Evaluation of Th1/Th2, regulatory cytokines and transcriptional factor FoxP3 in sheep immunized with a partially protective and non-protective vaccine and challenged with Fasciola hepatica.

Gene expression for Th1/Th2 cytokines (IL-4 and IFN-É£), regulatory cytokines (TGF-ß and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-É£ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-É£ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-ß in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.

Bioavailability of flumequine and diclofenac in mice exposed to a metal-drug chemical cocktail. Evaluation of the protective role of selenium.

BACKGROUND AND PURPOSE: Organisms, including humans, are subjected to the simultaneous action of a wide variety of pollutants, the effects of which should not be considered in isolation, as many synergies and antagonisms have been found between many of them. Therefore, this work proposes an in vivo study to evaluate the effect of certain metal contaminants on the bioavailability and metabolism of pharmacologically active compounds. Because the most frequent entry vector is through ingestion, the influence of the gut microbiota and the possible protective effects of selenium has been additionally evaluated. EXPERIMENTAL APPROACH: A controlled exposure experiment in mammals (Mus musculus) to a "chemical cocktail" consisting of metals and pharmaceuticals (diclofenac and flumequine). The presence of selenium has also been evaluated as an antagonist. Mouse plasma samples were measured by UPLC-QTOF. A targeted search of 48 metabolites was also performed. KEY RESULTS: Metals significantly affected the FMQ plasma levels when the gut microbiota was depleted. Hydroxy FMQ decreased if metals were present. Selenium minimized this decrease. The 3-hydroxy DCF metabolite was not found in any case. Changes in some metabolic pathways are discussed. CONCLUSIONS AND IMPLICATIONS: The presence of metals in the mouse diet as well as the prior treatment of mice with an antibiotic mixture (Abxs), which deplete the gut microbiota, has a decisive effect on the bioavailability and metabolism of the tested pharmaceuticals and dietary selenium minimize some of their effects.

Omics insights into the responses to dietary selenium.

Selenium is a well-known health-relevant element related with cancer chemoprevention, neuroprotective roles, beneficial in diabetes, and in several infectious diseases, among others. It is naturally present in some foods, but deficiency in people led to the production of nutraceuticals, supplements, and functional food enriched in this element. There is a U-shaped link between selenium levels and health and a narrow range between toxic and essential levels, and thus, supplementation should be performed carefully. Omics methodologies have become valuable approaches to delve into the responses of dietary selenium in mammals that allowed a deeper knowledge about the metabolism of this element as well as its biological role. In this review, we discuss omics approaches from the workflows to their applications that has been previously used to deep insight into the metabolism of dietary selenium. There is a special focus on selenoproteins, metabolomics responses in blood and tissues (e.g., brain, reproductive organs, etc.) as well as the impact on gut microbiota and its metabolites profile. Thus, we mainly reviewed heteroatom-tagged proteomics, metallomics, metabolomics, and metataxonomics, usually combined with transcriptomics, genomics, and other molecular methods.

Proteomic analysis of the hepatic response to a pollutant mixture in mice. The protective action of selenium.

Metals and pharmaceuticals contaminate water and food worldwide, forming mixtures where they can interact to enhance their individual toxicity. Here we use a shotgun proteomic approach to evaluate the toxicity of a pollutant mixture (PM) of metals (As, Cd, Hg) and pharmaceuticals (diclofenac, flumequine) on mice liver proteostasis. These pollutants are abundant in the environment, accumulate in the food chain, and are toxic to humans primarily through oxidative damage. Thus, we also evaluated the putative antagonistic effect of low-dose dietary supplementation with the antioxidant trace element selenium. A total of 275 proteins were affected by PM treatment. Functional analyses revealed an increased abundance of proteins involved in the integrated stress response that promotes translation, the inflammatory response, carbohydrate and lipid metabolism, and the sustained expression of the antioxidative response mediated by NRF2. As a consequence, a reductive stress situation arises in the cell that inhibits the RICTOR pathway, thus activating the early stage of autophagy, impairing xenobiotic metabolism, and potentiating lipid biosynthesis and steatosis. PM exposure-induced hepato-proteostatic alterations were significantly reduced in Se supplemented mice, suggesting that the use of this trace element as a dietary supplement may at least partially ameliorate liver damage caused by exposure to environmental mixtures.

The role of selenium in shaping mice brain metabolome and selenoproteome through the gut-brain axis by combining metabolomics, metallomics, gene expression, and amplicon sequencing.

Selenium (Se) is a trace element crucial for human health. Recently, the impact of Se supplementation on gut microbiota has been pointed out as well as its influence on the expression of certain selenoproteins and gut metabolites. This study aims to elucidate the link between Se supplementation, brain selenoproteins and brain metabolome as well as the possible connection with the gut-brain axis. To this end, an in vivo study with 40 BALB/c mice was carried out. The study included conventional (n=20) and mice model with microbiota depleted by antibiotics (n=20) under a regular or Se supplemented diet. Brain selenoproteome was determined by a transcriptomic/gene expression profile, while brain metabolome and gut microbiota profiles were accomplished by untargeted metabolomics and amplicon sequencing, respectively. The total content of Se in brain was also determined. The selenoproteins genes Dio and Gpx isoenzymes, SelenoH, SelenoI, SelenoT, SelenoV, and SelenoW and 31 metabolites were significantly altered in the brain after Se supplementation in conventional mice, while 11 selenoproteins and 26 metabolites were altered in microbiota depleted mice. The main altered brain metabolites were related to glyoxylate and dicarboxylate metabolism, amino acid metabolism, and gut microbiota that have been previously related with the gut-brain axis (e.g., members of Lachnospiraceae and Ruminococcaceae families). Moreover, specific associations were determined between brain selenoproteome and metabolome, which correlated with the same bacteria, suggesting an intertwined mechanism. Our results demonstrated the effect of Se on brain metabolome through specific selenoproteins gene expression and gut microbiota.

Fasciola hepatica primoinfections and reinfections in sheep drive distinct Th1/Th2/Treg immune responses in liver and hepatic lymph node at early and late stages.

The expression of proinflammatory (IL-1ß, IFN-γ, TNF-α) and regulatory (IL-10, TGF-ß, IL-4) cytokines, as well as the transcription factor FoxP3, was quantified in the liver and hepatic lymph node (HLN) of sheep primoinfected and reinfected with Fasciola hepatica at early (4, 8 and 16 days post-infection [dpi]) and late (100 dpi) stages. The liver exerted a Th2 immune response at very early stages after the primoinfection with F. hepatica that induced the downregulation of IFN-γ, followed by a Th1/Th2/Treg response although the late stages were characterised by the expression of Th1/Th2 immune mediators. Contrarily, in reinfected sheep a robust mixed Th1/Th2/Treg immune response was found at very early stages meanwhile at late stages we observed a Th2/Treg immune response overcoming the expression of Th1 immune mediators. However, the HLN displayed a completely different Th1/Th2/Treg expression profile compared to the liver. Primoinfections with F. hepatica in HLN induced a mixed Th1/Th2/Treg environment from early stages, establishing a Th2 immune response at a late stage. However, the reinfected sheep exerted a Th2 immune response at early stages led by the IL-4 expression in opposition to the Th1/Th2/Treg found in the liver, meanwhile at late stages the HLN of reinfected sheep exerted a mixed Th1/Th2/Treg immune response. This is the first work publishing the expression of immune mediators in the liver and HLN from reinfected sheep with F. hepatica. The study of the immune responses exerted by the natural host in the target organs directly implied in the development of F. hepatica are crucial to better understand the immunopathogenesis of the fasciolosis being a key factor to develop effective vaccines.

A selenium-enriched diet helps to recover liver function after antibiotic administration in mice.

Antibiotic (Abx) treatments or inadvertent exposure to Abx-contaminated food and water can adversely affect health. Many studies show strong correlations between Abx and liver damage pointing to gut dysbiosis as a contributing factor because the gut microbiota (GM) forms a complex network with liver. Selenium (Se) is a beneficial micronutrient able to shape the composition of the GM. We analyzed here the ability of a low dose (120 µg/kg bodyweight/day) Se-enriched diet to ameliorate the effects of a 7-day intervention with an Abx-cocktail over the global health and the homeostasis of cholesterol and bile acids in the mouse liver. We found that Se restored lipid metabolism preventing the increased synthesis and accumulation of cholesterol caused by Abx treatment. Integrating these results with previous metataxonomic and metabolomic data in same mice, we conclude that part of the effect of Se against liver dysfunction (cholesterol and bile acids metabolism and transport) could be mediated by the GM. We provide data that contribute to a more complete view of the molecular mechanisms underlying the beneficial action of Se on health, pointing to a possible use of low doses of Se as a functional food additive (prebiotic) to prevent the negative effects of antibiotics.

Evaluation of candidate reference genes for quantitative real-time PCR normalization in blood from red deer developing antlers.

Sexual selection favors male traits that increase their ability to monopolize the breeding access to several females. Deer antlers are cranial appendages that regenerate annually in males. Throughout life, the phenology of antler growth advances and antler mass increases until the stag reaches, between 8 and 10 years old, maximum body mass and highest reproductive success. The molecular mechanisms of antler development are of great interest in both evolutionary and regenerative medicine studies. To minimize errors in the assessment of gene expression levels by qRT-PCR, we analyzed the stability of a panel of eight candidate reference genes and concluded that qRT-PCR normalization to three stable genes is strongly convenient in experiments performed in red deer antler blood. To validate our proposal, we compared the expression level of three genes linked to red deer antler growth (ANXA2, APOD and TPM1) in fifteen male red deer classified as young (up to 4 years old) and adults (4-6 years old). Our data confirms that B2M, ACTB and RPLP0 are valuable reference genes for future gene expression studies in red deer antler blood, which would provide increased insight into the effects of intrinsic factors that determine antler development in red deer.

Selenium supplementation influences mice testicular selenoproteins driven by gut microbiota.

Selenium is a well-known essential element with important roles in human reproductive health mainly due to its antioxidant character. This study aimed to investigate the potential role of selenoproteins on gut microbiota and male reproductive health. A new assay for the absolute quantification of selenoproteins in testicular tissue based on two dimensional chromatography with inductively coupled plasma mass spectrometry was performed for the first time. The gut microbiota profile was obtained by 16S rRNA gene sequencing. Numerous associations were found between testicular selenoproteins and gut microbiota (e.g. Mucispirillum, related with sperm activity and testosterone, was associated with glutathione peroxidase (GPx) and selenoalbumin (SeAlb), while Escherichia/Shigella, related to sex hormones, correlated with GPx, selenoprotein P (SelP) and SeAlb). The effects of Se-supplementation on testicular selenoproteins only occur in conventional mice, suggesting a potential selenoproteins-microbiota interplay that underlies testicular function. The selenoproteins GPx and SelP have been quantified for the first time in the testicles, and the novel identification of SeAlb, a protein with nonspecifically incorporated Se, is also reported. These findings demonstrate the significant impact of Se-supplementation on gut microbiota and male reproductive health. In addition, the analytical methodology applied here in selenoprotein quantification in testicular tissue opens new possibilities to evaluate their role in gut microbiota and reproductive health axis.

Untargeted Gut Metabolomics to Delve the Interplay between Selenium Supplementation and Gut Microbiota.

Selenium (Se) is an essential trace element with important health roles due to the antioxidant properties of selenoproteins. To analyze the interplay between Se and gut microbiota, gut metabolomic profiles were determined in conventional (C) and microbiota depleted mice (Abx) after Se-supplementation (Abx-Se) by untargeted metabolomics, using an analytical multiplatform based on GC-MS and UHPLC-QTOF-MS (MassIVE ID MSV000087829). Gut microbiota profiling was performed by 16S rRNA gene amplicon sequencing. Significant differences in the levels of about 70% of the gut metabolites determined, including fatty acyls, glycerolipids, glycerophospholipids, and steroids, were found in Abx-Se compared to Abx, and only 30% were different between Abx-Se and C, suggesting an important effect of Se-supplementation on Abx mice metabolism. At genus level, the correlation analysis showed strong associations between metabolites and gut bacterial profiles. Likewise, higher abundance of Lactobacillus spp., a potentially beneficial genus enriched after Se-supplementation, was associated with higher levels of prenol lipids, phosphatidylglycerols (C-Se), steroids and diterpenoids (Abx-Se), and also with lower levels of fatty acids (Abx-Se). Thus, we observed a crucial interaction between Se intake-microbiota-metabolites, although further studies to clarify the specific mechanisms are needed. This is the first study about untargeted gut metabolomics after microbiota depletion and Se-supplementation.

Omic methodologies for assessing metal(-loid)s-host-microbiota interplay: A review.

Omic methodologies have become key analytical tools in a wide number of research topics such as systems biology, environmental analysis, biomedicine or food analysis. They are especially useful when they are combined providing a new perspective and a holistic view of the analytical problem. Methodologies for microbiota analysis have been mostly focused on genome sequencing. However, information provided by these metagenomic studies is limited to the identification of the presence of genes, taxa and their inferred functionality. To achieve a deeper knowledge of microbial functionality in health and disease, especially in dysbiosis conditions related to metal and metalloid exposure, the introduction of additional meta-omic approaches including metabolomics, metallomics, metatranscriptomics and metaproteomics results essential. The possible impact of metals and metalloids on the gut microbiota and their effects on gut-brain axis (GBA) only begin to be figured out. To this end new analytical workflows combining powerful tools are claimed such as high resolution mass spectrometry and heteroatom-tagged proteomics for the absolute quantification of metal-containing biomolecules using the metal as a "tag" in a sensitive and selective detector (e.g. ICP-MS). This review focus on current analytical methodologies related with the analytical techniques and procedures available for metallomics and microbiota analysis with a special attention on their advantages and drawbacks.

Proteomic profile of the effects of low-dose bisphenol A on zebrafish ovaries.

Human exposure to bisphenol-A (BPA) is largely unavoidable because BPA is an environmental contaminant found in soil, water, food and indoor dust. The safety of authorized BPA amounts in consumer products is under question because new studies have reported adverse effects of BPA at doses far below that previously established by the NOAEL (50 µg/kg per day). To protect public health, the consequences of low-dose BPA exposure in different organs and organismal functions must be further studied to generate relevant data. This study attempted to investigate the effects and potential molecular mechanisms of short-term exposure to 1 µg/L BPA on zebrafish ovarian follicular development. We observed only minor changes at the histopathological level with a small (3 %) increase in follicular atresia. However, a shotgun proteomics approach indicated deep alterations in BPA-exposed ovarian cells, including induction of the oxidative stress response, metabolic shifts and degradome perturbations, which could drive oocytes towards premature maturation. Based on these results, it could be suggested that inadvertent exposure to small concentrations of BPA on a continuous basis causes alteration in biological processes that are essential for healthy reproduction.

Impact of Antibiotic-Induced Depletion of Gut Microbiota and Selenium Supplementation on Plasma Selenoproteome and Metal Homeostasis in a Mice Model.

Selenium (Se) is a micronutrient involved in important health functions and it has been suggested to shape gut microbiota. Limited information on Se assimilation by gut microbes and the possible link with selenoproteins are available. For this purpose, conventional and gut microbiota-depleted BALB/c mice were fed a Se-supplemented diet. The absolute quantification of mice plasma selenoproteins was performed for the first time using heteroatom-tagged proteomics. The gut microbiota profile was analyzed by 16S rRNA gene sequencing. Se-supplementation modulated the concentration of the antioxidant glutathione peroxidase and the Se-transporter selenoalbumin as well as the metal homeostasis, being influenced by microbiota disruption, which suggests an intertwined mechanism. Se also modulated microbiota diversity and richness and increased the relative abundance of some health-relevant taxa (e.g., families Christensenellaceae, Ruminococcaceae, and Lactobacillus genus). This study demonstrated the potential beneficial effects of Se on gut microbiota, especially after antibiotic-treatment and the first associations between specific bacteria and plasma selenoproteins.

Assessment of pharmaceutical mixture (ibuprofen, ciprofloxacin and flumequine) effects to the crayfish Procambarus clarkii: A multilevel analysis (biochemical, transcriptional and proteomic approaches).

The knowledge about the effects of pharmaceuticals on aquatic organisms has been increasing in the last decade. However, due to the variety of compounds presents in the aquatic medium, exposure scenarios and exposed organisms, there are still many gaps in the knowledge on how mixtures of such bioactive compounds affect exposed non target organisms. The crayfish Procambarus clarkii was used to analyze the toxicity effects of mixtures of ciprofloxacin, flumequine and ibuprofen at low and high concentrations (10 and 100 µg/L) over 21 days of exposure and to assess the recovery capacity of the organism after a depuration phase following exposure during additional 7 days in clean water. The crayfish accumulated the three compounds throughout the entire exposure in the hepatopancreas. The exposure to the mixture altered the abundance of proteins associated with different cells functions such as biotransformation and detoxification processes (i.e. catalase and glutathione transferase), carbohydrate metabolism and immune responses. Additionally changes in expression of genes encoding antioxidant enzymes and in activity of the corresponding enzymes (i.e. superoxide dismutase, glutathione peroxidase and glutathione transferase) were reported. Alterations at different levels of biological organization did not run in parallel under all circumstances and can be related to changes in the redox status of the target tissue. No differences were observed between control and exposed organisms for most of selected endpoints after a week of depuration, indicating that exposure to the drug mixture did not produce permanent damage in the hepatopancreas of P. clarkii.

Proteomic profile and protease activity in the skin mucus of greater amberjack (Seriola dumerili) infected with the ectoparasite Neobenedenia girellae - An immunological approach.

Skin mucus is considered the first barrier against diseases in fish. The skin mucus protein profile of the greater amberjack (Seriola dumerili) and its changes due to experimental infection with Neobenedenia girellae were studied by combining 2-DE-MS/MS and gel-free LC-MS/MS proteomic approaches. The 2-DE results led to the identification of 69 and 55 proteins in noninfected and infected fish, respectively, and revealed that keratins were specifically cleaved in parasitized fish. Therefore, the skin mucus of the infected fish showed a higher protease activity due to, at least in part, an increase of metal-dependent protease and serine-type protease activities. Additionally, through a gel-free LC-MS/MS analysis, 1377 and 1251 different proteins were identified in the skin mucus of healthy and parasitized fish, respectively. The functional analysis of these proteins demonstrated a statistical overrepresentation of ribosomal proteins (a well-known source of antimicrobial peptides) in N. girellae-infected fish. In contrast, the components of membranes and protein transport GO categories were underrepresented after infection. Immune system process-related proteins constituted 2.5% of the total skin mucosal proteins. Among these skin mucosal proteins, 14 and 15 proteins exclusive to non-parasitized and parasitized fish were found, respectively, including specific serine-type proteases and metalloproteases in the parasitized fish. Moreover, the finding of tryptic peptides exclusive to some bacterial genera, obtained by gel-free LC-MS/MS, allowed us to construct a preliminary map of the microbiota living in the mucus of S. dumerili, with Pseudomonas and Paracoccus the most represented genera in both noninfected and infected fish.

Pedro Ximenez sun-dried grape must: a dietary supplement for a healthy longevity.

Polyphenols in red wine are bioactive compounds with positive effects on health and disease prevention. White grape musts and wines have a lower concentration of phenolic compounds compared to the red ones and are therefore considered less beneficial to health. In Andalucía, a region located in the South West of Spain, Pedro Ximenez white grapes are desiccated under the sun for a week before they are pressed and the juice (must) is obtained. This ancient procedure increases the variety and content of polyphenols present in the Pedro Ximenez must (PXM). We incorporated PXM into the daily diet of aged Mus spretus mice (24 months) and investigated their properties by comparing several parameters determined in these old mice with those measured in young mice (two months old). Biochemical, histological, and transcriptional analyses indicated that PXM exhibited potent antioxidant properties, promoted the normalization of the biotransforming ability of several cytochromes, i.e., the P450 family, in the liver, and regularized hepatic apoptosis, promoting proliferation instead. Our data indicated that PXM possesses a profound ability to promote liver regeneration in terms of both the structure and the function, thus contributing to a healthy aging process.

Protective effect of Pedro-Ximénez must against p,p'-DDE-induced liver damages in aged Mus spretus mice.

Aging is characterized by deterioration of biomolecules and impaired stress responses that make the elderly especially vulnerable to environmental pollutants. The pesticide p,p'-DDE is a DDT derivative that generates great concern because of its wide distribution and its harmful effects on both human health and the environment. We analyzed here the biological responses elicited by p,p'-DDE exposure in the liver of aged Mus spretus mice. Data demonstrate that the elderly constitute a population especially sensitive to this noxious environmental pollutant. We also demonstrated here that the daily consumption of sun-dried Pedro Ximénez (PX) white-grape must (PXM) protects the liver of aged mice from both the age and the damages caused by p,p'-DDE exposure. The PXM activity was exerted through the restoration of the hepatic metabolisms of lipids and carbohydrates and, probably, is a consequence of the ability of this polyphenol-rich mixture to avoid oxidative stress. Nutritional interventions including PXM, which ameliorates the effects of unavoidable exposure to pesticides in our food, are helpful tools that can help elderly populations to enjoy a healthy and expanded lifetime.

Metabolic Impairments Caused by a "Chemical Cocktail" of DDE and Selenium in Mice Using Direct Infusion Triple Quadrupole Time-of-Flight and Gas Chromatography-Mass Spectrometry.

Among organic contaminants, pesticides are one of the most important groups of chemicals due to their persistent character and toxicity. However, the biological systems are exposed to a complex environment in which the contaminants can interact in a synergistic/antagonistic fashion, and for this reason, the study of "chemical cocktails" is of great interest to fully understand the final biological effect. In this way, selenium is known for its antagonistic action against several toxicants. In this paper, metabolic impairments caused by the joint exposure of p,p'-dichloro diphenyl trichloroethane (DDE) and selenium (Se) have been issued for the first time. A metabolomic workflow was applied to mice fed DDE and DDE with Se diet, on the basis of the complementary use of two organic mass spectrometric techniques, combining direct infusion mass spectrometry (DI-ESI-QqQ-TOF MS) and gas chromatography-mass spectrometry (GC-MS). The results show a good classification between the studied groups caused by about 70 altered metabolites in the liver, kidney, or brain, including the pathways of energy metabolism, degradation of phospholipidic membrane, ß-oxidation, and oxidative stress, which confirm the potential of combined metabolomic platforms in environmental studies.

Germination and Early Seedling Development in Quercus ilex Recalcitrant and Non-dormant Seeds: Targeted Transcriptional, Hormonal, and Sugar Analysis.

Seed germination and early seedling development have been studied in the recalcitrant species Quercus ilex using targeted transcriptional, hormonal, and sugar analysis. Embryos and seedlings were collected at eight morphologically defined developmental stages, S0-S7. A typical triphasic water uptake curve was observed throughout development, accompanied by a decrease in sucrose and an increase in glucose and fructose. Low levels of abscisic acid (ABA) and high levels of gibberellins (GAs) were observed in mature seeds. Post-germination, indole-3-acetic acid (IAA), increased, whereas GA remained high, a pattern commonly observed during growth and development. The abundance of transcripts from ABA-related genes was positively correlated with the changes in the content of the phytohormone. Transcripts of the drought-related genes Dhn3 and GolS were more abundant at S0, then decreased in parallel with increasing water content. Transcripts for Gapdh, and Nadh6 were abundant at S0, supporting the occurrence of an active metabolism in recalcitrant seeds at the time of shedding. The importance of ROS during germination is manifest in the high transcript levels for Sod and Gst, found in mature seeds. The results presented herein help distinguish recalcitrant (e.g., Q. ilex) seeds from their orthodox counterparts. Our results indicate that recalcitrance is established during seed development but not manifest until germination (S1-S3). Post-germination the patterns are quite similar for both orthodox and recalcitrant seeds.

Metabolomic alterations and oxidative stress are associated with environmental pollution in Procambarus clarkii.

Soils contaminated by toxic metallic elements from agricultural activities raise grave concern about their potential risk to human health through direct intake, bioaccumulation through the food chain, and their impacts on ecological systems. We have measured here the lipid and protein oxidation status and used metabolomic methodologies to identify and characterize the changes caused by metal pollution exposure in the digestive glands and gills of Procambarus clarkii, the red swamp crayfish. Specimens captured at two sites with intensive agriculture practices using diverse types of agrochemicals, located in the borders of Doñana Natural Park, were compared to ones caught in the core of the Park, a proven non-polluted place. As a highly metabolically active organ, the digestive gland accumulated more metallic elements than the gills and was consequently more affected at the metabolic level. Results also indicate that chronic pollution exposure generates oxidative stress and mitochondrial dysfunction that imposes a metabolic shift to enhanced aerobic glycolysis and lipid metabolism alteration. The integration of metabolomics with previous proteomic data gives a comprehensive vision of the metabolic disorders caused by chronic metal exposure to P. clarkii and identifies potential biomarkers useful for routine risk assessment of the aquatic ecosystems health.