RESUMO
Microplastics are widespread contaminants in terrestrial environments but comparatively little is known about interactions between microplastics and common terrestrial contaminants such as zinc (Zn). In adsorption experiments fragmented HDPE bags c. one mm2 in size showed similar sorption characteristics to soil. However, when present in combination with soil, concentrations of adsorbed Zn on a per mass basis were over an order of magnitude lower on microplastics. Desorption of the Zn was minimal from both microplastics and soil in synthetic soil solution (0.01 M CaCl2), but in synthetic earthworm guts desorption was higher from microplastics (40-60%) than soil (2-15%), suggesting microplastics could increase Zn bioavailability. Individual Lumbricus terrestris earthworms exposed for 28 days in mesocosms of 260 g moist soil containing 0.35 wt % of Zn-bearing microplastic (236-4505 mg kg-1) ingested the microplastics, but there was no evidence of Zn accumulation, mortality, or weight change. Digestion of the earthworms showed that they did not retain microplastics in their gut. These findings indicate that microplastics could act as vectors to increase metal exposure in earthworms, but that the associated risk is unlikely to be significant for essential metals such as Zn that are well regulated by metabolic processes.
Assuntos
Metais , Oligoquetos , Plásticos/toxicidade , Animais , Exposição Ambiental , Oligoquetos/efeitos dos fármacos , Oligoquetos/metabolismo , Solo , Zinco/metabolismoRESUMO
Many regulations are beginning to explicitly require investigation of a chemical's endocrine-disrupting properties as a part of the safety assessment process for substances already on or about to be placed on the market. Different jurisdictions are applying distinct approaches. However, all share a common theme requiring testing for endocrine activity and adverse effects, typically involving in vitro and in vivo assays on selected endocrine pathways. For ecotoxicological evaluation, in vivo assays can be performed across various animal species, including mammals, amphibians, and fish. Results indicating activity (i.e., that a test substance may interact with the endocrine system) from in vivo screens usually trigger further higher-tier in vivo assays. Higher-tier assays provide data on adverse effects on relevant endpoints over more extensive parts of the organism's life cycle. Both in vivo screening and higher-tier assays are animal- and resource-intensive and can be technically challenging to conduct. Testing large numbers of chemicals will inevitably result in the use of large numbers of animals, contradicting stipulations set out within many regulatory frameworks that animal studies be conducted as a last resort. Improved strategies are urgently required. In February 2020, the UK's National Centre for the 3Rs and the Health and Environmental Sciences Institute hosted a workshop ("Investigating Endocrine Disrupting Properties in Fish and Amphibians: Opportunities to Apply the 3Rs"). Over 50 delegates attended from North America and Europe, across academia, laboratories, and consultancies, regulatory agencies, and industry. Challenges and opportunities in applying refinement and reduction approaches within the current animal test guidelines were discussed, and utilization of replacement and/or new approach methodologies, including in silico, in vitro, and embryo models, was explored. Efforts and activities needed to enable application of 3Rs approaches in practice were also identified. This article provides an overview of the workshop discussions and sets priority areas for follow-up. Integr Environ Assess Manag 2022;18:442-458. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).
Assuntos
Disruptores Endócrinos , Anfíbios , Animais , Ecotoxicologia , Disruptores Endócrinos/análise , Sistema Endócrino/química , Medição de Risco/métodosRESUMO
Microplastic (MP) pollution is everywhere. In terrestrial environments, microfibres (MFs) generated from textile laundering are believed to form a significant component of MPs entering soils, mainly through sewage sludge and compost applications. The aim of this study was to assess the effect of MFs on a keystone soil organism. We exposed the earthworm Lumbricus terrestris to soil with polyester MFs incorporated at rates of 0, 0.1 and 1.0 %w/w MF for a period of 35 days (in the dark at 15⯰C; nâ¯=â¯4 for each treatment). Dried plant litter was applied at the soil surface as a food source for the earthworms. We assessed earthworm vitality through mortality, weight change, depurate production and MF avoidance testing. In addition, we measured stress biomarker responses via the expression of metallothionein-2 (mt-2), heat shock protein (hsp70) and superoxide dismutase (sod-1). Our results showed that exposure and ingestion of MFs (as evidenced by subsequent retrieval of MFs within earthworm depurates) were not lethal to earthworms, nor did earthworms actively avoid MFs. However, earthworms in the MF1.0% treatment showed a 1.5-fold lower cast production, a 24.3-fold increase in expression of mt-2 (pâ¯<â¯0.001) and a 9.9-fold decline in hsp70 expression (pâ¯<â¯0.001). Further analysis of soil and MF samples indicated that metal content was not a contributor to the biomarker results. Given that burrowing and feeding behaviour, as well as molecular genetic biomarkers, were modulated in earthworms exposed to MFs, our study highlights potential implications for soil ecosystem processes due to MF contamination.
Assuntos
Oligoquetos/efeitos dos fármacos , Plásticos/toxicidade , Poliésteres/toxicidade , Poluentes do Solo/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Biomarcadores Ambientais/efeitos dos fármacos , Biomarcadores Ambientais/genética , Oligoquetos/genética , Oligoquetos/metabolismo , Plásticos/metabolismo , Poliésteres/metabolismo , Poluentes do Solo/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
The knowledge that exposure to estrogenic wastewater treatment work (WwTW) effluents induces a range of reproductive abnormalities in fish has highlighted the need to understand the wider health effects of effluents. Access to laboratory-based testing systems for WwTW effluents could greatly facilitate this endeavour. In this investigation, a laboratory-based test system was developed and applied for WwTW effluents using fathead minnows (Pimephales promelas). Sexually maturing fathead minnows were exposed, under flow-through conditions in the laboratory, for up to 21 days to graded concentrations of effluent from three different UK (temperate) WwTWs. The stability of the estrogenic component within the test system was assessed via measurements for estradiol and estrone concentrations in the effluent, and through determining estrogenic responses in an in vitro recombinant yeast estrogen screen (rYES) and in fish (plasma vitellogenin induction). The estrogen component of the effluents was stable within the holding system used (chilled <10 degrees C) for up to 7 days and measured concentrations of estradiol and estrone were shown to differ by less than 20% between the first and final day of use for each batch of effluent. Total estrogenic activity as measured in the rYES was found to be more variable (up to 66% variance between measurements for the two time points) but there was no consistent trend for a reduction in estrogenic activity. Vitellogenin was induced in males in a concentration-dependent manner and the magnitude of the response observed was proportional to the average measured concentrations of estradiol and estrone in the exposure effluent. The system described, thus, provides a robust test method for evaluating the estrogenic effects of temperate WwTW effluents that could be further applied to assess wider health effects, including population-relevant endpoints such as reproduction, using model OECD warm-water fish species such as the fathead minnow.
Assuntos
Cyprinidae/metabolismo , Estradiol/farmacologia , Estrona/farmacologia , Poluentes Químicos da Água/farmacologia , Animais , Cyprinidae/sangue , Relação Dose-Resposta a Droga , Estradiol/análise , Estrona/análise , Feminino , Masculino , Maturidade Sexual/efeitos dos fármacos , Vitelogênese/efeitos dos fármacos , Vitelogeninas/sangue , Movimentos da Água , Poluentes Químicos da Água/análiseRESUMO
Oil sands-affected water from mining must eventually be incorporated into the reclaimed landscape or treated and released. However, this material contains petrogenic organic compounds, such as naphthenic acids and traces of polycyclic aromatic hydrocarbons. This has raised concerns for impacts of oil sands process-affected waters on the heath of wildlife and humans downstream of receiving environments. The objective of this study was to evaluate the temporal association of disease states in fish with water chemistry of oil sands-affected waters over more than a decade and determine the pathogens associated with disease pathologies. Yellow perch (Perca flavescens) captured from nearby lakes were stocked into two experimental ponds during 1995-1997 and 2008-2010. South Bison Pond is a drainage basin that has received unextracted oil sands-contaminated material. Demonstration Pond is a constructed pond containing mature fine tailings capped with fresh water. Two disease pathologies, fin erosion for which a suspected bacterial pathogen (Acinetobacter Iwoffi) is identified, and lymphocystis (confirmed using a real-time PCR) were associated with oil sands-affected water exposure. From 1995 to 1997 pathologies were most prevalent in the South Bison Pond; however, from 2008 to 2009, disease was more frequently observed in the Demonstration Pond. CYP1A activity was 3-16 fold higher in fish from experimental ponds as compared to reference populations and this pattern was consistent across all sampling years. Bile fluorescence displayed a gradient of exposure with experimental ponds being elevated over local perch populations. Naphthenic acids decreased in the Bison Pond from approximately 12 mg/L to <4 mg/L while naphthenic acids increased in the Demonstration Pond from 6 mg/L to 12 mg/L due to tailings densification. Temporal changes in naphthenic acid levels, CYP1A activity and bile fluorescent metabolites correlate positively with incidence of disease pathologies whereas all inorganic water quality changes (major ions, pH, metals) were not associated with disease responses.
Assuntos
Infecções por Acinetobacter/microbiologia , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/virologia , Hidrocarbonetos Policíclicos Aromáticos/química , Poluentes Químicos da Água/química , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Acinetobacter/fisiologia , Infecções por Acinetobacter/etiologia , Animais , Infecções por Vírus de DNA/etiologia , Doenças dos Peixes/etiologia , Iridoviridae/genética , Iridoviridae/isolamento & purificação , Iridoviridae/fisiologia , Lagos/química , Metais/efeitos adversos , Metais/análise , Mineração , Campos de Petróleo e Gás , Percas/microbiologia , Percas/virologia , Hidrocarbonetos Policíclicos Aromáticos/efeitos adversos , Lagoas/química , Poluentes Químicos da Água/efeitos adversos , Qualidade da ÁguaRESUMO
BACKGROUND: Environmental estrogens in wastewater treatment work (WwTW) effluents are well established as the principal cause of reproductive disruption in wild fish populations, but their possible role in the wider health effects of effluents has not been established. OBJECTIVES: We assessed the contribution of estrogens to adverse health effects induced in a model fish species by exposure to WwTW effluents and compared effects of an estrogen alone and as part of a complex mixture (i.e., spiked into effluent). METHODS: Growth, genotoxic, immunotoxic, metabolic, and endocrine (feminized) responses were compared in fathead minnows (Pimephales promelas) exposed for 21 days to a potent estrogenic effluent, a weakly estrogenic effluent before and after spiking with a steroidal estrogen [17 alpha-ethinyl-estradiol (EE2)], and to EE2 alone. RESULTS: In addition to endocrine disruption, effluent exposure induced genotoxic damage, modulated immune function, and altered metabolism; many of these effects were elicited in a sex-specific manner and were proportional to the estrogenic potencies of the effluents. A key finding was that some of the responses to EE2 were modified when it was present in a complex mixture (i.e., spiked into effluent), suggesting that mixture effects may not be easily modeled for effluent discharges or when the chemicals impact on a diverse array of biological axes. CONCLUSION: These data reveal a clear link between estrogens present in effluents and diverse, adverse, and sex-related health impacts. Our findings also highlight the need for an improved understanding of interactive effects of chemical toxicants on biological systems for understanding health effects of environmental mixtures.
Assuntos
Misturas Complexas/toxicidade , Cyprinidae/metabolismo , Meio Ambiente , Estrogênios/toxicidade , Saúde , Animais , Cyprinidae/crescimento & desenvolvimento , Cyprinidae/imunologia , Dano ao DNA/efeitos dos fármacos , Sistema Endócrino/efeitos dos fármacos , Água/química , Poluentes Químicos da Água/toxicidadeRESUMO
Mechanism specific biomarkers are used in ecotoxicology to identify classes of chemicals and to inform on their presence in the environment, but their use in signalling for adverse effects has been limited by a poor understanding of their associated links with health. In this study an experimental analysis was undertaken to investigate how induction or suppression of an estrogen-dependent biomarker, vitellogenin (VTG), related to health effects in fathead minnow (Pimephales promelas, FHM). Exposure to an oestrogen agonist, estradiol (29 and 60 ng/L), resulted in rapid induction of VTG (elevated plasma concentrations within 2 days of exposure) in male FHM that was subsequently slow to clear from the plasma (concentrations remained elevated 70 days after cessation of exposure). The induction of VTG to concentrations of 0.5 mg/mL, however, and its continued presence in the plasma were not associated with any overt adverse health effects to the males. In contrast, induction of higher concentrations of VTG (>1 mg/mL) in reproductively active FHM exposed to estrone (307 and 781 ng/L), were associated with impacts on male survival (>33% male mortality) and an inhibitory effect on egg production in females (>51% decrease in egg number). Exposure of reproductively active FHM to a chemical that disrupts estrogen biosynthesis (an aromatase inhibitor; fenarimol 497 microg/L) also reduced reproductive success (40% decrease in egg number), and this was associated with a reduction in plasma VTG concentrations in females (36% decrease). These findings show that high level induction or suppression (in females) of plasma VTG are associated with alterations in health status and reproductive fitness. VTG, therefore, has the potential to act as a health measure as well as a biomarker for exposure, for chemicals that alter the oestrogen signalling pathway.
Assuntos
Cyprinidae/fisiologia , Estrona/toxicidade , Pirimidinas/toxicidade , Vitelogeninas/sangue , Poluentes Químicos da Água/toxicidade , Animais , Inibidores da Aromatase/toxicidade , Biomarcadores/análise , Relação Dose-Resposta a Droga , Feminino , Fertilidade/efeitos dos fármacos , Fertilidade/fisiologia , Fungicidas Industriais/toxicidade , Masculino , Mortalidade , Reprodução/efeitos dos fármacos , Reprodução/fisiologia , Diferenciação Sexual , Fatores de TempoRESUMO
The production of viable offspring is fundamental to the survival of any population. Tests that quantify effects on reproduction can, therefore, inform on the potential for long-term health effects of exposure to endocrine active chemicals. Surprisingly little is known, however, about the reproductive capacity of laboratory fish species used for chemical testing. As an example, the fathead minnow, Pimephales promelas, is widely used in chronic assessments of reproductive toxicology, and is readily induced to reproduce in captivity, yet there is little agreement on the reproductive capacity (egg number) of this species. For this species, the notable variation in reported estimates of egg number might relate to differences in the methods of egg collection adopted by many laboratories. To investigate this hypothesis, reproduction was assessed in a total of 200 pair-breeding fathead minnow, using egg collection methods that included the addition of trays placed beneath an inverted U-shaped PVC tile that is conventionally used alone for egg collection. The results demonstrated that the placement of a mesh-screened egg collection tray, beneath the spawning tile, increased estimates of the egg number by 25-67%. In addition, adopting the mesh-screened tray reduced variation in egg number between pairs, within an experiment, from >50% to <30% and variation between experiments was reduced from 53% to 7%. Adoption of the revised system for egg collection shows that egg number in the fathead minnow is considerably more consistent than frequently reported and is a highly robust endpoint against which chemical effects can be challenged effectively.
Assuntos
Tamanho da Ninhada/fisiologia , Cyprinidae/fisiologia , Testes de Toxicidade/veterinária , Animais , Tamanho da Ninhada/efeitos dos fármacos , Feminino , Masculino , Distribuição Aleatória , Valores de Referência , Fatores de Tempo , Testes de Toxicidade/instrumentação , Testes de Toxicidade/métodos , Poluentes Químicos da Água/toxicidadeRESUMO
Environmental anti-androgens are increasingly being recognized as potential contributing factors in the chemically induced feminization of wild fish because, by blocking androgen action, they can produce phenotypic effects similar to environmental estrogens. The molecular mechanisms by which anti-androgens and estrogens exert feminizing effects, however, have not been systematically compared. Using a targeted approach, we profiled the expression responses of a suite of 22 genes involved in reproduction, growth and development (processes controlled by androgens and estrogens) in the liver and gonad in adult male and female fathead minnow (Pimephales promelas) exposed to the model anti-androgen flutamide and the model synthetic estrogen 17alpha-ethinylestradiol (EE(2)). Both flutamide (320 microg/L) and EE(2) (10ng/L) produced phenotypic effects indicative of feminization (induction of plasma vitellogenin, reduced gonadosomatic index, and reduced secondary sex characters), although for the chosen test concentrations EE(2) was the more potent. For the genes studied, flutamide and EE(2) produced distinct expression profiles, suggesting that they largely operate via distinct molecular mechanisms. As examples, in liver EE(2) (but not flutamide) exposure up-regulated estrogen receptor (ER) alpha mRNA, whereas flutamide exposure increased ERbeta and ERgamma mRNAs in males and resulted in decreased androgen receptor (AR) mRNA in females. In the testis, flutamide up-regulated genes coding for enzymes involved in androgen biosynthesis (cytochrome P450 17 [CYP17] and 11beta-hydroxysteroid dehydrogenase [11beta-HSD]) implying an inhibitory action on androgen negative feedback pathways. EE(2), in contrast, inhibited the expression of enzymes involved in androgen biosynthesis (CYP17, 11beta-HSD and 17beta-hydroxysteroid dehydrogenase [17beta-HSD]). There were also some commonalities in the molecular mechanisms of flutamide and EE(2) action, including the down-regulation of gonadal sex steroid receptor expression (gonadal AR and ovarian ERalpha), increased expression of genes coding for estrogen-producing enzymes (cytochrome P450 19A and B [CYP19A and CYP19B]), decreased expression of genes involved in testis differentiation (anti-Mullerian hormone [AMH] and doublesex and mab-3 related transcription factor 1 [DMRT1]), and decreased expression of hepatic genes which mediate wider physiological processes such as somatic growth (growth hormone [GH], GH receptor [GHR], insulin-like growth factor-I [IGF-I], IGF-I receptor [IGF-IR], thyroid hormone receptor alpha [TRalpha] and beta [TRbeta]).
Assuntos
Antagonistas de Androgênios/toxicidade , Cyprinidae/fisiologia , Etinilestradiol/toxicidade , Feminização/veterinária , Expressão Gênica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Cyprinidae/genética , Cyprinidae/crescimento & desenvolvimento , Feminino , Feminização/induzido quimicamente , Flutamida/toxicidade , Perfilação da Expressão Gênica/veterinária , Gônadas/efeitos dos fármacos , Hormônios/genética , Hormônios/metabolismo , Fígado/efeitos dos fármacos , Masculino , Receptores Androgênicos/biossíntese , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/genética , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/genética , Reprodução/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Caracteres Sexuais , Vitelogeninas/sangue , Vitelogeninas/efeitos dos fármacosRESUMO
Complex interrelationships in the signalling of oestrogenic effects mean that environmental oestrogens present in the aquatic environment have the potential to disrupt physiological function in fish in a more complex manner than portrayed in the present literature. Taking a broader approach to investigate the possible effect pathways and the likely consequences of environmental oestrogen exposure in fish, the effects of 17beta-oestradiol (E(2)) were studied on the expression of a suite of genes which interact to mediate growth, development and thyroid and interrenal function (growth hormone GH (gh), GH receptor (ghr ), insulin-like growth factor (IGF-I) (igf1), IGF-I receptor (igf1r ), thyroid hormone receptors-alpha (thra) and -beta (thrb) and glucocorticoid receptor (gr )) together with the expression analyses of sex-steroid receptors and ten other genes centrally involved in sexual development and reproduction in fathead minnow (fhm; Pimephales promelas). Exposure of adult fhm to 35 ng E(2)/l for 14 days induced classic oestrogen biomarker responses (hepatic oestrogen receptor 1 and plasma vitellogenin), and impacted on the reproductive axis, feminising "male" steroidogenic enzyme expression profiles and suppressing genes involved in testis differentiation. However, E(2) also triggered a cascade of responses for gh, ghr, igf1, igf1r, thra, thrb and gr in the pituitary, brain, liver, gonad and gill, with potential consequences for the functioning of many physiological processes, not just reproduction. Molecular responses to E(2) were complex, with most genes showing differential responses between tissues and sexes. For example, igf1 expression increased in brain but decreased in gill on exposure to E(2), and responded in an opposite way in males compared with females in liver, gonad and pituitary. These findings demonstrate the importance of developing a deeper understanding of the endocrine interactions for unravelling the mechanisms of environmental oestrogen action and predicting the likely health consequences.
Assuntos
Cyprinidae , Exposição Ambiental , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Feminino , Masculino , Dados de Sequência MolecularRESUMO
The effects of simple mixtures of chemicals, with similar mechanisms of action, can be predicted using the concentration addition model (CA). The ability of this model to predict the estrogenic effects of more complex mixtures such as effluent discharges, however, has yet to be established. Effluents from 43 U.K. wastewater treatment works were analyzed for the presence of the principal estrogenic chemical contaminants, estradiol, estrone, ethinylestradiol, and nonylphenol. The measured concentrations were used to predict the estrogenic activity of each effluent, employing the model of CA, based on the relative potencies of the individual chemicals in an in vitro recombinant yeast estrogen screen (rYES) and a short-term (14-day) in vivo rainbow trout vitellogenin induction assay. Based on the measured concentrations of the four chemicals in the effluents and their relative potencies in each assay, the calculated in vitro and in vivo responses compared well and ranged between 3.5 and 87 ng/L of estradiol equivalents (E2 EQ) for the different effluents. In the rYES, however, the measured E2 EQ concentrations in the effluents ranged between 0.65 and 43 ng E2 EQ/L, and they varied against those predicted by the CA model. Deviations in the estimation of the estrogenic potency of the effluents by the CA model, compared with the measured responses in the rYES, are likely to have resulted from inaccuracies associated with the measurement of the chemicals in the extracts derived from the complex effluents. Such deviations could also result as a consequence of interactions between chemicals present in the extracts that disrupted the activation of the estrogen response elements in the rYES. E2 EQ concentrations derived from the vitellogenic response in fathead minnows exposed to a series of effluent dilutions were highly comparable with the E2 EQ concentrations derived from assessments of the estrogenic potency of these dilutions in the rYES. Together these data support the use of bioassays for determining the estrogenic potency of WwTW effluents, and they highlight the associated problems for modeling approaches that are reliant on measured concentrations of estrogenic chemicals.
Assuntos
Estrogênios/toxicidade , Previsões/métodos , Resíduos Industriais/efeitos adversos , Modelos Teóricos , Concentração Osmolar , Poluentes Químicos da Água/toxicidade , Relação Dose-Resposta a Droga , Estradiol/farmacocinéticaRESUMO
Fish in vivo screening methods to detect endocrine active substances, specifically interacting with the hypothalamic-pituitary-gonadal axis, have been developed by both the Organization for Economic Co-operation and Development (OECD) and United States Environmental Protection Agency (US-EPA). In application of these methods, i.e. regulatory testing, this paper provides a proposal on the setting of test concentrations using all available acute and chronic data and also discusses the importance of avoiding the confounding effects of systemic toxicity on endocrine endpoints. This guidance is aimed at reducing the number of false positives and subsequently the number of inappropriate definitive vertebrate studies potentially triggered by effects consequent to systemic, rather than endocrine, toxicity. At the same time it provides a pragmatic approach that maximizes the probability of detecting an effect, if it exists, thus limiting the potential for false negative outcomes.
Assuntos
Disruptores Endócrinos/toxicidade , Androstenodiona/química , Androstenodiona/toxicidade , Animais , Disruptores Endócrinos/química , Etilenoglicóis/química , Etilenoglicóis/toxicidade , Peixes , Flutamida/química , Flutamida/toxicidade , Imidazóis/química , Imidazóis/toxicidade , Cetoconazol/química , Cetoconazol/toxicidade , Dose Máxima Tolerável , Octanóis/química , Octanóis/toxicidade , Fenóis/química , Fenóis/toxicidade , Permanganato de Potássio/química , Permanganato de Potássio/toxicidade , Dodecilsulfato de Sódio/química , Dodecilsulfato de Sódio/toxicidadeRESUMO
The evolution of TLR-mediated innate immunity is a fundamental question in immunology. Here, we report the characterization and functional analysis of four TLR members in the lophotrochozoans Crassostreagigas (CgTLRs). All CgTLRs bear a conserved domain organization and have a close relationship with TLRs in ancient non-vertebrate chordates. In HEK293 cells, every CgTLR could constitutively activate NF-κB responsive reporter, but none of the PAMPs tested could stimulate CgTLR-activated NF-κB induction. Subcellular localization showed that CgTLR members have similar and dual distribution on late endosomes and plasma membranes. Moreover, CgTLRs and CgMyD88 mRNA show a consistent response to multiple PAMP challenges in oyster hemocytes. As CgTLR-mediated NF-κB activation is dependent on CgMyD88, we designed a blocking peptide for CgTLR signaling that would inhibit CgTLR-CgMyD88 dependent NF-κB activation. This was used to demonstrate that a Vibrio parahaemolyticus infection-induced enhancement of degranulation and increase of cytokines TNF mRNA in hemocytes, could be inhibited by blocking CgTLR signaling. In summary, our study characterized the primitive TLRs in the lophotrocozoan C. gigas and demonstrated a fundamental role of TLR signaling in infection-induced hemocyte activation. This provides further evidence for an ancient origin of TLR-mediated innate immunity.
Assuntos
Crassostrea/imunologia , Crassostrea/metabolismo , Imunidade Inata , Receptores Toll-Like/metabolismo , Sequência de Aminoácidos , Animais , Degranulação Celular/imunologia , Linhagem Celular , Crassostrea/classificação , Crassostrea/genética , Citocinas/genética , Citocinas/metabolismo , Evolução Molecular , Expressão Gênica , Perfilação da Expressão Gênica , Genes Reporter , Hemócitos/imunologia , Hemócitos/metabolismo , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Filogenia , Ligação Proteica , Conformação Proteica , Transporte Proteico , Transdução de Sinais , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/química , Receptores Toll-Like/genéticaRESUMO
Naphthenic acids are the major organic constituents in waters impacted by oil sands. To investigate their immunotoxicity, rainbow trout (Oncorhynchus mykiss) were injected with naphthenic acids extracted from aged oil sands tailings water. In two experiments, rainbow trout were injected intraperitoneally with 0, 10, or 100 mg/kg of naphthenic acids, and sampled after 5 or 21 d. Half of the fish from the 21 d exposure were co-exposed to inactivated Aeromonas salmonicida (A.s.) to induce an immune response. A positive control experiment was conducted using an intraperitoneal injection of 100 mg/kg of benzo[a]pyrene, a known immune suppressing compound. T-lymphocytes, B-lymphocytes, thrombocytes, and myeloid cells were counted in blood and lymphatic tissue using flow cytometry. In the 5d exposure, there was a reduction in blood leucocytes and spleen thrombocytes at the 100 mg/kg dose. However, at 21 d, leucocyte populations showed no effects of exposure with the exception that spleen thrombocyte populations increase at the 100 mg/kg dose. In the 21 d exposure, B- and T-lymphocytes in blood showed a significant Dose × A.s. interaction, indicating stimulated blood cell proliferation due to naphthenic acids alone as well as due to A.s. Naphthenic acid injections did not result in elevated bile fluorescent metabolites or elevated hepatic EROD activity. In contrast to naphthenic acids exposures, as similar dose of benzo[a]pyrene caused a significant decrease in B- and T-lymphocyte absolute counts in blood and relative B-lymphocyte counts in spleen. Results suggest that the naphthenic acids may act via a generally toxic mechanism rather than by specific toxic effects on immune cells.
Assuntos
Ácidos Carboxílicos/toxicidade , Sistema Imunitário/efeitos dos fármacos , Campos de Petróleo e Gás , Oncorhynchus mykiss/fisiologia , Poluentes Químicos da Água/toxicidade , Aeromonas salmonicida , Animais , Doenças dos Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterináriaRESUMO
Exogenous treatment of fish with natural sex hormones and their mimics has been shown to influence gonadal differentiation resulting in biased phenotypic sex-ratios. This has lead to the development of the Fish Sexual Development Test (FSDT) as a method for the detection of endocrine active chemicals. Proposed test organisms include the medaka, zebrafish (ZF) and stickleback, although the guideline also allows for inclusion of species such as the fathead minnow (FHM), provided the test duration allows for sufficient sexual differentiation. However, although the processes underlying sexual differentiation are known to differ for each of these species, it is not known how, or if, these differences would influence the results of the FSDT. In the experiments reported here, responses of the ZF and FHM to prochloraz, a sterol biosynthesis inhibitor and androgen antagonist, were characterized and compared. Exposure to 320 µg/L of prochloraz, from embryo until 60 (ZF) or 95-125 (FHM) days post hatch inhibited somatic growth of both species, but while a negative impact on ZF larval survival was observed (LOEC 32 µg/L) there was no evidence for an effect on FHM larval survival. Prochloraz influenced sexual differentiation in both species by decreasing the proportion of females (LOEC 100 µg/L (ZF), 320 µg/L (FHM)) and delaying completion of sexual differentiation; manifest as an increased incidence of ovotestis in the ZF (LOEC 100 µg/L) and as an increased number of fish with undifferentiated gonads in the FHM (LOEC 320 µg/L). However, while exposure to 320 µg/L prochloraz delayed maturation of the differentiated FHM testis, there was no such effect in the ZF. These results demonstrate that the different strategy of sexual differentiation in the ZF and FHM influences the profile of responses of their gonads to the masculinising effects of prochloraz, but does not affect their overall sensitivity.
Assuntos
Antagonistas de Androgênios/toxicidade , Cyprinidae/fisiologia , Disruptores Endócrinos/toxicidade , Imidazóis/toxicidade , Desenvolvimento Sexual/efeitos dos fármacos , Testes de Toxicidade/métodos , Poluentes Químicos da Água/toxicidade , Animais , Relação Dose-Resposta a Droga , Feminino , Larva/efeitos dos fármacos , Larva/fisiologia , Masculino , Nível de Efeito Adverso não Observado , Distribuição Aleatória , Sensibilidade e Especificidade , Diferenciação Sexual/efeitos dos fármacos , Vitelogeninas/metabolismo , Peixe-Zebra/fisiologiaRESUMO
Zearalenone (ZON) is one of the worldwide most common mycotoxins and exhibits estrogenic activity in the range of natural steroid estrogens such as 17ß-estradiol (E2). The occurrence of ZON has been reported in drainage water, soil, wastewater effluents and rivers, but its ecotoxicological effects on fish have hardly been investigated. In this study the estrogenic potency of the ZON was compared to E2 in a recombinant yeast estrogen screen (rYES) and the effects of waterborne ZON exposure on reproduction, physiology and morphology of zebrafish (Danio rerio) were investigated in a 42-day reproduction experiment. E2 as well as ZON evoked a sigmoid concentration-response curve in the rYES with a mean EC(50) of 2 and 500 µg/L, respectively, resulting in an E2:ZON EC(50) ratio of 1:250. Exposure to ZON for 21 days reduced relative spawning frequency at 1000 and 3200 ng/L to 38.9 and 37.6%, respectively, and relative fecundity at 100, 320, 1000 and 3200 ng/L to 74.2, 41.7, 43.8 and 16.7%, respectively, in relation to the 21-day pre-exposure period. A 4.4 and 8.1 fold induction of plasma vitellogenin (VTG) was observed in male zebrafish at 1000 and 3200 ng/L ZON, respectively. Exposure to ZON did not affect fertility, hatch, embryo survival and gonad morphology of zebrafish. The results of this study demonstrate that although ZON possesses a moderate estrogenic potency in vitro, it exhibits a comparably strong effect on induction of VTG and reproduction in vivo. This indicates that ZON might contribute to the overall estrogenic activity in the environment and could therefore pose a risk for wild fish in their natural habitat.
Assuntos
Estrogênios/toxicidade , Reprodução/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Zearalenona/toxicidade , Peixe-Zebra/fisiologia , Animais , Feminino , Fertilidade/efeitos dos fármacos , Masculino , Vitelogeninas/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Estrogenic chemicals found within wastewater treatment work (WwTW) effluents have been shown individually to inhibit reproduction in fish, but the impact of the WwTW effluents themselves and the complex mixtures of environmental estrogens and other endocrine disrupting chemicals they contain has not been established. In this investigation, the effect of exposure to three WwTW effluents, with differing levels of estrogenic activity, was assessed on egg production in pair-breeding fathead minnow. Exposure to two of the three effluents tested resulted in a reduced egg production (by 28% for effluent I at a dilution of 50% and by 44% for effluent III at full strength), which was proportional to the estrogenic content of the effluents. The test effluents, however, had a greater effect on egg production than might be expected, on the basis of both the response they induced for induction of vitellogenin (an estrogen exposure biomarker) and when compared with an equivalent estrogen exposure to EE2. These data show that reliance on relatively simple biomarker responses for estrogenic activity alone, such as vitellogenin, can significantly underestimate the impacts of estrogenic WwTW effluents on fitness parameters such as reproductive health that are regulated by more complex estrogenic (and other endocrine) signaling mechanisms.
Assuntos
Cyprinidae/fisiologia , Disruptores Endócrinos/toxicidade , Estrogênios/toxicidade , Óvulo/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , AnimaisRESUMO
Gene expression profiling offers considerable potential for identifying chemical causation of effects induced in exposures to complex mixtures, and for understanding the mechanistic basis for their phenotypic effects. We characterized gene expression responses in livers and gonads of fathead minnow (Pimephales promelas) exposed (for 14-21 days) to estrogenic wastewater treatment works final effluents with varying potencies and assessed the extent to which these expression profiles mapped with those induced by individual steroid estrogens present in the effluents (17beta-estradiol and 17alpha-ethinylestradiol) and, thus, were diagnostic of estrogen exposure. For these studies, we adopted a targeted approach (via real-time PCR) with a suite of 12 genes in liver and 21 genes in gonad known to play key roles in reproduction, growth and development (processes controlled by estrogens) and responses were compared with effects on phenotypic end points indicative of feminization. Gene responses to effluent were induced predominantly in a linear (monotonic) concentration-dependent manner but were complex with many genes responding differently between tissue types and sexes. The gene expression profiles for the estrogenic effluents and the individual steroid estrogens had many common features. There were marked differences in the profiles between the two effluents, however, that were not explained by differences in their estrogenic potencies, suggesting that these may have arisen as a consequence of differences in the contents of other chemicals, which may act directly or indirectly with the estrogen-response pathway to alter estrogen-induced gene expression. These data demonstrate that the patterns of gene expression induced by estrogenic effluents, although complex, can be diagnostic for some of the estrogens they contain and provide insights into the mechanistic basis for the phenotypic effects seen.
Assuntos
Cyprinidae/genética , Estrogênios/toxicidade , Perfilação da Expressão Gênica/métodos , Animais , Relação Dose-Resposta a Droga , Feminino , Gônadas/efeitos dos fármacos , Gônadas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Vitelogeninas/sangueRESUMO
The natural steroids estradiol-17beta (E2) and estrone (E1) and the synthetic steroid ethynylestradiol-17alpha (EE2) have frequently been measured in waters receiving domestic effluents. All of these steroids bind to the estrogen receptor(s) and have been shown to elicit a range of estrogenic responses in fish at environmentally relevant concentrations. At present, however, no relative potency estimates have been derived for either the individual steroidal estrogens or their mixtures in vivo. In this study the estrogenic activity of E2, E1, and EE2, and the combination effects of a mixture of E2 and EE2 (equi-potent fixed-ratio mixture), were assessed using vitellogenin induction in a 14-day in vivo juvenile rainbow trout screening assay. Median effective concentrations, relative to E2, for induction of vitellogenin were determined from the concentration-response curves and the relative estrogenic potencies of each of the test chemicals calculated. Median effective concentrations were between 19 and 26 ng L(-1) for E2, 60 ng L(-1) for E1, and between 0.95 and 1.8 ng L(-1) for EE2, implying that EE2 was approximately 11 to 27 times more potent than E2, while E2 was 2.3 to 3.2 times more potent than E1. The median effective concentration, relative to E2, for the binary mixture of E2 and EE2 was 15 ng L(-1) (comprising 14.4 ng L(-1) E2 and 0.6 ng L(-1) EE2). Using the model of concentration addition it was shown that this activity of the binary mixture could be predicted from the activity of the individual chemicals. The ability of each individual steroid to contribute to the overall effect of a mixture, even at individual no-effect concentrations, combined with the high estrogenic potency of the steroids, particularly the synthetic steroid EE2, emphasizes the need to consider the total estrogenic load of these chemicals in our waterways.