A protocol for wide-scope non-target analysis of contaminants in small amounts of biota using bead beating tissuelyser extraction and LC-HRMS

This work describes a robust and powerful method for wide-scope target and non-target analysis of xenobiotics in biota samples based on bead beating tissuelyser extraction, solid phase extraction (SPE) clean-up and further detection by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Unlike target methodologies, non-target methods usually aim at determining a wide range of still unknown substances with different physicochemical properties. Therefore, losses during the extraction process were minimised. Apart from that, the reduction of possible interferences showed to be necessary to expand the number of compounds that can be detected. This was achieved with an additional SPE clean-up step carried out with mixed-bed multi-layered cartridges. The method was validated with a set of 27 compounds covering a wide range of physicochemical properties, and further applied to the analysis of krill and fish samples. •The bead beating extraction was efficient for a wide range of organic pollutants in small quantities of biota samples. •Multi-layered solid phase extraction clean-up yield a wide xenobiotics coverage reducing matrix effects. •Method validation with 27 compounds led to a suitable method for non-target analysis of organic pollutants in biota.ICRA researchers thank funding from CERCA program. This work has been funded by the Spanish Ministry of Economy and Competitiveness (MINECO): project PLAS-MED (CTM2017-89701-C3-2-R). SRM acknowledges the Ramon y Cajal program (RYC-2014-16707). This project has been funded by the Chilean Antarctic Institute through project RT_12_17. The Chilean Agency for Research and Development through the FONDAP initiative (grant no. 15150003) “Centro de Investigación Dinámica de Ecosistemas Marinos de Altas Latitudes (IDEAL)”; and “Anillos de Investigación en Ciencia Antartica (grant no. ANID-PIA-ACT-INACH Anillo ACT192057). IDAEA researchers acknowledge the Spanish Ministry of Science and Innovation (Severo Ochoa, Project CEX2018-000794-S).Peer reviewe

Tumoral and normal brain tissue extraction protocol for wide-scope screening of organic pollutants

Little is known about the presence of organic pollutants in human brain (and even less in brain tumors). In this regard, it is necessary to develop new analytical protocols capable of identify-ing a wide range of exogenous chemicals in this type of samples (by combining target, suspect and non-target strategies). These methodologies should be robust and simple. This is particularly challenging for solid samples, as reliable extraction and clean-up techniques should be combined to obtain an optimal result. Hence, the present study focuses on the development of an analytical methodology that allows the screening of a wide range of organic chemicals in brain and brain tumor samples. This protocol was based on a solid-liquid extraction based on bead beating, solid-phase extraction clean-up with multi-layer mixed-mode cartridges, reconstitution and LC -HRMS analysis. To evaluate the performance of the extraction methodology, a set of 66 chemicals (e.g., pharmaceuticals, biocides, or plasticizers, among others) with a wide range of physicochemical properties was employed. Quality control parameters (i.e., linear range, sensitivity, matrix effect (ME%), and recoveries (R%)) were calculated and satisfactory results were obtained for them (e.g., R% within 60-120% for 32 chemicals, or ME% higher than 50% (signal suppression) for 79% of the chemicals)

Disruption of gut integrity and permeability contributes to enteritis in a fish‑parasite model: a story told from serum metabolomics

Background In the animal production sector, enteritis is responsible for serious economic losses, and intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and animal production efficiency. The effect of enteric parasites on the gut function of teleost fish, which represent the most ancient bony vertebrates, is far from being understood. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe enteritis in gilthead sea bream (Sparus aurata), anorexia, cachexia, growth impairment, reduced marketability and increased mortality. Methods This study aimed to outline the gut failure in this fish-parasite model using a multifaceted approach and to find and validate non-lethal serum markers of gut barrier dysfunction. Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits. Results The immunodetection of Tjp1 and Cldn3 was significantly lower in the intestine of parasitized fish, while no strong differences were found in E-cadherin. Parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses. Of these compounds, serum inosine (decreased) and creatine (increased) were identified as relevant and validated with commercial kits. Conclusions The results demonstrate the disruption of tight junctions and the loss of gut barrier function, a metabolomic profile of absorption dysfunction and anorexia, which further outline the pathophysiological effects of E. leei

Serum metabolomics tells the story of disease degree in a fish enteritis model

Comunicación presentada en la 19th International Conference on Diseases of Fish and Shellfish, celebrada en Oporto (Portugal) del 9 al 12 de septiembre de 2019.[Introduction]: In animal production, enteritis is responsible for serious economic losses, being intestinal parasitism a major stress factor leading to malnutrition and lowered performance and production efficiency. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe desquamative enteritis in gilthead sea bream (Sparus aurata) that impairs nutrient absorption causing anorexia, cachexia, growth impairment, reduced marketability and increased mortality. This study aimed to outline the gut failure produced in this fish-parasite model using a multifaceted approach and to find and validate serum non-lethal markers of gut barrier dysfunction.[Methodology]: Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp-1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits.[Results]: The expression of the tight junction proteins Tjp-1 and Cldn3 was significantly lower in parasitized fish along all the intestine, while no differences were found in E-cadherin labeling. Some parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology studies showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile when compared to the normal absorptive profile of the control animals. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses (PLS-DA), and even separated groups by intensity of infection. Of these compounds, inosine and creatine were identified as relevant and tested with commercial kits in serum samples.[Conclusion]: This study demonstrates the loss of barrier function induced by the enteric parasite E. leei and underlines key markers to differentiate control and infected fish. The untargeted serum metabolomics approach did not reveal specific effects by the parasite, but more a profile typical of absorption dysfunction and anorexia, which are, of course, part of the disease signs.Funding: ParaFishControl H2020 project (634429), Aquaexcel2020 (652831, TNA AE10004-INTEBREAM), AGL2013-48560-R

The Potential of Sewage Sludge to Predict and Evaluate the Human Chemical Exposome

Chemicals are part of our daily lives, and we are exposed to numerous chemicals through multiple pathways. Relevant scientific evidence contributing to the regulation of hazardous chemicals require a holistic approach to assess simultaneous exposure to multiple compounds. Biomonitoring provides an accurate estimation of exposure to chemicals through very complex and costly sampling campaigns. Finding efficient proxies to predict the risk of chemical exposure in humans is an urgent need to cover large areas and populations at a reasonable cost. We conducted an exploratory study to characterize the human chemical exposome in maternal blood and placenta samples of a population-based birth cohort in Barcelona (2018-2021). Ultimate HRMS-based approaches were applied including wide-scope target, suspect, and nontarget screening. Forty-two chemicals were identified including pesticides, personal care products, or industrial compounds, among others, in the range of ng/mL and ng/g. In parallel, sewage sludge from the wastewater treatment plants serving the residence areas of the studied population were also screened, showing correlations with the type and concentrations of chemicals found in humans. Our findings were suggestive for the potential use of sewage sludge as a proxy of the human exposure and its application in early warning systems to prevent bioaccumulation of hazardous chemicals.This work received support from the “La Caixa” Foundation (ID 100010434), fellowship code LCF/BQ/PR20/11770013, and Barcelona Council (Expo-Bar). The BiSC cohort study is funded by the European Research Council (ERC) under Grant Agreement No. 785994 (AIR-NB), and the Health Effects Institute (HEI) with Grant Agreement No. 4959-RFPA15-1/ 18-1 (FRONTIER). IDAEA-CSIC and ISGlobal are Centres of Excellence Severo Ochoa (Spanish Ministry of Science and Innovation).Peer reviewe

Desarrollo de metodologías metabolómicas no dirigidas basadas en cromatografía de líquidos de ultra alto rendimiento acoplado a espectometría de masas de alta resolución en el campo de seguridad alimentaria, sanidad y nutrición

La tesis está basada en el estudio del proceso y flujo de trabajo metabolómico en diferentes ámbitos de trabajo, como la obtención de marcadores para procesos de malnutricion y/o cambios de dieta en doradas, obtención de modelos de autentificación de alimentos y la obtención de biomarcadores indirectos de consumo de canabinoides sintéticos.The doctoral thesis is based on the study of the process and workflow of the metabolomics workflow in the different working scopes, as the ob tention of biomarkers for malnourishment and/or diet changes in sparus aurata, the obtention of food authentication models and the elucidation of indirect synthetic cannabinoid consumption biomarkers.Programa de Doctorat en Cièncie

(Xeno)metabolomics for the evaluation of aquatic organism’s exposure to field contaminated water

Environmental (xeno)metabolomics offers a major advantage compared to other approaches for the evaluation of aquatic organism’s exposure to contaminated water because its allows the simultaneous profiling of the xenometabolome (chemical xenobiotics and their metabolites accumulated in an organism exposed to environmental contaminants) and the metabolome (endogenous metabolites whose levels are altered due to an external stressor). This approach has been widely explored in lab exposure experiments, however in field studies environmental (xeno)metabolomics has only started in the last years. In this review, the papers published so far that have performed different (xeno)metabolomics approaches for the evaluation of aquatic organisms exposed to contaminated water are presented, together with their main achievements, current limitations, and future perspectives. The different analytical methods applied including sample pre-treatment (considering matrix type), platforms used (Nuclear Magnetic Resonance (NMR) and low- or high-resolution Mass Spectrometry (MS or HRMS)), and the analytical strategy (target vs non-target analysis) are discussed. The application of (xeno)metabolomics to provide information of xenobiotics mixtures accumulated in exposed organisms, either in lab or field studies, as well as biomarkers of exposure and biomarkers of effect are debated, and finally, the most commonly metabolic pathways disrupted by chemical contamination are highlighted.This study was supported by the Spanish Ministry of Economy and Competitiveness, State Research Agency, and by the European Union through the European Regional Development Fund through the projects XENOMETABOLOMIC (CTM2015-73179-JIN) (AEI/FEDER/UE) and PLAS-MED (CTM2017-89701-C3-2-R). Authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Groups ICRA-ENV 2017 SGR 1124 and 2017 SGR 01404. Sara Rodriguez-Mozaz acknowledges the Ramon y Cajal program (RYC-2014-16707).Peer reviewe

Blurred lines: Crossing the boundaries between the chemical exposome and the metabolome

The aetiology of every human disease lies in a combination of genetic and environmental factors, each contributing in varying proportions. While genomics investigates the former, a comparable holistic paradigm was proposed for environmental exposures in 2005, marking the onset of exposome research. Since then, the exposome definition has broadened to include a wide array of physical, chemical, and psychosocial factors that interact with the human body and potentially alter the epigenome, the transcriptome, the proteome, and the metabolome. The chemical exposome, deeply intertwined with the metabolome, includes all small molecules originating from diet as well as pharmaceuticals, personal care and consumer products, or pollutants in air and water. The set of techniques to interrogate these exposures, primarily mass spectrometry and nuclear magnetic resonance spectroscopy, are also extensively used in metabolomics. Recent advances in untargeted metabolomics using high resolution mass spectrometry have paved the way for the development of methods able to provide in depth characterisation of both the internal chemical exposome and the endogenous metabolome simultaneously. Herein we review the available tools, databases, and workflows currently available for such work, and discuss how these can bridge the gap between the study of the metabolome and the exposome.CB, LM and HCK received funding from European Union's Horizon 2020 research and innovation programme under grant agreement no. 874583 [ATHLETE]. YX is supported by the President's PhD Scholarship from Imperial College London.Peer reviewe

HRMS-based suspect screening of pharmaceuticals and their transformation products in multiple environmental compartments: An alternative to target analysis?

The comprehensive monitoring of pharmaceutically active compounds (PhACs) in the environment is challenging given the myriad of substances continuously discharged, the increasing number of new compounds being produced (and released), or the variety of the associated human metabolites and transformation products (TPs). Approaches such as high-resolution mass spectrometry (HRMS)-based suspect analysis have emerged to overcome the drawbacks of classical target analytical methods, e.g., restricted chemical coverage. In this study, we assess the readiness of HRMS-based suspect screening to replace or rather complement target methodologies by comparing the performance of both approaches in terms of i) detection of PhACs in various environmental samples (water, sediments, biofilm, fish plasma, muscle and liver) in a field study; ii) PhACs (semi)quantification and iii) prediction of their environmental risks. Our findings revealed that target strategies alone significantly underestimate the variety of PhACs potentially impacting the environment. However, relying solely on suspect strategies can misjudge the presence and risk of low-level but potentially risky PhACs. Additionally, semiquantitative approaches, despite slightly overestimating concentrations, can provide a realistic overview of PhACs concentrations. Hence, it is recommended to adopt a combined strategy that first evaluates suspected threats and subsequently includes the relevant ones in the established target methodologies.This work was supported by the Spanish Inter-Ministerial Science and Technology Commission through the PLAS-MED project (CICYT, CTM2017-89701-C3), the European Union through the European Regional Development Fund (ERDF), and the Spanish State Research Agency of the Spanish Ministry of Science, and Next Generation EU/PRTR (Project PCI2021_121929: ARENA). J.M. Castaño Ortiz acknowledges the predoctoral grant from AGAUR (2019 FI_B 00881REF). P. Gago-Ferrero acknowledges his Ramón y Cajal fellowship (RYC2019-027913-I) from the AEI-MICI. The authors are grateful for funding from the CERCA Programme, Generalitat de Catalunya. The Ultra-Performance Liquid Chromatograph coupled to a triple Quadrupole hybrid Linear Ion Trap Detector (Acquity UPLC-MS QTRAP 5500, Waters-SCIEX) received support from the CERCAGINYS programme, funded by the Spanish Ministry of Science and Innovation. The authors acknowledge the support of the Economy and Knowledge Department of the Catalan Government through a Consolidated Research Group (ICRA-ENV – 2021 SGR 01282).Peer reviewe