin (Calatayud Basin, NE Spain)

Diagenetic carbonates formed through early calcitization of evaporite (gypsum, anhydrite) and/or magnesium carbonate (dolomite, magnesite) deposits have been identified throughout the Tertiary Calatayud Basin (NE Spain). The diagenetic carbonates consist in all cases of low magnesian calcite and are related to main sedimentary discontinuities, which record episodes of significant subaerial exposure in the basin. Early calcite replacements predominate in sediments of the Intermediate Unit in central areas of the basin. Two types of diagenetic carbonates have been distinguished: (1) laminated diagenetic carbonates in which the original structure of sequences formed of laminated magnesite, dolomite and primary gypsum is preserved, though transformed in calcite; (2) brecciated diagenetic carbonates, which resulted from extensive dissolution of lenticular gypsum macrocrystals accompanied by dedolomitization processes of dolomite host rock. The replacement by calcite resulted from rapid transformation of evaporite deposits either at the contact of these deposits with overlying freshwaters or by percolating freshwater at very shallow burial depth. Stable isotope (carbon and oxygen) analyses of the diagenetic carbonates support influence of meteoric-derived carbonate-rich groundwater for their formation. The meteoric isotope signature is supported by its comparison with the stable isotope values determined from diagenetic calcites formed after magnesite in evaporite sequences during the Late Pleistocene and Holocene. The diagenetic carbonate bodies formed as a result of a progressive change from highly to moderately concentrated saline lake waters. This gradual evolution was related to a climatic shift from dry and hot to cooler and more humid conditions throughout the Miocene, a trend that has been also recognized in other large Tertiary basins of the Iberian Peninsula in this period

Geochemical Fingerprinting of Rising Deep Endogenous Gases in an Active Hypogenic Karst System

The hydrothermal caves linked to active faulting can potentially harbour subterranean atmospheres with a distinctive gaseous composition with deep endogenous gases, such as carbon dioxide (CO2) and methane (CH4). In this study, we provide insight into the sourcing, mixing, and biogeochemical processes involved in the dynamic of deep endogenous gas formation in an exceptionally dynamic hypogenic karst system (Vapour Cave, southern Spain) associated with active faulting. The cave environment is characterized by a prevailing combination of rising warm air with large CO2 outgassing (>1%) and highly diluted CH4 with an endogenous origin. The δ13CCO2 data, which ranges from −4.5 to −7.5‰, point to a mantle-rooted CO2 that is likely generated by the thermal decarbonation of underlying marine carbonates, combined with degassing from CO2-rich groundwater. A pooled analysis of δ13CCO2 data from exterior, cave, and soil indicates that the upwelling of geogenic CO2 has a clear influence on soil air, which further suggests a potential for the release of CO2 along fractured carbonates. CH4 molar fractions and their δD and δ13C values (ranging from −77 to −48‰ and from −52 to −30‰, respectively) suggest that the methane reaching Vapour Cave is the remnant of a larger source of CH4, which was likely generated by microbial reduction of carbonates. This CH4 has been affected by a postgenetic microbial oxidation, such that the gas samples have changed in both molecular and isotopic composition after formation and during migration through the cave environment. Yet, in the deepest cave locations (i.e., 30m below the surface), measured concentration values of deep endogenous CH4 are higher than in atmospheric with lighter δ13C values with respect to those found in the local atmosphere, which indicates that Vapour Cave may occasionally act as a net source of CH4 to the open atmosphere

Petrographic and geochemical evidence for the formation of primary, bacterially induced lacustrine dolomite: La Roda 'white earth' (Pliocene, central Spain)

Upper Pliocene dolomites (‘white earth’) from La Roda, Spain, offer a good opportunity to evaluate the process of dolomite formation in lakes. The relatively young nature of the deposits could allow a link between dolomites precipitated in modern lake systems and those present in older lacustrine formations. The La Roda Mg‐carbonates (dolomite unit) occur as a 3·5‐ to 4‐m‐thick package of poorly indurated, white, massive dolomite beds with interbedded thin deposits of porous carbonate displaying root and desiccation traces as well as local lenticular gypsum moulds. The massive dolomite beds consist mainly of loosely packed 1‐ to 2‐μm‐sized aggregates of dolomite crystals exhibiting poorly developed faces, which usually results in a subrounded morphology of the crystals. Minute rhombs of dolomite are sparse within the aggregates. Both knobbly textures and clumps of spherical bodies covering the crystal surfaces indicate that bacteria were involved in the formation of the dolomites. In addition, aggregates of euhedral dolomite crystals are usually present in some more clayey (sepiolite) interbeds. The thin porous carbonate (mostly dolomite) beds exhibit both euhedral and subrounded, bacterially induced dolomite crystals. The carbonate is mainly Ca‐dolomite (51–54 mol% CaCO3), showing a low degree of ordering (degree of ordering ranges from 0·27 to 0·48). Calcite is present as a subordinate mineral in some samples. Sr, Mn and Fe contents show very low correlation coefficients with Mg/Ca ratios, whereas SiO2 and K contents are highly correlated. δ18O‐ and δ13C‐values in dolomites range from −3·07‰ to 5·40‰ PDB (mean=0·06, σ=1·75) and from −6·34‰ to −0·39‰ PDB (mean=−3·55, σ=1·33) respectively. Samples containing significant amounts of both dolomite and calcite do not in general show significant enrichment or depletion in 18O and 13C between the two minerals. The correlation coefficient between δ18O and δ13C for dolomite is extremely low and negative (r=−0·05), whereas it is higher and positive (r=0·47) for calcite. The lacustrine dolomite deposit from La Roda is interpreted mainly as a result of primary precipitation of dolomite in a shallow, hydrologically closed perennial lake. The lake was supplied by highly saturated HCO3−/CO32− groundwater that leached dolomitic Mesozoic formations. Precipitation of dolomite from alkaline lake waters took place under a semi‐arid to arid climate. However, according to our isotopic data, strong evaporative conditions were not required for the formation of the La Roda dolomite. A significant contribution by bacteria to the formation of the dolomites is assumed in view of both petrographic and geochemical evidence

c‑MYC Triggers Lipid Remodelling During Early Somatic Cell Reprogramming to Pluripotency

Metabolic rewiring and mitochondrial dynamics remodelling are hallmarks of cell reprogramming, but the roles of the reprogramming factors in these changes are not fully understood. Here we show that c-MYC induces biosynthesis of fatty acids and increases the rate of pentose phosphate pathway. Time-course profiling of fatty acids and complex lipids during cell reprogramming using lipidomics revealed a profound remodelling of the lipid content, as well as the saturation and length of their acyl chains, in a c-MYC-dependent manner. Pluripotent cells displayed abundant cardiolipins and scarce phosphatidylcholines, with a prevalence of monounsaturated acyl chains. Cells undergoing cell reprogramming showed an increase in mitochondrial membrane potential that paralleled that of mitochondrial-specific cardiolipins. We conclude that c-MYC controls the rewiring of somatic cell metabolism early in cell reprogramming by orchestrating cell proliferation, synthesis of macromolecular components and lipid remodelling, all necessary processes for a successful phenotypic transition to pluripotency