Benjamin Piña | CSIC (Consejo Superior de Investigaciones Científicas-Spanish National Research Council) (original) (raw)
Papers by Benjamin Piña
Analytical Chemistry, 2018
Scientific Reports, 2018
Acute exposure to acrylamide (ACR), a type-2 alkene, may lead to a ataxia, skeletal muscles weakn... more Acute exposure to acrylamide (ACR), a type-2 alkene, may lead to a ataxia, skeletal muscles weakness and numbness of the extremities in human and laboratory animals. In the present manuscript, ACR acute neurotoxicity has been characterized in adult zebrafish, a vertebrate model increasingly used in human neuropharmacology and toxicology research. At behavioral level, ACR-treated animals exhibited "depression-like" phenotype comorbid with anxiety behavior. At transcriptional level, ACR induced down-regulation of regeneration-associated genes and up-regulation of oligodendrocytes and reactive astrocytes markers, altering also the expression of genes involved in the presynaptic vesicle cycling. ACR induced also significant changes in zebrafish brain proteome and formed adducts with selected cysteine residues of specific proteins, some of them essential for the presynaptic function. Finally, the metabolomics analysis shows a depletion in the monoamine neurotransmitters, consistent with the comorbid depression and anxiety disorder, in the brain of the exposed fish. Acrylamide (ACR) is a water-soluble alkene widely used in the paper and textile industries, as flocculant in the wastewater treatment and municipal drinking water, as soil conditioner, as chemical grout in tunnels, sewers and wells, in ore processing, and in cosmetics 1,2. Reports of ACR poisoning after occupational or accidental exposure to ACR indicated major symptoms related to polyneuropathy, including lethargy, ataxia, skeletal muscle weakness and numbness of the extremities 3-5. Although early studies suggested that ACR neurotoxicity was associated with central-peripheral distal axonopathy 6 , it is currently well-established that the molecular initiating event (MIE) of ACR neurotoxicity is the disruption of presynaptic vesicle cycling by selectively forming adducts with thiolate sites located on proteins specifically involved in vesicle docking (synaptotagmin, synaptophysin, and syntaxin), vesicle priming (complexin-2), SNARE core dissolution (N-ethylmaleimide sensitive factor), endocytosis (clathrin), neurotransmitter re-uptake (membrane dopamine transporter) and vesicular storage (vesicular monoamine transporter) at the nerve terminals 7. Consistently with this adverse effect of ACR on the presynaptic terminals function, a decrease in the monoamine neurotransmitters serotonin, norepinephrine and dopamine content in rat brain has been reported 8,9. As monoamine depletion is believed to result in depression and anxiety 10 , the potential of ACR to induce these psychological disorders should be conveniently addressed. Zebrafish (Danio rerio) is a vertebrate model increasingly used in biomedical research, including neurotoxicology studies 11-16 , as this animal species exhibits a similar overall nervous system organization to humans and similar neurotransmitter systems, including glutaminergic, cholinergic, serotonergic, dopaminergic, adrenergic, GABAergic, and histaminergic 11,15,17. Recently we characterized the ACR acute neurotoxicity in zebrafish larvae by exposing 5 days post-fertilization (dpf) animals to 1 mM ACR for 3 days 15. The reported results suggest that the adverse outcome pathways behind the ACR acute neurotoxicity in zebrafish larvae are similar to those described in humans and mammalian models 15. However, adult zebrafish is more feasible than larvae for modeling complex brain disorders due to their well-developed central nervous system (CNS) and more complex behaviors 18,19. Moreover, as zebrafish are a less sentient species, the use of adult zebrafish for modeling brain disorders fully meets the 3Rs principles (Replacement, Reduction and Refinement) 18. Adult zebrafish have been used to model affective disorders, including stress-related, anxiety spectrum, depression, post-traumatic and phobic disorders, and multiple behavioral paradigms, resembling well-established rodent tests, have been developed 18,20 .
Scientific reports, Jan 14, 2017
The impact of organic pollutants on oceanic ecosystem functioning is largely unknown. Prochloroco... more The impact of organic pollutants on oceanic ecosystem functioning is largely unknown. Prochlorococcus, the most abundant known photosynthetic organism on Earth, has been suggested to be especially sensible to exposure to organic pollutants, but the sub-lethal effects of organic pollutants on its photosynthetic function at environmentally relevant concentrations and mixtures remain unexplored. Here we show the modulation of the expression of two photosynthetic genes, rbcL (RuBisCO large subunit) and psbA (PSII D1 protein), of oceanic populations of Prochlorococcus from the Atlantic, Indian and Pacific Oceans when exposed to mixtures of organic pollutants consisting of the non-polar fraction of a seawater extract. This mixture included most persistent organic pollutants, semivolatile aromatic-like compounds, and the unresolved complex mixture of hydrocarbons. Prochlorococcus populations in the controls showed the expected diel cycle variations in expression of photosynthetic genes. Ho...
Molecular and Cellular Biology, 1990
Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thy... more Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexameth...
PLOS ONE, 2015
Newly designed primers targeting rbcL (CO 2 fixation), psbA (photosystem II) and rnpB (reference)... more Newly designed primers targeting rbcL (CO 2 fixation), psbA (photosystem II) and rnpB (reference) genes were used in qRT-PCR assays to assess the photosynthetic capability of natural communities of Prochlorococcus, the most abundant photosynthetic organism on Earth and a major contributor to primary production in oligotrophic oceans. After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths. Sequence and quantitative analyses of the corresponding amplicons showed the presence of highlight (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences. Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons. Laboratory exposure of Prochlorococcus MED4 (HL) and MIT9313 (LL) strains to organic pollutants (PAHs and organochlorine compounds) showed a decrease of rbcL transcript abundances, and of the rbcL to psbA ratios for both strains. We propose this technique as a convenient assay to evaluate effects of environmental stressors, including pollution, on the oceanic Prochlorococcus photosynthetic function.
Freshwater Biology, 2009
1. Sediment extracts from 63 European remote lakes were tested for the presence of ligands of the... more 1. Sediment extracts from 63 European remote lakes were tested for the presence of ligands of the aryl hydrocarbon receptor (AhR), using a yeast-based bioassay. The lakes belong to different European mountain regions in Central Norway, Scotland, Piedmont (Italy), Tyrol (Austria and Italy) and the Julian Alps (Slovenia). 2. AhR ligand activity varied about 40-fold among the examined lakes, with Scotland having the highest proportion of lakes with high AhR ligand activity, whereas samples with low AhR ligand activity predominate in the Julian Alps. 3. AhR ligand activity in sediment samples correlated with the concentration of several environmentally relevant compounds, including polycyclic aromatic hydrocarbons, hexachlorobenzene and several polychlorobiphenyl ether congeners. These correlations suggest an anthropogenic origin for the detected AhR ligands, rather than being generated from natural processes. 4. We conclude that pollutants deposited in remote lakes by atmospheric processes are active AhR ligands at the concentrations found in these lakes. Given the high toxicity associated with ectopic activation of AhR in different animal systems, this activity may represent a significant risk for the exposed fauna.
Bulletin of Environmental Contamination and Toxicology, 2009
Estrogenic activity of kraft pulp mill effluents (P. radiata, E. globulus and mixed-50% E. globul... more Estrogenic activity of kraft pulp mill effluents (P. radiata, E. globulus and mixed-50% E. globulus and 50% P. radiata) was evaluated by the yeast estrogen screen assay. The estrogenic activity values were relatively low, ranking between 1.475 and 0.383 ng/L of EE2 eq. (Estrogenic equivalent of 17 a-ethynylestradiol), where the highest value corresponds to the E. globulus effluent and the lowest value to the P. radiata effluent. Analysis by solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) of chemical compounds present in all three effluents detected at least five major groups of organic compounds, corresponding to fatty acids, hydrocarbons, phenols, sterols and triterpenes. Comparison of analytical and biological data suggests that sterols could be the cause of the estrogenic activity in the evaluated effluent.
This work was supported by the European Research Council under the European Union's Seventh F... more This work was supported by the European Research Council under the European Union's Seventh Framework Programme (FP/ 2007-2013)/ERC Grant Agreement n. 320737. Some part of this study was also supported by grants from the Spanish Ministry of Economy and Competitiveness (CTQ2014-56777-R) and the Spanish Ministry of Science, Innovation and University (RTI2018-096175-B-I00). RM was supported by a FPU predoctoral fellow from the Spanish Ministry of Education and Science (ref. FPU15/03332). IF was supported by a FPI predoctoral fellow from the Spanish Ministry of Economy and Competitiveness (FPI-MICINN BES-2015-075023). We thank Juan Carlos Navarro and Josefina Casas for his valuable advice and expertise in the optimization of the thin-layer chromatography technique and liquid chromatography/ mass spectrometry, respectively.
Journal of Biophotonics, 2018
Aquatic Toxicology, 2019
Understanding the mode of action of the different pollutants in human and wildlife health is a ke... more Understanding the mode of action of the different pollutants in human and wildlife health is a key step in environmental risk assessment. The aim of this study was to determine signatures that could link morphological phenotypes to the toxicity mechanisms of four Endocrine Disrupting Chemicals (EDCs): bisphenol A (BPA), perfluorooctanesulfonate potassium salt (PFOS), tributyltin chloride (TBT), and 17-ß-estradiol (E2). Zebrafish (Danio rerio) eleutheroembryos were exposed from 2 to 5 dpf to a wide range of BPA, PFOS, TBT and E2 concentrations. At the end of the exposures several morphometric features were assessed. Common and non-specific effects on larvae pigmentation or swim bladder area were observed after exposures to all compounds. BPA specifically induced yolk sac malabsorption syndrome and altered craniofacial parameters, whereas PFOS had specific effects on the notochord formation presenting higher rates of scoliosis and kyphosis. The main effect of E2 was an increase in the body length of the exposed eleutheroembryos. In the case of TBT, main alterations on the morphological traits were related to developmental delays. When integrating all morphometrical parameters, BPA showed the highest rates of malformations in terms of equilethality, followed by PFOS and, distantly, by TBT and E2. In the case of BPA and PFOS, we were able to relate our results with effects on the transcriptome and metabolome, previously reported. We propose that methodized morphometric analyses in zebrafish embryo model can be used as an inexpensive and easy screening tool to predict modes of action of a wide-range number of contaminants.
Science of The Total Environment, 2019
The escape response evoked by vibrational stimuli and its habituation, essential behaviors for fi... more The escape response evoked by vibrational stimuli and its habituation, essential behaviors for fish larvae survival, can be altered by neurotoxic environmental pollutants commonly found in our aquatic ecosystems. In this study we have analyzed the suitability of the Vibrational Startle Response Assay (VSRA) to obtain mechanistic information about the mode of action (MoA) of the chemicals impairing the escape response and its habituation. As a proof of concept, the pathophysiological mechanisms behind the action of two common neurotoxic pesticides, chlorpyrifos-oxon (CPO) and imidacloprid, over their effects on arousal and habituation of the escape response were studied by using pharmacological antagonists of the nicotinic and muscarinic acetylcholine receptors, mecamylamine (MCA) and scopolamine, respectively. Furthermore, potential changes in the neurotransmitter profile were analyzed. Results revealed that whereas the effect of CPO on arousal was mainly mediated by the activation of nAChRs, its effect on habituation was mainly mediated by mAChRs. On the other hand, imidacloprid only affected larvae arousal which was found to be mediated by a cholinergic independent mechanism. No association between behavioral effects on arousal or habituation in affected larvae was found with their corresponding neurotransmitter profile. These results confirm the suitability of VSRA to provide mechanistic information about the potential MoA of neuroactive compounds.
Frontiers in Marine Science, 2018
Search for articles, people, events and more. Login Register Login Register About Submit Journals... more Search for articles, people, events and more. Login Register Login Register About Submit Journals Research Topics Search for articles, people, events and more.
Environmental pollution (Barking, Essex : 1987), 2018
Despite the abundant literature on the adverse effects of Bisphenol A (BPA) as endocrine disrupto... more Despite the abundant literature on the adverse effects of Bisphenol A (BPA) as endocrine disruptor, its toxicity mechanisms are still poorly understood. We present here a study of its effects on the zebrafish eleutheroembryo transcriptome at concentrations ranging from 0.1 to 4 mg L, this latter representing the lowest observed effect concentration (LOEC) found in our study at three different macroscopical endpoints (survival, hatching and swim bladder inflation). Multivariate data analysis methods identified both monotonic and bi-phasic patterns of dose-dependent responses. Functional analyses of genes affected by BPA exposure suggest an interaction of BPA with different signaling pathways, being the estrogenic and retinoid receptors two likely targets. In addition, we identified an apparently unrelated inhibitory effect on, among others, visual function genes. We interpret our data as the result of a sum of underlying, independent molecular mechanisms occurring simultaneously at t...
Methods in molecular biology (Clifton, N.J.), 2018
The genome revolution represents a complete change on our view of biological systems. The quantit... more The genome revolution represents a complete change on our view of biological systems. The quantitative determination of changes in all major molecular components of the living cells, the "omics" approach, opened whole new fields for all health sciences. Genomics, transcriptomics, proteomics, metabolomics, and others, together with appropriate prediction and modeling tools, will mark the future of developmental toxicity assessment both for wildlife and humans. This is especially true for disciplines, like teratology, which rely on studies in model organisms, as studies at lower levels of organization are difficult to implement. Rodents and frogs have been the favorite models for studying human reproductive and developmental disorders for decades. Recently, the study of the development of zebrafish embryos (ZE) is becoming a major alternative tool to adult animal testing. ZE intrinsic characteristics makes this model a unique system to analyze in vivo developmental alteratio...
Scientific reports, Jan 24, 2018
Tryptophan hydroxylase (TRH) is the rate limiting enzyme in the serotonin synthesis. CRISPR-Cas9 ... more Tryptophan hydroxylase (TRH) is the rate limiting enzyme in the serotonin synthesis. CRISPR-Cas9 technology was used to generate seven indel TRH mutants in Daphnia magna. Mono-allelic indel TRH-/+ clones showed normal levels of serotonin, measured by both immunohistochemistry and mass spectrometry (LC-MS/MS), whereas bi-allelic indel TRH-/- clones showed no detectable levels of serotonin. Life history and behavioral responses of TRH-/- clones showed the anti-phenotype of those exposed to selective serotonin reuptake inhibitors (SSRI). Mutants lacking serotonin grew less and hence reproduced latter, produced smaller clutches of smaller offspring and responded to a greater extent to light than wild type individuals. Mono-allelic indel TRH-/+ individuals showed the intermediate phenotype. The SSRI fluoxetine enhanced offspring production in all clones and decreased the response to light only in those clones having serotonin, thus indication that behavioral effects of this drug in D. ma...
Molecular and Cellular Biology, 1997
The ADA genes encode factors which are proposed to function as transcriptional coactivators. Here... more The ADA genes encode factors which are proposed to function as transcriptional coactivators. Here we describe the cloning, sequencing, and initial characterization of a novel ADA gene, ADA1. Similar to the previously isolated ada mutants, ada1 mutants display decreases in transcription from various reporters. Furthermore, ADA1 interacts with the other ADAs in the ADA/GCN5 complex as demonstrated by partial purification of the complex and immunoprecipitation experiments. We estimate that the complex has a molecular mass of approximately 2 MDa. Previously, it had been demonstrated that ada5 mutants displayed more severe phenotypic defects than the other ada mutants (G. A. Marcus, J. Horiuchi, N. Silverman, and L. Guarente, Mol. Cell. Biol. 16:3197-3205, 1996; S. M. Roberts and F. Winston, Mol. Cell. Biol. 16:3206-3213, 1996). ada1 mutants display defects similar to those of ada5 mutants and different from those of the other mutants with respect to promoters affected, inositol auxotrop...
Bulletin of Environmental Contamination and Toxicology, 2016
Scientific Reports, 2016
Disruption of specific metabolic pathways constitutes the mode of action of many known toxicants ... more Disruption of specific metabolic pathways constitutes the mode of action of many known toxicants and it is responsible for the adverse phenotypes associated to human genetic defects. Conversely, many industrial applications rely on metabolic alterations of diverse microorganisms, whereas many therapeutic drugs aim to selectively disrupt pathogens' metabolism. In this work we analyzed metabolic changes induced by auxotrophic starvation conditions in yeast in a non-targeted approach, using onedimensional proton Nuclear Magnetic Resonance spectroscopy (1 H NMR) and chemometric analyses. Analysis of the raw spectral datasets showed specific changes linked to the different stages during unrestricted yeast growth, as well as specific changes linked to each of the four tested starvation conditions (L-methionine, L-histidine, L-leucine and uracil). Analysis of changes in concentrations of more than 40 metabolites by Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) showed the normal progression of key metabolites during lag, exponential and stationary unrestricted growth phases, while reflecting the metabolic blockage induced by the starvation conditions. In this case, different metabolic intermediates accumulated over time, allowing identification of the different metabolic pathways specifically affected by each gene disruption. This synergy between NMR metabolomics and molecular biology may have clear implications for both genetic diagnostics and drug development. Metabolomics aims to identify the specific cellular processes undergoing in biological organisms by the identification and quantitation of dozens to thousands metabolites with high-throughput techniques, by using a non-aprioristic approach 1. Metabolomic analyses have been performed in many organisms, including human and mammalian tissues 2,3 , different animal species, both vertebrates 4 and invertebrates 5 , plants 6 , and microorganisms, both Eukaryotes (yeasts 7 , protists 8) and Prokaryotes (bacteria 9 , archea 10). Among the eukaryotic microorganisms, the yeast Saccharomyces cerevisiae is widely used in many biological fields, such as biotechnology 11 or food industry 12 , and it constitutes an excellent model organism for metabolomics 13 and other "omic" approaches 14. We present here an NMR analysis of the metabolome variations induced by auxotrophic starvation in yeast, which occurs when a strain lacking specific genes (in this case, HIS3, LEU2, MET15 and URA3, also called genetic markers) is confronted with a medium devoid of one or more of the essential metabolites it can no longer synthesize (L-histidine, L-leucine, L-methionine and/or uracil, respectively). Reports of metabolic disruption in yeast have previously focused on the triggered proteins 15 or on the transcriptomic 16 evidences. The general consensus is that starvation is controlled through the RAS/protein kinase A (PKA) and TOR pathways 17 that mediate the transcriptional, translational, and metabolic state of the cell. However, most studies analyzed the effects of the depletion in a requiring nutrient, like carbon, nitrogen, phosphate or
Analytical Chemistry, 2018
Scientific Reports, 2018
Acute exposure to acrylamide (ACR), a type-2 alkene, may lead to a ataxia, skeletal muscles weakn... more Acute exposure to acrylamide (ACR), a type-2 alkene, may lead to a ataxia, skeletal muscles weakness and numbness of the extremities in human and laboratory animals. In the present manuscript, ACR acute neurotoxicity has been characterized in adult zebrafish, a vertebrate model increasingly used in human neuropharmacology and toxicology research. At behavioral level, ACR-treated animals exhibited "depression-like" phenotype comorbid with anxiety behavior. At transcriptional level, ACR induced down-regulation of regeneration-associated genes and up-regulation of oligodendrocytes and reactive astrocytes markers, altering also the expression of genes involved in the presynaptic vesicle cycling. ACR induced also significant changes in zebrafish brain proteome and formed adducts with selected cysteine residues of specific proteins, some of them essential for the presynaptic function. Finally, the metabolomics analysis shows a depletion in the monoamine neurotransmitters, consistent with the comorbid depression and anxiety disorder, in the brain of the exposed fish. Acrylamide (ACR) is a water-soluble alkene widely used in the paper and textile industries, as flocculant in the wastewater treatment and municipal drinking water, as soil conditioner, as chemical grout in tunnels, sewers and wells, in ore processing, and in cosmetics 1,2. Reports of ACR poisoning after occupational or accidental exposure to ACR indicated major symptoms related to polyneuropathy, including lethargy, ataxia, skeletal muscle weakness and numbness of the extremities 3-5. Although early studies suggested that ACR neurotoxicity was associated with central-peripheral distal axonopathy 6 , it is currently well-established that the molecular initiating event (MIE) of ACR neurotoxicity is the disruption of presynaptic vesicle cycling by selectively forming adducts with thiolate sites located on proteins specifically involved in vesicle docking (synaptotagmin, synaptophysin, and syntaxin), vesicle priming (complexin-2), SNARE core dissolution (N-ethylmaleimide sensitive factor), endocytosis (clathrin), neurotransmitter re-uptake (membrane dopamine transporter) and vesicular storage (vesicular monoamine transporter) at the nerve terminals 7. Consistently with this adverse effect of ACR on the presynaptic terminals function, a decrease in the monoamine neurotransmitters serotonin, norepinephrine and dopamine content in rat brain has been reported 8,9. As monoamine depletion is believed to result in depression and anxiety 10 , the potential of ACR to induce these psychological disorders should be conveniently addressed. Zebrafish (Danio rerio) is a vertebrate model increasingly used in biomedical research, including neurotoxicology studies 11-16 , as this animal species exhibits a similar overall nervous system organization to humans and similar neurotransmitter systems, including glutaminergic, cholinergic, serotonergic, dopaminergic, adrenergic, GABAergic, and histaminergic 11,15,17. Recently we characterized the ACR acute neurotoxicity in zebrafish larvae by exposing 5 days post-fertilization (dpf) animals to 1 mM ACR for 3 days 15. The reported results suggest that the adverse outcome pathways behind the ACR acute neurotoxicity in zebrafish larvae are similar to those described in humans and mammalian models 15. However, adult zebrafish is more feasible than larvae for modeling complex brain disorders due to their well-developed central nervous system (CNS) and more complex behaviors 18,19. Moreover, as zebrafish are a less sentient species, the use of adult zebrafish for modeling brain disorders fully meets the 3Rs principles (Replacement, Reduction and Refinement) 18. Adult zebrafish have been used to model affective disorders, including stress-related, anxiety spectrum, depression, post-traumatic and phobic disorders, and multiple behavioral paradigms, resembling well-established rodent tests, have been developed 18,20 .
Scientific reports, Jan 14, 2017
The impact of organic pollutants on oceanic ecosystem functioning is largely unknown. Prochloroco... more The impact of organic pollutants on oceanic ecosystem functioning is largely unknown. Prochlorococcus, the most abundant known photosynthetic organism on Earth, has been suggested to be especially sensible to exposure to organic pollutants, but the sub-lethal effects of organic pollutants on its photosynthetic function at environmentally relevant concentrations and mixtures remain unexplored. Here we show the modulation of the expression of two photosynthetic genes, rbcL (RuBisCO large subunit) and psbA (PSII D1 protein), of oceanic populations of Prochlorococcus from the Atlantic, Indian and Pacific Oceans when exposed to mixtures of organic pollutants consisting of the non-polar fraction of a seawater extract. This mixture included most persistent organic pollutants, semivolatile aromatic-like compounds, and the unresolved complex mixture of hydrocarbons. Prochlorococcus populations in the controls showed the expected diel cycle variations in expression of photosynthetic genes. Ho...
Molecular and Cellular Biology, 1990
Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thy... more Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexameth...
PLOS ONE, 2015
Newly designed primers targeting rbcL (CO 2 fixation), psbA (photosystem II) and rnpB (reference)... more Newly designed primers targeting rbcL (CO 2 fixation), psbA (photosystem II) and rnpB (reference) genes were used in qRT-PCR assays to assess the photosynthetic capability of natural communities of Prochlorococcus, the most abundant photosynthetic organism on Earth and a major contributor to primary production in oligotrophic oceans. After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths. Sequence and quantitative analyses of the corresponding amplicons showed the presence of highlight (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences. Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons. Laboratory exposure of Prochlorococcus MED4 (HL) and MIT9313 (LL) strains to organic pollutants (PAHs and organochlorine compounds) showed a decrease of rbcL transcript abundances, and of the rbcL to psbA ratios for both strains. We propose this technique as a convenient assay to evaluate effects of environmental stressors, including pollution, on the oceanic Prochlorococcus photosynthetic function.
Freshwater Biology, 2009
1. Sediment extracts from 63 European remote lakes were tested for the presence of ligands of the... more 1. Sediment extracts from 63 European remote lakes were tested for the presence of ligands of the aryl hydrocarbon receptor (AhR), using a yeast-based bioassay. The lakes belong to different European mountain regions in Central Norway, Scotland, Piedmont (Italy), Tyrol (Austria and Italy) and the Julian Alps (Slovenia). 2. AhR ligand activity varied about 40-fold among the examined lakes, with Scotland having the highest proportion of lakes with high AhR ligand activity, whereas samples with low AhR ligand activity predominate in the Julian Alps. 3. AhR ligand activity in sediment samples correlated with the concentration of several environmentally relevant compounds, including polycyclic aromatic hydrocarbons, hexachlorobenzene and several polychlorobiphenyl ether congeners. These correlations suggest an anthropogenic origin for the detected AhR ligands, rather than being generated from natural processes. 4. We conclude that pollutants deposited in remote lakes by atmospheric processes are active AhR ligands at the concentrations found in these lakes. Given the high toxicity associated with ectopic activation of AhR in different animal systems, this activity may represent a significant risk for the exposed fauna.
Bulletin of Environmental Contamination and Toxicology, 2009
Estrogenic activity of kraft pulp mill effluents (P. radiata, E. globulus and mixed-50% E. globul... more Estrogenic activity of kraft pulp mill effluents (P. radiata, E. globulus and mixed-50% E. globulus and 50% P. radiata) was evaluated by the yeast estrogen screen assay. The estrogenic activity values were relatively low, ranking between 1.475 and 0.383 ng/L of EE2 eq. (Estrogenic equivalent of 17 a-ethynylestradiol), where the highest value corresponds to the E. globulus effluent and the lowest value to the P. radiata effluent. Analysis by solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) of chemical compounds present in all three effluents detected at least five major groups of organic compounds, corresponding to fatty acids, hydrocarbons, phenols, sterols and triterpenes. Comparison of analytical and biological data suggests that sterols could be the cause of the estrogenic activity in the evaluated effluent.
This work was supported by the European Research Council under the European Union's Seventh F... more This work was supported by the European Research Council under the European Union's Seventh Framework Programme (FP/ 2007-2013)/ERC Grant Agreement n. 320737. Some part of this study was also supported by grants from the Spanish Ministry of Economy and Competitiveness (CTQ2014-56777-R) and the Spanish Ministry of Science, Innovation and University (RTI2018-096175-B-I00). RM was supported by a FPU predoctoral fellow from the Spanish Ministry of Education and Science (ref. FPU15/03332). IF was supported by a FPI predoctoral fellow from the Spanish Ministry of Economy and Competitiveness (FPI-MICINN BES-2015-075023). We thank Juan Carlos Navarro and Josefina Casas for his valuable advice and expertise in the optimization of the thin-layer chromatography technique and liquid chromatography/ mass spectrometry, respectively.
Journal of Biophotonics, 2018
Aquatic Toxicology, 2019
Understanding the mode of action of the different pollutants in human and wildlife health is a ke... more Understanding the mode of action of the different pollutants in human and wildlife health is a key step in environmental risk assessment. The aim of this study was to determine signatures that could link morphological phenotypes to the toxicity mechanisms of four Endocrine Disrupting Chemicals (EDCs): bisphenol A (BPA), perfluorooctanesulfonate potassium salt (PFOS), tributyltin chloride (TBT), and 17-ß-estradiol (E2). Zebrafish (Danio rerio) eleutheroembryos were exposed from 2 to 5 dpf to a wide range of BPA, PFOS, TBT and E2 concentrations. At the end of the exposures several morphometric features were assessed. Common and non-specific effects on larvae pigmentation or swim bladder area were observed after exposures to all compounds. BPA specifically induced yolk sac malabsorption syndrome and altered craniofacial parameters, whereas PFOS had specific effects on the notochord formation presenting higher rates of scoliosis and kyphosis. The main effect of E2 was an increase in the body length of the exposed eleutheroembryos. In the case of TBT, main alterations on the morphological traits were related to developmental delays. When integrating all morphometrical parameters, BPA showed the highest rates of malformations in terms of equilethality, followed by PFOS and, distantly, by TBT and E2. In the case of BPA and PFOS, we were able to relate our results with effects on the transcriptome and metabolome, previously reported. We propose that methodized morphometric analyses in zebrafish embryo model can be used as an inexpensive and easy screening tool to predict modes of action of a wide-range number of contaminants.
Science of The Total Environment, 2019
The escape response evoked by vibrational stimuli and its habituation, essential behaviors for fi... more The escape response evoked by vibrational stimuli and its habituation, essential behaviors for fish larvae survival, can be altered by neurotoxic environmental pollutants commonly found in our aquatic ecosystems. In this study we have analyzed the suitability of the Vibrational Startle Response Assay (VSRA) to obtain mechanistic information about the mode of action (MoA) of the chemicals impairing the escape response and its habituation. As a proof of concept, the pathophysiological mechanisms behind the action of two common neurotoxic pesticides, chlorpyrifos-oxon (CPO) and imidacloprid, over their effects on arousal and habituation of the escape response were studied by using pharmacological antagonists of the nicotinic and muscarinic acetylcholine receptors, mecamylamine (MCA) and scopolamine, respectively. Furthermore, potential changes in the neurotransmitter profile were analyzed. Results revealed that whereas the effect of CPO on arousal was mainly mediated by the activation of nAChRs, its effect on habituation was mainly mediated by mAChRs. On the other hand, imidacloprid only affected larvae arousal which was found to be mediated by a cholinergic independent mechanism. No association between behavioral effects on arousal or habituation in affected larvae was found with their corresponding neurotransmitter profile. These results confirm the suitability of VSRA to provide mechanistic information about the potential MoA of neuroactive compounds.
Frontiers in Marine Science, 2018
Search for articles, people, events and more. Login Register Login Register About Submit Journals... more Search for articles, people, events and more. Login Register Login Register About Submit Journals Research Topics Search for articles, people, events and more.
Environmental pollution (Barking, Essex : 1987), 2018
Despite the abundant literature on the adverse effects of Bisphenol A (BPA) as endocrine disrupto... more Despite the abundant literature on the adverse effects of Bisphenol A (BPA) as endocrine disruptor, its toxicity mechanisms are still poorly understood. We present here a study of its effects on the zebrafish eleutheroembryo transcriptome at concentrations ranging from 0.1 to 4 mg L, this latter representing the lowest observed effect concentration (LOEC) found in our study at three different macroscopical endpoints (survival, hatching and swim bladder inflation). Multivariate data analysis methods identified both monotonic and bi-phasic patterns of dose-dependent responses. Functional analyses of genes affected by BPA exposure suggest an interaction of BPA with different signaling pathways, being the estrogenic and retinoid receptors two likely targets. In addition, we identified an apparently unrelated inhibitory effect on, among others, visual function genes. We interpret our data as the result of a sum of underlying, independent molecular mechanisms occurring simultaneously at t...
Methods in molecular biology (Clifton, N.J.), 2018
The genome revolution represents a complete change on our view of biological systems. The quantit... more The genome revolution represents a complete change on our view of biological systems. The quantitative determination of changes in all major molecular components of the living cells, the "omics" approach, opened whole new fields for all health sciences. Genomics, transcriptomics, proteomics, metabolomics, and others, together with appropriate prediction and modeling tools, will mark the future of developmental toxicity assessment both for wildlife and humans. This is especially true for disciplines, like teratology, which rely on studies in model organisms, as studies at lower levels of organization are difficult to implement. Rodents and frogs have been the favorite models for studying human reproductive and developmental disorders for decades. Recently, the study of the development of zebrafish embryos (ZE) is becoming a major alternative tool to adult animal testing. ZE intrinsic characteristics makes this model a unique system to analyze in vivo developmental alteratio...
Scientific reports, Jan 24, 2018
Tryptophan hydroxylase (TRH) is the rate limiting enzyme in the serotonin synthesis. CRISPR-Cas9 ... more Tryptophan hydroxylase (TRH) is the rate limiting enzyme in the serotonin synthesis. CRISPR-Cas9 technology was used to generate seven indel TRH mutants in Daphnia magna. Mono-allelic indel TRH-/+ clones showed normal levels of serotonin, measured by both immunohistochemistry and mass spectrometry (LC-MS/MS), whereas bi-allelic indel TRH-/- clones showed no detectable levels of serotonin. Life history and behavioral responses of TRH-/- clones showed the anti-phenotype of those exposed to selective serotonin reuptake inhibitors (SSRI). Mutants lacking serotonin grew less and hence reproduced latter, produced smaller clutches of smaller offspring and responded to a greater extent to light than wild type individuals. Mono-allelic indel TRH-/+ individuals showed the intermediate phenotype. The SSRI fluoxetine enhanced offspring production in all clones and decreased the response to light only in those clones having serotonin, thus indication that behavioral effects of this drug in D. ma...
Molecular and Cellular Biology, 1997
The ADA genes encode factors which are proposed to function as transcriptional coactivators. Here... more The ADA genes encode factors which are proposed to function as transcriptional coactivators. Here we describe the cloning, sequencing, and initial characterization of a novel ADA gene, ADA1. Similar to the previously isolated ada mutants, ada1 mutants display decreases in transcription from various reporters. Furthermore, ADA1 interacts with the other ADAs in the ADA/GCN5 complex as demonstrated by partial purification of the complex and immunoprecipitation experiments. We estimate that the complex has a molecular mass of approximately 2 MDa. Previously, it had been demonstrated that ada5 mutants displayed more severe phenotypic defects than the other ada mutants (G. A. Marcus, J. Horiuchi, N. Silverman, and L. Guarente, Mol. Cell. Biol. 16:3197-3205, 1996; S. M. Roberts and F. Winston, Mol. Cell. Biol. 16:3206-3213, 1996). ada1 mutants display defects similar to those of ada5 mutants and different from those of the other mutants with respect to promoters affected, inositol auxotrop...
Bulletin of Environmental Contamination and Toxicology, 2016
Scientific Reports, 2016
Disruption of specific metabolic pathways constitutes the mode of action of many known toxicants ... more Disruption of specific metabolic pathways constitutes the mode of action of many known toxicants and it is responsible for the adverse phenotypes associated to human genetic defects. Conversely, many industrial applications rely on metabolic alterations of diverse microorganisms, whereas many therapeutic drugs aim to selectively disrupt pathogens' metabolism. In this work we analyzed metabolic changes induced by auxotrophic starvation conditions in yeast in a non-targeted approach, using onedimensional proton Nuclear Magnetic Resonance spectroscopy (1 H NMR) and chemometric analyses. Analysis of the raw spectral datasets showed specific changes linked to the different stages during unrestricted yeast growth, as well as specific changes linked to each of the four tested starvation conditions (L-methionine, L-histidine, L-leucine and uracil). Analysis of changes in concentrations of more than 40 metabolites by Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) showed the normal progression of key metabolites during lag, exponential and stationary unrestricted growth phases, while reflecting the metabolic blockage induced by the starvation conditions. In this case, different metabolic intermediates accumulated over time, allowing identification of the different metabolic pathways specifically affected by each gene disruption. This synergy between NMR metabolomics and molecular biology may have clear implications for both genetic diagnostics and drug development. Metabolomics aims to identify the specific cellular processes undergoing in biological organisms by the identification and quantitation of dozens to thousands metabolites with high-throughput techniques, by using a non-aprioristic approach 1. Metabolomic analyses have been performed in many organisms, including human and mammalian tissues 2,3 , different animal species, both vertebrates 4 and invertebrates 5 , plants 6 , and microorganisms, both Eukaryotes (yeasts 7 , protists 8) and Prokaryotes (bacteria 9 , archea 10). Among the eukaryotic microorganisms, the yeast Saccharomyces cerevisiae is widely used in many biological fields, such as biotechnology 11 or food industry 12 , and it constitutes an excellent model organism for metabolomics 13 and other "omic" approaches 14. We present here an NMR analysis of the metabolome variations induced by auxotrophic starvation in yeast, which occurs when a strain lacking specific genes (in this case, HIS3, LEU2, MET15 and URA3, also called genetic markers) is confronted with a medium devoid of one or more of the essential metabolites it can no longer synthesize (L-histidine, L-leucine, L-methionine and/or uracil, respectively). Reports of metabolic disruption in yeast have previously focused on the triggered proteins 15 or on the transcriptomic 16 evidences. The general consensus is that starvation is controlled through the RAS/protein kinase A (PKA) and TOR pathways 17 that mediate the transcriptional, translational, and metabolic state of the cell. However, most studies analyzed the effects of the depletion in a requiring nutrient, like carbon, nitrogen, phosphate or