RESUMO
In the coming decade, Europe will dedicate billions of euros to the necessary research and innovation (R&I) to support a transition to safe and sustainable food systems. EU Agencies, individually and even more so collectively, can make a difference in supporting the European research agenda. EU Agencies are knowledge centres, bringing together know-how to inform policy makers. EU Agencies that have traditionally dealt with aspects of human health, animal health, plant health and ecosystem health in silos, now need to take a broader perspective and move towards a One Health (OH) approach. In this paper, the authors highlight the need for more transdisciplinary cooperation in support of the One Health approach, identify challenges in strengthening interagency cooperation and provide recommendations to address them. EU Agencies are natural bridges between the scientific community and policy-makers and need to dedicate time and effort in fostering this dialogue, e.g. by engaging with relevant initiatives, research projects and European Partnerships. Research generates evidence that can be used also for regulatory science, in support of policy-making. It is urgent to define transdisciplinary research needs and formulate a One Health research agenda. This would be facilitated by establishing transdisciplinary One Health Research & Innovation governance, both at national and EU levels. Ongoing large initiatives, such as the One Health European Joint Programme, have demonstrated that active dialogue with national ministries and EU agencies is beneficial for all parties. Involvement of EU Agencies in the programming of the EU Research Framework programmes is beneficial, because of their regulatory science perspective, their expertise and current or future tasks on research topics. It is timely for EU Agencies to demonstrate leadership in moving the One Health agenda forward and it is encouraging that EU Agencies have committed to establish a cross-agency task force on One Health.
RESUMO
In 2013 the Organisation for Economic Co-operation and Development (OECD) test guideline (236) for fish embryo acute toxicity (FET) was adopted. It determines the acute toxicity of chemicals to embryonic fish. Previous studies show a good correlation of FET with the standard acute fish toxicity (AFT) test; however, the potential of the FET test to predict AFT, which is required by the Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) regulation (EC 1907/2006) and the Classification, Labelling and Packaging (CLP) Regulation (EC 1272/2008), has not yet been fully clarified. In 2015 the European Chemicals Agency (ECHA) requested that a consultant perform a scientific analysis of the applicability of FET to predict AFT. The purpose was to compare the toxicity of substances to fish embryos and to adult fish, and to investigate whether certain factors (e.g., physicochemical properties, modes of action, or chemical structures) could be used to define the applicability boundaries of the FET test. Given the limited data availability, the analysis focused on organic substances. The present critical review summarizes the main findings and discusses regulatory application of the FET test under REACH. Given some limitations (e.g., neurotoxic mode of action) and/or remaining uncertainties (e.g., deviation of some narcotic substances), it has been found that the FET test alone is currently not sufficient to meet the essential information on AFT as required by the REACH regulation. However, the test may be used within weight-of-evidence approaches together with other independent, relevant, and reliable sources of information. The present review also discusses further research needs that may overcome the remaining uncertainties and help to increase acceptance of FET as a replacement for AFT in the future. For example, an increase in the availability of data generated according to OECD test guideline 236 may provide evidence of a higher predictive power of the test. Environ Toxicol Chem 2018;37:657-670. © 2017 SETAC.
Assuntos
Embrião não Mamífero/metabolismo , Peixes/embriologia , Controle Social Formal , Testes de Toxicidade Aguda , Animais , Organização para a Cooperação e Desenvolvimento Econômico , Reprodutibilidade dos TestesRESUMO
REACH(1) entered into force in June 2007 and has hence been operational for six years. With the first registration deadline in November 2010, the European Chemicals Agency (ECHA(2)) has received a large amount of scientific and administrative information related to chemical substances. In order to understand what type of data on ecotoxicity endpoints was submitted under the REACH framework a detailed analysis of the availability and content of relevant information was performed. To avoid unnecessary testing, the REACH Regulation provides registrants with the possibility to build testing strategies and to adopt the standard information requirements based on the specific conditions listed in the regulation. The types of information submitted by registrants to fulfil data requirements for aquatic, sediment and terrestrial toxicity endpoints were analysed. The REACH database analysis confirms large differences in the availability of experimental aquatic versus sediment and soil ecotoxicity data. Information requirements on aquatic organisms are mainly covered by experimental data, while those for sediment and soil are mostly waived.
Assuntos
Bases de Dados de Compostos Químicos , Política Ambiental , Poluição Ambiental/legislação & jurisprudência , Substâncias Perigosas/toxicidade , Ecotoxicologia , Testes de ToxicidadeRESUMO
This paper summarises the aquatic ecotoxicity data submitted in the REACH(1) registration dossiers and disseminated by the European Chemicals Agency (ECHA(2)). The analysis describes both the guidelines and the species mostly used by registrants. Non-OECD guidelines have been extensively used, in particular in covering of fish and aquatic invertebrate studies, but the main concern is that in 22-36% of the cases, depending on the endpoint, no information on the methodological approach and potential equivalences to test guidelines has been provided. As expected, most studies were conducted with those species typically used in laboratory ecotoxicity testing; nevertheless, the database provides a broad range of available species, covering the most relevant taxonomic groups for both freshwater and marine systems, although most are just occasionally used. This species diversity is essential for higher tier testing strategies, including the use of Species Sensitivity Distribution approaches. The assessment suggests that collecting available information has been the main approach used by registrants to fulfil their REACH information requirements for this first REACH registration deadline. Many studies are disclosed for the first time, and all are available through searchable web tools.
Assuntos
Substâncias Perigosas/toxicidade , Testes de Toxicidade/métodos , Poluentes Químicos da Água/toxicidade , Poluição Química da Água/legislação & jurisprudência , Animais , União Europeia , Peixes , Invertebrados , Medição de Risco , Poluição Química da Água/estatística & dados numéricosRESUMO
This paper summarises the terrestrial ecotoxicity data submitted in the REACH registration dossiers and disseminated by ECHA. The analysis describes both the guidelines and the test species mostly used by registrants. REACH information requirements in relation to the effects on terrestrial organisms encompass three trophic levels; invertebrates, plants and micro-organisms, and the study of both long and short-term exposure. The results observed for soil invertebrates showed that on one hand there was a clear prevalence for testing on the species recommended by the standard test guidelines. On the other, the reporting included a large variety of species from very different families, demonstrating the feasibility for conducting toxicity tests on a number of relevant groups e.g. for species sensitivity distribution approaches. Standard toxicity testing with terrestrial plants under REACH follows a different approach and requires simultaneous testing on several species, using the same test conditions, adapted to each species, if needed. The test methods used to conduct the studies were only reported for 30% of cases. The most extensively reported test guidelines for terrestrial plants were OECD 208, ISO 11269-1 and ISO 11269-1. Information requirements for soil micro-organisms under REACH are related to the analysis of functional endpoints instead of on species or taxa. As recommended in REACH, OECD 216 and OECD 217 were the most often used test methods for soil micro-organisms. But overall, the test method was reported for only about 40% of the experimental studies. Moreover, it is noted that information on potential effects on soil micro-organisms is available for a limited number of REACH registered substances. The assessment suggests that providing waiving justifications and collecting available information, which in many cases might be well used for covering standard REACH data requirements, have been the main approaches used by registrants for the first REACH registration deadline.
Assuntos
Monitoramento Ambiental/legislação & jurisprudência , Política Ambiental , Sedimentos Geológicos/química , Substâncias Perigosas/toxicidade , Testes de Toxicidade/normas , Ecotoxicologia/legislação & jurisprudência , Ecotoxicologia/normas , Monitoramento Ambiental/métodos , Medição de Risco , Poluentes Químicos da Água/toxicidadeRESUMO
For the first REACH registration deadline, companies have submitted registrations with relevant hazard and exposure information for substances at the highest tonnage level (above 1000 tonnes per year). At this tonnage level, information on the long-term toxicity of a substance to sediment organisms is required. There are a number of available test guidelines developed and accepted by various national/international organisations, which can be used to investigate long-term toxicity to sediment organisms. However instead of testing, registrants may also use other options to address toxicity to sediment organisms, e.g. weight of evidence approach, grouping of substances and read-across approaches, as well as substance-tailored exposure-driven testing. The current analysis of the data provided in ECHA database focuses on the test methods applied and the test organisms used in the experimental studies to assess long-term toxicity to sediment organisms. The main guidelines used for the testing of substances registered under REACH are the OECD guidelines and OSPAR Protocols on Methods for the Testing of Chemicals used in the Offshore Oil Industry: "Part A: A Sediment Bioassay using an Amphipod Corophium sp." explaining why one of the mostly used test organisms is the marine amphipod Corophium sp. In total, testing results with at least 40 species from seven phyla are provided in the database. However, it can be concluded that the ECHA database does not contain a high enough number of available experimental data on toxicity to sediment organisms for it to be used extensively by the scientific community (e.g. for development of non-testing methods to predict hazards to sediment organisms).
Assuntos
Monitoramento Ambiental/legislação & jurisprudência , Política Ambiental , Sedimentos Geológicos/química , Substâncias Perigosas/toxicidade , Testes de Toxicidade/normas , Ecotoxicologia/legislação & jurisprudência , Ecotoxicologia/normas , Monitoramento Ambiental/métodos , Medição de Risco , Poluentes Químicos da Água/toxicidadeRESUMO
The effects of copper exposure at five different concentrations on the freshwater alga Chlamydomonas reinhardtii were studied at the biochemical (metabolite), physiological (uptake kinetics and flow cytometry) and growth level. Changes at the physiological level were evident at the lowest exposure concentration while effects on the metabolome and on growth only occurred at the highest copper concentration tested. Flow cytometry revealed the presence of higher reactive oxygen species concentrations in algae exposed to higher copper concentrations and this was confirmed by a significant reduction in glutathione levels as part of the metabolomics assessment. Cu(2+) uptake kinetic data contributed information on possible mechanisms of copper toxicity, revealing that, a decrease in efflux pumping might be at the basis of an increased metal accumulation at higher exposure levels. This study demonstrates the value of using a comparative approach to investigating the mechanisms of toxicity rather than focusing on a single level of organization or effect.
Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Cobre/toxicidade , Chlamydomonas reinhardtii/citologia , Cobre/química , Relação Dose-Resposta a Droga , Citometria de Fluxo , Cinética , Relação Estrutura-AtividadeRESUMO
Cellular energy allocation (CEA) is a measure of the energy status of an organism. The effects of Cd and Zn (reproduction EC(50)s and EC(90)s) on the total energy budget of Enchytraeus albidus (Oligochaeta) were assessed through CEA determination, over periods of time from 0 to 8 d. Results showed reduction on the energy reserves for both metals after 2 d exposure. Lipids were the first reserves to be used and carbohydrates were reduced exclusively after Cd exposure. Electron transport system (ETS) activities were enhanced, suggesting increased metabolism and higher energy requirements for metal detoxification. This was supported by previous results at transcription level, where an up-regulation of genes involved in the mitochondrial oxidative phosphorylation was verified. Additionally, the reduction of CEA may be related with the decrease on the reproductive output. These results showed the relevance of integrating various endpoints, which enabled an overview of various processes and to unravel mechanisms of action of chemicals.
Assuntos
Cádmio/metabolismo , Oligoquetos/fisiologia , Poluentes do Solo/metabolismo , Zinco/metabolismo , Animais , Metabolismo Energético/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Oligoquetos/genética , Reprodução/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The effects of cadmium were assessed in the freshwater alga Chlamydomonas reinhardtii. Algae were exposed to concentrations of 0, 8.1 or 114.8 µM of cadmium and growth rates, gene transcription and metabolite profiles were examined after 48 and 72 h of exposure. In algae exposed to 8.1 µM Cd, several genes were differentially transcribed after 48 h but no adverse growth related effects were detected. A transient effect on both gene transcription patterns and metabolite profiles could be discerned after 48 h of exposure but the majority of these changes disappeared after 72 h. In contrast, all effects were more pronounced at the 114.8 µM cadmium exposure. Here growth was clearly reduced and transcription of a large number of genes involved in oxidative stress defense mechanisms was differentially increased. Metabolites involved in the glutathione synthesis pathway (an important antioxidant defense) were also affected but the effects of cadmium were found to be more pronounced at the transcript level than in the metabolome, suggesting that the former exhibits greater sensitivity toward cadmium exposure.
Assuntos
Cádmio/toxicidade , Chlamydomonas reinhardtii/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Cádmio/análise , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Água/químicaRESUMO
Metal ecotoxicity to soil organisms (for example, in enchytraeids) has been addressed mainly by assessing effects on survival and reproduction, but very little is known about the underlying molecular mechanisms of responses. The main purpose of the present study was to assess and compare the transcriptional responses of Enchytraeus albidus to an essential (Zn) and a nonessential (Cd) metal. Exposure was performed with two concentrations with a known effect on reproduction (effective concentration for 50% [EC50] and 90% [EC90]) at three time points (2, 4, and 8 d). Results showed that transcriptional responses were influenced by exposure duration but, independently of that, the mechanisms of response to Cd and Zn were consistently different. Both metals affected pathways related to the regulation of gene expression, calcium homeostasis, and cellular respiration. Mechanisms of toxicity that were exclusively associated with Cd exposures were the inhibition of DNA repair and the impairment of ubiquitin-mediated proteolysis. The microarray for E. albidus was a useful tool for detecting molecular pathways affected by metal exposures. Transcriptional responses strongly correlated with known mechanisms of Cd and Zn responses in other organisms, suggesting cross-species conserved mechanisms of action. It should be highlighted not only that the authors could retrieve mechanistic information but also that genes responded within 2 to 8 d of exposure. This represents an additional advantage of using such molecular endpoints as a complement to the traditional, more time-consuming endpoints.
Assuntos
Cádmio/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Zinco/toxicidade , Animais , Ecotoxicologia/métodos , Expressão Gênica , Biblioteca Genômica , Oligoquetos/efeitos dos fármacos , Oligoquetos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reprodução/efeitos dos fármacos , Solo , Transcrição GênicaRESUMO
BACKGROUND: Molecular mechanisms of response to pesticides are scarce and information on such responses from soil invertebrates is almost inexistent. Enchytraeus albidus (Oligochaeta) is a standard soil ecotoxicology model species for which effects of many pesticides are known on survival, reproduction and avoidance behaviour. With the recent microarray development additional information can be retrieved on the molecular effects. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed to investigate the transcription responses of E. albidus when exposed to three pesticides - dimethoate (insecticide), atrazine (herbicide) and carbendazim (fungicide) - in a range of concentrations that inhibited reproduction by 10%, 20%, 50% and 90% (EC(10), EC(20), EC(50) and EC(90), respectively). The goal of this study was to further identify key biological processes affected by each compound and if dose-related. All three pesticides significantly affected biological processes like translation, regulation of the cell cycle or general response to stress. Intracellular signalling and microtubule-based movement were affected by dimethoate and carbendazim whereas atrazine affected lipid and steroid metabolism (also by dimethoate) or carbohydrate metabolism (also by carbendazim). Response to DNA damage/DNA repair was exclusively affected by carbendazim. CONCLUSIONS: Changes in gene expression were significantly altered after 2 days of exposure in a dose-related manner. The mechanisms of response were comparable with the ones for mammals, suggesting across species conserved modes of action. The present results indicate the potential of using gene expression in risk assessment and the advantage as early markers.
Assuntos
Atrazina/toxicidade , Benzimidazóis/toxicidade , Carbamatos/toxicidade , Dimetoato/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Oligoquetos/genética , Oligoquetos/fisiologia , Animais , Análise por Conglomerados , Exposição Ambiental , Perfilação da Expressão Gênica , Oligoquetos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodução/efeitos dos fármacos , Reprodução/genéticaRESUMO
Enchytraeus albidus (Oligochaeta) is an ecologically relevant species used as standard test organisms for risk assessment. Effects of stressors in this species are commonly determined at the population level using reproduction and survival as endpoints. The assessment of transcriptomic responses can be very useful e.g. to understand underlying mechanisms of toxicity with gene expression fingerprinting. In the present paper the following is being addressed: 1) development of suppressive subtractive hybridization (SSH) libraries enriched for differentially expressed genes after metal and pesticide exposures; 2) sequencing and characterization of all generated cDNA inserts; 3) development of a publicly available genomic database on E. albidus. A total of 2100 Expressed Sequence Tags (ESTs) were isolated, sequenced and assembled into 1124 clusters (947 singletons and 177 contigs). From these sequences, 41% matched known proteins in GenBank (BLASTX, e-value ≤ 10(-5)) and 37% had at least one Gene Ontology (GO) term assigned. In total, 5.5% of the sequences were assigned to a metabolic pathway, based on KEGG. With this new sequencing information, an Agilent custom oligonucleotide microarray was designed, representing a potential tool for transcriptomic studies. EnchyBASE (http://bioinformatics.ua.pt/enchybase/) was developed as a web freely available database containing genomic information on E. albidus and will be further extended in the near future for other enchytraeid species. The database so far includes all ESTs generated for E. albidus from three cDNA libraries. This information can be downloaded and applied in functional genomics and transcription studies.
Assuntos
Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Metais Pesados/toxicidade , Oligoquetos/genética , Praguicidas/toxicidade , Animais , Sequência de Bases , Análise por Conglomerados , Biologia Computacional , DNA Complementar/genética , Internet , Análise em Microsséries , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNARESUMO
Reporter gene assays are commonly used in applied toxicology to measure the transcription of genes involved in toxic responses. In these reporter gene assays, transgenic cells are used, which contain a promoter-operator region of a gene of interest fused to a reporter gene. The transcription of the gene of interest can be measured by the detection of the reporter protein. Chloramphenicol acetyl transferase (CAT) is frequently used as a reporter protein in mammalian reporter gene assays. Although CAT can be measured by different detection systems, like enzymatic and immune assays, most of these tests are expensive, time-consuming and labor-intensive. The excellent characteristics of phages, like their high affinity and specificity, their fast, cheap and animal-friendly manufacturing process with low batch-to-batch variations and their stability, make them appropriate as alternatives for antibodies in detection assays. Therefore, in this study single-chain variable fragment (scFv) phages were selected with affinity for CAT. Several scFv phages were selected that showed affinity towards CAT in a screening ELISA. Surface plasmon resonance analyses showed that the tested scFv phages have an affinity for CAT with a dissociation constant (K(d)) around 1 µM. The selected scFv phages in this study could be used as capture elements in a highly sensitive sandwich ELISA to detect CAT concentration as low as 0.1 ng ml⻹ or 4 pM. This low detection limit demonstrates the potential of the scFv phages as an alternative for capturing antibodies in a highly sensitive detection test to measure CAT concentrations in reporter gene assays.
Assuntos
Especificidade de Anticorpos , Bacteriófagos/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/metabolismo , Genes Reporter , Anticorpos de Cadeia Única/metabolismo , Toxicologia/métodos , Transcrição Gênica , Afinidade de Anticorpos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/metabolismo , Cloranfenicol O-Acetiltransferase/antagonistas & inibidores , Cloranfenicol O-Acetiltransferase/genética , Ensaio de Imunoadsorção Enzimática/métodos , Biblioteca Gênica , Genes Reporter/efeitos dos fármacos , Humanos , Limite de Detecção , Regiões Operadoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Análise Serial de Proteínas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Solubilidade , Ressonância de Plasmônio de Superfície , Transcrição Gênica/efeitos dos fármacosRESUMO
The soil oligochaete Enchytraeus albidus is a standard test organism used in biological testing for Environmental Risk Assessment (ERA). Although effects are known at acute and chronic level through survival, reproduction and avoidance behaviour endpoints, very little is known at the sub-cellular and molecular levels. In this study, the effects of soil properties (clay, organic matter and pH) and of the chemicals copper and phenmedipham were studied on E. albidus gene expression, during exposure periods of 2, 4 and 21 days, using DNA microarrays based on a normalised cDNA library for this test species (Amorim et al. 2011). The main objectives of this study were: (1) to assess changes in gene expression of E. albidus over time, and (2) to identify molecular markers for natural and chemical exposures. Results showed an influence of exposure time on gene expression. Transcriptional responses to phenmedipham were seen at 2 days while the responses to copper and the different soils were more pronounced at 4 days of exposure. Some genes were differentially expressed in a stress specific manner and, in general, the responses were related with effects in the energy metabolism and cell growth.
Assuntos
Monitoramento Ambiental/métodos , Oligoquetos/efeitos dos fármacos , Oligoquetos/genética , Poluentes do Solo/análise , Solo/análise , Silicatos de Alumínio , Animais , Carbamatos , Argila , Biologia Computacional , Cobre/análise , Primers do DNA , Expressão Gênica , Biblioteca Gênica , Oligoquetos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Reprodução/efeitos dos fármacos , Análise de Sequência de RNARESUMO
Despite increased utilization of copper (Cu) nanoparticles, their behaviour and effect in the environment is largely unknown. Enchytraeids are extensively used in studies of soil ecotoxicology. Ecotoxicogenomic tools have shown to be valuable in nanotoxicity interpretation. A cDNA microarray for Enchytraeus albidus has recently been developed, which was used in this study. We compared the gene expression profiles of E. albidus when exposed to Cu-salt (CuCl(2)) and Cu-nanoparticles (Cu-NP) spiked soil. Exposure time was 48 h with a concentration range of 400 to 1000 mg Cu/kg. There were more down-regulated than up-regulated genes. The number of differently expressed genes (DEG) decreased with increasing concentration for CuCl(2) exposure, whereas for Cu-NP, the number did not change. The number of common DEG decreased with increasing concentration. Differences were mainly related to transcripts involved in energy metabolism (e.g. monosaccharide transporting ATPase, NADH dehydrogenase subunit 1, cytochrome c). Overall, our results indicated that Cu-salt and Cu-NP exposure induced different gene responses. Indirect estimates of Cu-NP related ion-release indicated little or no free Cu(2+) activity in soil solutions. Hence, it was concluded that the Cu-NP effects were probably caused by the nanoparticles themselves and not by released ions.
Assuntos
Cobre/toxicidade , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Oligoquetos/genética , Animais , Análise por Conglomerados , Cobre/química , Ecotoxicologia/métodos , Nanopartículas Metálicas/química , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Poluentes do Solo/toxicidade , Toxicogenética/métodosRESUMO
The need for implementation of effects on steroid synthesis and hormone processing in screening batteries of endocrine disruptive compounds is widely acknowledged. In this perspective, hormone profiling in the H295R adrenocortical cell system is extensively examined and recently OECD validated (TG 456) as a replacement of the minced testis assay. To further elucidate the complete mechanisms and endocrine responsiveness of this cell system, microarray-based gene expression profiling of the cAMP response pathway, one of the major pathways in steroidogenesis regulation, was examined in H295R cells. Next to the steroid synthesis pathway, a broader lipid metabolic pathway, including cholesterol uptake/biosynthesis, hormone metabolization and many hormone and nuclear receptors, are sensitive towards cAMP stimulation in this cell system. Moreover, these pathways were clearly dose and time responsive, indicating early regulation (10 h) of cholesterol uptake and mobilization genes and later expression (24-48 h) of cholesterol biosynthesis and steroid synthesis. Transcription network analysis suggested several important transcription factors that could be involved in regulation of the steroid hormone pathway, of which HNF4α, a broader lipid metabolism related transcription factor, might indicate some new transcription regulation patterns in this cell line. Overall we can conclude that the time dependent gene expression patterns of the strongly coordinated cholesterol supply and steroidogenesis pathways in the H295R cell system seem to reflect well the in vivo ACTH/cAMP signalling cascade in adrenal cells. Moreover, the completeness of the steroidogenic related pathways in terms of gene expression sensitivity, indicates the H295R cell line as a promising cell line in omics-based endocrine disruption screening.
Assuntos
Corticosteroides/biossíntese , Córtex Suprarrenal/efeitos dos fármacos , Testes de Toxicidade/métodos , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/biossíntese , Vias Biossintéticas/efeitos dos fármacos , Linhagem Celular , Colesterol/biossíntese , AMP Cíclico/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Testes de Toxicidade/normasRESUMO
In this study, gene transcription profiling in combination with the assessment of systemic parameters at individual and population levels were applied to study the (toxic) effects induced through temperature stress in the presence or the absence of an additional chemical stressor (nickel) in Daphnia magna. It was illustrated that lower temperatures were mainly characterized by a reduction of growth and lipid content, while higher temperatures caused an increase of both endpoints. Many of the differentially regulated transcripts could be correlated with processes affected at higher hierarchical levels of biological organization. Gene clusters with probable roles in producing offspring (peak expression at 22°C), enhancing the metabolic rate (temperature related expression) and translational processes (increased expression at 14°C) were identified. However, it was not possible to pinpoint a specific subset of genes, exclusively responding to temperature or nickel and allowing a retrospective identification of the particular stressor. Overall, extreme temperatures caused a higher level of stress in the organisms in comparison to nickel exposure. Moreover, organisms subjected to the natural stressor appeared to be less capable of dealing with the additional chemical stressor and as a result activate or repress more gene pathways.
Assuntos
Daphnia/efeitos dos fármacos , Daphnia/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Níquel/farmacologia , Estresse Fisiológico/genética , Temperatura , Animais , Perfilação da Expressão Gênica , Análise em Microsséries , Família MultigênicaRESUMO
To better understand chemical modes of action, emphasis has been given to stress responses at lower levels of biological organization. Cholinesterases and antioxidant defenses are among the most used biomarkers due to their crucial role in the neurocholinergic transmission and in cell homeostasis preventing DNA damage, enzymatic inactivation and lipid peroxidation. The main goal of this study was to investigate the effects of zinc and cadmium on survival and reproduction of E. albidus and to assess metals oxidative stress potential and neurotoxic effects at concentrations that affected reproduction. Both metals affected the enchytraeids' survival and reproduction and induced significant changes in the antioxidant defenses as well as increased lipid peroxidation, indicating oxidative damage. This study demonstrates that determining effects at different levels of biological organization can give better information on the physiological responses of enchytraeids in metal contamination events and further unravel the mechanistic processes dealing with metal stress.
Assuntos
Cádmio/toxicidade , Oligoquetos/efeitos dos fármacos , Oligoquetos/fisiologia , Zinco/toxicidade , Animais , Monitoramento Ambiental , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Reprodução/efeitos dos fármacosRESUMO
Standard bioassays allow hazard assessment at the population level, but much remains to be learned about the molecular level response of organisms to stressors. The main aim of this study was the development of a DNA microarray for Enchytraeus albidus, a common soil worm species. Further, this microarray was tested using worms exposed to Cu, phenmedipham, and different soil types. Hybridization onto the developed microarray revealed several genes with homology to known sequences. Genes of interest were confirmed through real-time polymerase chain reaction. It was possible to discriminate between natural and chemical stressors and chemical concentrations. Gene responses were detected under conditions known to have effects in the reproduction of individuals. It was confirmed that the integration of different endpoints improves the assessment process and enhances the understanding of the modes of action of stressors. The chemical stress-induced genes were related to factors such as immune response, stress response, metabolic processes, and/or signal transduction. The present study represents the first step of a gene-level study in the ecologically relevant and standard test species E. albidus. It demonstrates the usefulness of cDNA normalization in the production of cDNA libraries of ecotoxicological standard organisms that are not genome models like E. albidus.
Assuntos
Oligoquetos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Poluentes do Solo/toxicidade , Animais , Carbamatos/toxicidade , Cobre/toxicidade , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Oligoquetos/genética , Oligoquetos/metabolismo , Reprodução/efeitos dos fármacos , Medição de Risco/métodos , Solo/análiseRESUMO
Decapod crustaceans are characterized by multiple ecdysteroid receptor (EcR) and retinoid-X-receptor (RXR) isoforms, which likely exhibit variant dimerization and transactivation interactions. In the brown shrimp C. crangon we cloned C-terminally truncated CrcEcR and CrcRXR isoforms and isoforms exhibiting deletions within the hinge region. For the former, in silico modeling of the CrcEcR indicated that, where the conserved helices H10 and H11 of the ligand-binding domain (LBD) are missing, an alternative C-terminal α-helix repairs the ligand-binding pocket (LBP). The truncated CrcRXR isoforms lack a major part of the LBD (H4-H12), thereby compromising ligand binding and dimerization. Through an in vitro ecdysteroid responsive reporter assay, we showed that these natural receptor variations do not impair receptor functioning but probably alter the receptor dimerization preferences. By the same in vitro assay, using full-length CrcEcR and CrcRXR, the effect of tributyltin (TBT) on ecdysteroid-induced transactivation was evaluated. The transactivation by 10nM PonA was reduced with 64% by 20 nM TBT. In silico modeling confirmed that TBT fits in the full-length CrcRXR-LBD. Furthermore, semi-quantitative PCR indicated altered expression of CrcEcR and CrcRXR isoforms after in vivo acute exposure to TBT, especially in the ovaries.