Facilitating microplastic ingestion in aquatic models: A verified protocol for daphnia magna as a trojan horse vector.

Microplastic pollution poses a significant environmental threat due to its persistence, widespread distribution, and inherent toxic potential. Despite the increasing number of publications in this field, a standardized protocol for the laboratory intake of microplastics by Daphnia magna has yet to be established. In this study, we introduce a verified protocol designed to facilitate the ingestion of microplastic particles (MPs) by D. magna, ranging in size from 5-55 µm. This protocol can be further applied to evaluate the toxicity of MPs on D. magna, a crucial organism model in ecotoxicology. Furthermore, this protocol can be used to assess toxicity of MPs in other aquatic species, such as fish, by using daphnids as a vehicle for ensuring the ingestion of these particles. Consequently, this protocol can be applied to study also one of the most pressing concerns regarding exposure to MPs, the transfer of MPs through different trophic levels, which has a great potential for ecotoxicological studies.•The influence of MPs concentration, duration and exposure dynamics and D. magna age/size in MPs intake were tested.•We have determined the optimal conditions for promoting microplastic ingestion by D. magna.

Efficient removal of toxicity associated to wastewater treatment plant effluents by enhanced Soil Aquifer Treatment.

The regeneration of wastewater has been recognized as an effective strategy to counter water scarcity. Nonetheless, Wastewater Treatment Plant (WWTP) effluents still contain a wide range of contaminants of emerging concern (CECs) even after water depuration. Filtration through Soil Aquifer Treatment (SAT) systems has proven efficient for CECs removal although the attenuation of their associated biological effects still remains poorly understood. To evaluate this, three pilot SAT systems were monitored, two of them enhanced with different reactive barriers. SATs were fed with secondary effluents during two consecutive campaigns. Fifteen water samples were collected from the WWTP effluent, below the barriers and 15 m into the aquifer. The potential attenuation of effluent-associated biological effects by SATs was evaluated through toxicogenomic bioassays using zebrafish eleutheroembryos and human hepatic cells. Transcriptomic analyses revealed a wide range of toxic activities exerted by the WWTP effluents that were reduced by more than 70% by SAT. Similar results were observed when HepG2 hepatic cells were tested for cytotoxic and dioxin-like responses. Toxicity reduction appeared partially determined by the barrier composition and/or SAT managing and correlated with CECs removal. SAT appears as a promising approach to efficiently reduce effluent-associated toxicity contributing to environmental and human health preservation.

Impact of organic soil amendments in antibiotic levels, antibiotic resistance gene loads, and microbiome composition in corn fields and crops.

The potential spreading of antibiotic resistance genes (ARG) into agricultural fields and crops represent a fundamental limitation on the use of organic fertilization in food production systems. We present here a study of the effect of spreading four types of organic soil amendments (raw pig slurry, liquid and solid fractions, and a digested derivative) on demonstrative plots in two consecutive productive cycles of corn harvest (Zea mays), using a mineral fertilizer as a control, following the application of organic amendments at 32-62 T per ha (150 kg total N/ha) and allowing 5-8 months between fertilization and harvest. A combination of qPCR and high-throughput 16S rDNA sequencing methods showed a small, but significant impact of the fertilizers in both ARG loads and microbiomes in soil samples, particularly after the second harvesting cycle. The slurry solid fraction showed the largest impact on both ARG loads and microbiome variation, whereas its digestion derivatives showed a much smaller impact. Soil samples with the highest ARG loads also presented increased levels of tetracyclines, indicating a potential dual hazard by ARG and antibiotic residues linked to some organic amendments. Unlike soils, no accumulation of ARG or antibiotics was observed in corn leaves (used as fodder) or grains, and no grain sample reached detection limits for neither parameter. These results support the use of organic soil amendments in corn crops, while proposing the reduction of the loads of ARGs and antibiotics from the fertilizers to greatly reduce their potential risk.

Implications of the use of organic fertilizers for antibiotic resistance gene distribution in agricultural soils and fresh food products. A plot-scale study.

The spread of antibiotic resistance genes (ARG) into agricultural soils, products, and foods severely limits the use of organic fertilizers in agriculture. In order to help designing agricultural practices that minimize the spread of ARG, we fertilized, sown, and harvested lettuces and radish plants in experimental land plots for two consecutive agricultural cycles using four types of fertilizers: mineral fertilization, sewage sludge, pig slurry, or composted organic fraction of municipal solid waste. The analysis of the relative abundances of more than 200,000 ASV (Amplicon Sequence Variants) identified a small, but significant overlap (<10%) between soil's and fertilizer microbiomes. Clinically relevant ARG were found in higher loads (up to 100 fold) in fertilized soils than in the initial soil, particularly in those treated with organic fertilizers, and their loads grossly correlated to the amount of antibiotic residues found in the corresponding fertilizer. Similarly, low, but measurable ARG loads were found in lettuce (tetM, sul1) and radish (sul1), corresponding the lowest values to samples collected from minerally fertilized fields. Comparison of soil samples collected along the total period of the experiment indicated a relatively year-round stability of soil microbiomes in amended soils, whereas ARG loads appeared as unstable and transient. The results indicate that ARG loads in soils and foodstuffs were likely linked to the contribution of bacteria from organic fertilizer to the soil microbiomes, suggesting that an adequate waste management and good pharmacological and veterinarian practices may significantly reduce the presence of these ARGs in agricultural soils and plant products.

Towards regulation of Endocrine Disrupting chemicals (EDCs) in water resources using bioassays - A guide to developing a testing strategy.

Endocrine disrupting chemicals (EDCs) are found in every environmental medium and are chemically diverse. Their presence in water resources can negatively impact the health of both human and wildlife. Currently, there are no mandatory screening mandates or regulations for EDC levels in complex water samples globally. Bioassays, which allow quantifying in vivo or in vitro biological effects of chemicals are used commonly to assess acute toxicity in water. The existing OECD framework to identify single-compound EDCs offers a set of bioassays that are validated for the Estrogen-, Androgen-, and Thyroid hormones, and for Steroidogenesis pathways (EATS). In this review, we discussed bioassays that could be potentially used to screen EDCs in water resources, including in vivo and in vitro bioassays using invertebrates, fish, amphibians, and/or mammalians species. Strengths and weaknesses of samples preparation for complex water samples are discussed. We also review how to calculate the Effect-Based Trigger values, which could serve as thresholds to determine if a given water sample poses a risk based on existing quality standards. This work aims to assist governments and regulatory agencies in developing a testing strategy towards regulation of EDCs in water resources worldwide. The main recommendations include 1) opting for internationally validated cell reporter in vitro bioassays to reduce animal use & cost; 2) testing for cell viability (a critical parameter) when using in vitro bioassays; and 3) evaluating the recovery of the water sample preparation method selected. This review also highlights future research avenues for the EDC screening revolution (e.g., 3D tissue culture, transgenic animals, OMICs, and Adverse Outcome Pathways (AOPs)).

Emerging concepts and opportunities for endocrine disruptor screening of the non-EATS modalities.

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and involve diverse chemical-receptor interactions that can perturb hormone signaling. The Organization for Economic Co-operation and Development has validated several EDC-receptor bioassays to detect endocrine active chemicals and has established guidelines for regulatory testing of EDCs. Focus on testing over the past decade has been initially directed to EATS modalities (estrogen, androgen, thyroid, and steroidogenesis) and validated tests for chemicals that exert effects through non-EATS modalities are less established. Due to recognition that EDCs are vast in their mechanisms of action, novel bioassays are needed to capture the full scope of activity. Here, we highlight the need for validated assays that detect non-EATS modalities and discuss major international efforts underway to develop such tools for regulatory purposes, focusing on non-EATS modalities of high concern (i.e., retinoic acid, aryl hydrocarbon receptor, peroxisome proliferator-activated receptor, and glucocorticoid signaling). Two case studies are presented with strong evidence amongst animals and human studies for non-EATS disruption and associations with wildlife and human disease. This includes metabolic syndrome and insulin signaling (case study 1) and chemicals that impact the cardiovascular system (case study 2). This is relevant as obesity and cardiovascular disease represent two of the most significant health-related crises of our time. Lastly, emerging topics related to EDCs are discussed, including recognition of crosstalk between the EATS and non-EATS axis, complex mixtures containing a variety of EDCs, adverse outcome pathways for chemicals acting through non-EATS mechanisms, and novel models for testing chemicals. Recommendations and considerations for evaluating non-EATS modalities are proposed. Moving forward, improved understanding of the non-EATS modalities will lead to integrated testing strategies that can be used in regulatory bodies to protect environmental, animal, and human health from harmful environmental chemicals.

Antibiotic and antibiotic-resistant gene loads in swine slurries and their digestates: Implications for their use as fertilizers in agriculture.

The spread of antibiotic resistance in bacteria is a matter of global concern, and the identification of possible sources of the associated genetic elements (antibiotic resistance genes -ARGs-, components of the horizontal gene transfer mechanism), is becoming an urgent need. While the transmission of ARGs in medical settings have been adequately characterized, ARG propagation in agroecosystems remains insufficiently studied. Particularly crucial is the determination of potential risks associated to the use of swine slurries and related products as component of organic fertilizers, an increasingly used farming practice. We determined ARGs and antibiotic loads analysed from swine slurries and digestates from eight farms from Catalonia (NE Spain), and compared the results with their microbiome composition. Both ARGs and antibiotic were conspicuous in farm organic wastes, and the levels of some antibiotics exceeded currently accepted minimum inhibitory concentrations. Particularly, the presence of high loads of fluoroquinolones was directly correlated to the prevalence of the related qnrS1 ARG in the slurry. We also found evidence that ARG loads were directly correlated to the prevalence of determined bacterial taxa (Actinobacteria, Proteobacteria, Spirochaeta), a parameter that could be potentially modulated by the processing of the raw slurry prior to their use as fertilizer.

Comparative epigenetics in animal physiology: An emerging frontier.

The unprecedented access to annotated genomes now facilitates the investigation of the molecular basis of epigenetic phenomena in phenotypically diverse animals. In this critical review, we describe the roles of molecular epigenetic mechanisms in regulating mitotically and meiotically stable spatiotemporal gene expression, phenomena that provide the molecular foundation for the intra-, inter-, and trans-generational emergence of physiological phenotypes. By focusing principally on emerging comparative epigenetic roles of DNA-level and transcriptome-level epigenetic mark dynamics in the emergence of phenotypes, we highlight the relationship between evolutionary conservation and innovation of specific epigenetic pathways, and their interplay as a priority for future study. This comparative approach is expected to significantly advance our understanding of epigenetic phenomena, as animals show a diverse array of strategies to epigenetically modify physiological responses. Additionally, we review recent technological advances in the field of molecular epigenetics (single-cell epigenomics and transcriptomics and editing of epigenetic marks) in order to (1) investigate environmental and endogenous factor dependent epigenetic mark dynamics in an integrative manner; (2) functionally test the contribution of specific epigenetic marks for animal phenotypes via genome and transcript-editing tools. Finally, we describe advantages and limitations of emerging animal models, which under the Krogh principle, may be particularly useful in the advancement of comparative epigenomics and its potential translational applications in animal science, ecotoxicology, ecophysiology, climate change science and wild-life conservation, as well as organismal health.

Dose effect of Zn and Cu in sludge-amended soils on vegetable uptake of trace elements, antibiotics, and antibiotic resistance genes: Human health implications.

The application of sewage sludge to agricultural fields reduces the need for mineral fertilizers by increasing soil organic matter, but may also increase soil pollution. Previous studies indicate that zinc and copper, as the most abundant elements in sewage sludge, affect plant uptake of other contaminants. This paper aims to investigate and compare the effect of increasing amounts of Zn and Cu in sludge-amended soils on the accumulation of trace elements (TEs), antibiotics (ABs), and antibiotic resistance genes (ARGs) in lettuce and radish. The vegetables were grown under controlled conditions, and the influence on plant physiology and human health were also evaluated. The results show that the addition of Zn and Cu significantly increased the concentration of TEs in the edible tissue of both vegetables. According to the hazard quotient (HQ) of the TEs, the human health risk increased 2 to 3 times and was 3-4 times greater in lettuce than in radish. In contrast to the TEs, the occurrence of ABs and most of the ARGs was higher in radish roots than lettuce leaves. ABs were not detected in lettuce leaves, and the amount of all ARGs except blaTEM was 10 times lower than in radish roots. On the other hand, the addition of Zn and Cu had no significant effect on the occurrence of ABs and ARGs in the edible part of the vegetables, and no damage was found to plant productivity or physiology. The results show that the consumption of lettuce and radish grown in sewage-sludge-amended soils under tested doses of Cu and Zn does not pose an adverse human health effect, as the total HQ value was always less than 1, and the presence of ABs and ARGs was not found to have any potential impact. Nevertheless, further studies are needed to estimate the long-term effect on human health of crops grown under frequent application of biosolids in arable soil.

Transcriptomic effects of tributyltin (TBT) in zebrafish eleutheroembryos. A functional benchmark dose analysis.

Exposure to the antifouling tributyltin (TBT) has been related to imposex in mollusks and to obesogenicity, adipogenesis and masculinization in fish. To understand the underlying molecular mechanisms, we evaluated dose-response effects of TBT (1.7-56 nM) in zebrafish eleutheroembryos transcriptome exposed from 2 to 5 days post-fertilization. RNA-sequencing analysis identified 3238 differentially expressed transcripts in eleutheroembryos exposed to TBT. Benchmark dose analyses (BMD) showed that the point of departure (PoD) for transcriptomic effects (9.28 nM) was similar to the metabolomic PoD (11.5 nM) and about one order of magnitude lower than the morphometric PoD (67.9 nM) or the median lethal concentration (LC50: 93.6 nM). Functional analysis of BMD transcriptomic data identified steroid metabolism and cholesterol and vitamin D3 biosynthesis as the most sensitive pathways to TBT (<50% PoD). Conversely, transcripts related to general stress and DNA damage became affected only at doses above the PoD. Therefore, our results indicate that transcriptomes can act as early molecular indicators of pollutant exposure, and illustrates their usefulness for the mechanistic identification of the initial toxic events. As the estimated molecular PoDs are close to environmental levels, we concluded that TBT may represent a substantial risk in some natural environments.

Agrochemicals disrupt multiple endocrine axes in amphibians.

Concern over global amphibian declines and possible links to agrochemical use has led to research on the endocrine disrupting actions of agrochemicals, such as fertilizers, fungicides, insecticides, acaricides, herbicides, metals, and mixtures. Amphibians, like other species, have to partition resources for body maintenance, growth, and reproduction. Recent studies suggest that metabolic impairments induced by endocrine disrupting chemicals, and more particularly agrichemicals, may disrupt physiological constraints associated with these limited resources and could cause deleterious effects on growth and reproduction. Metabolic disruption has hardly been considered for amphibian species following agrichemical exposure. As for metamorphosis, the key thyroid hormone-dependent developmental phase for amphibians, it can either be advanced or delayed by agrichemicals with consequences for juvenile and adult health and survival. While numerous agrichemicals affect anuran sexual development, including sex reversal and intersex in several species, little is known about the mechanisms involved in dysregulation of the sex differentiation processes. Adult anurans display stereotypical male mating calls and female phonotaxis responses leading to successful amplexus and spawning. These are hormone-dependent behaviours at the foundation of reproductive success. Therefore, male vocalizations are highly ecologically-relevant and may be a non-invasive low-cost method for the assessment of endocrine disruption at the population level. While it is clear that agrochemicals disrupt multiple endocrine systems in frogs, very little has been uncovered regarding the molecular and cellular mechanisms at the basis of these actions. This is surprising, given the importance of the frog models to our deep understanding of developmental biology and thyroid hormone action to understand human health. Several agrochemicals were found to have multiple endocrine effects at once (e.g., targeting both the thyroid and gonadal axes); therefore, the assessment of agrochemicals that alter cross-talk between hormonal systems must be further addressed. Given the diversity of life-history traits in Anura, Caudata, and the Gymnophiona, it is essential that studies on endocrine disruption expand to include the lesser known taxa. Research under ecologically-relevant conditions will also be paramount. Closer collaboration between molecular and cellular endocrinologists and ecotoxicologists and ecologists is thus recommended.

Acute and long-term metabolic consequences of early developmental Bisphenol A exposure in zebrafish (Danio rerio).

Bisphenol A (BPA) is an estrogenic contaminant linked to metabolic disruption. Developmental BPA exposure is of particular concern, as organizational effects may irreversibly disrupt metabolism at later life-stages. While BPA exposures in adult fish elicit metabolic perturbations similar to effects described in rodents, the metabolic effects of developmental BPA exposure in juvenile fish remain largely unknown. Following embryonic zebrafish exposure to BPA (0.1, 1 and 4 mg/L) and EE2 (10 ng/L) from 2 to 5 dpf, we assessed the metabolic phenotype in larvae (4-6 dpf) and juveniles (43-49 dpf) which had been divided into regular-fed and overfed groups at 29 dpf. Developmental BPA exposure in larvae dose-dependently reduced food-intake and locomotion and increased energy expenditure. Juveniles (29 dpf) exhibited a transient increase in body weight after developmental BPA exposure and persistent diet-dependent locomotion changes (43-49 dpf). At the molecular level, glucose and lipid metabolism-related transcript abundance clearly separated BPA exposed fish from controls and EE2 exposed fish at the larval stage, in juveniles on a regular diet and, to a lesser extent, in overfed juveniles. In general, the metabolic endpoints affected by BPA exposure were not mimicked by EE2 treatment. We conclude that developmental BPA exposure elicits acute metabolic effects in zebrafish larvae and fewer transient and persistent effects in juveniles and that these metabolic effects are largely independent of BPA's estrogenicity.

Compensatory indirect effects of an herbicide on wetland communities.

The direct effects of large-scale disturbances are readily studied because their effects are often apparent and result in large changes to ecosystems. Direct effects can cascade through the ecosystem, leading to indirect effects that are often subtle and difficult to detect. Managing anthropogenic disturbances, such as chemical contamination, requires an understanding of both direct and indirect effects to predict, measure, and characterize the impact. Using a replicated whole-ecosystem experiment and path analyses (assesses the effects of a set of variables on a specified outcome, similar to multiple regression), we examined the direct and indirect effects of a glyphosate-based herbicide and nutrient enrichment on wetland communities. The latter did not impact any measured endpoints. The strongest drivers of macrophyte, benthic invertebrate, and amphibian assemblages were the ephemerality and the size of wetlands, factors which were not altered by herbicide applications. The herbicide had a direct negative effect on macrophyte cover, amphibian larval abundance, and the proportion of predatory benthic invertebrates. However, both amphibians and invertebrates were positively affected by the reduction in the macrophyte cover caused by the herbicide applications. The opposing directions of the direct and indirect effects lead to no net change in either group. The compensatory dynamics observed herein highlight the need for a better understanding of indirect effects pathways to determine whether common anthropogenic disturbances alter the ecological communities in small wetland ecosystems.

Changes in lipid profiles induced by bisphenol A (BPA) in zebrafish eleutheroembryos during the yolk sac absorption stage.

Bisphenol A (BPA; 4,4'-(propane-2,2-diyl)diphenol) has been shown to act as an obesogen and to disrupt lipid metabolism in zebrafish eleutheroembryos (ZE). To characterize the consequences of this disruption, we performed a detailed lipidomic study using ZE exposed to different BPA concentrations (0, 4, 6 and 8 mg/L of BPA) from day 2 to up to day 6 post fertilization (dpf). Total lipids at 4, 5 and 6 dpf were extracted by Folch method and analyzed by high-performance thin layer chromatography (HPTLC) as wide-range preliminary screening. Selected conditions (0 and 6 mg/L of BPA) were used to obtain a high-quality lipid profile using ultra high-performance liquid chromatography/time-of-flight mass spectrometry (UHPLC-TOFMS). BPA exposed ZE exhibited increased amounts of triglycerides (TG), diglycerides (DG), phosphatidylcholines (PC) and phosphatidylinositols (PI), regarding the control group. Analysis of time- and BPA exposure-related patterns of specific lipid species showed a clear influence of unsaturation degree (mostly in DG and PC) and/or fatty acid chain length (mostly in TG and PC derivatives) on their response to the presence of BPA. A decreased yolk-sac and energy consumption in exposed individuals appeared as the main reason for the observed BPA-driven effects. Integration of these results with previous morphological, biochemical, transcriptomic, metabolomic and behavioral data suggests a disruption of different signalling pathways by BPA that starts at very low BPA concentrations, whose effects propagate across different organization levels, and that cannot be only explained by the relatively weak estrogenic effect of BPA.

Bioconcentration and Metabolic Effects of Emerging PFOS Alternatives in Developing Zebrafish.

The novel PFOS alternatives, 6:2 chlorinated polyfluorinated ether sulfonate (F-53B) and sodium p-perfluorous nonenoxybenzenesulfonate (OBS), are emerging in the Chinese market, but little is known about their ecological risks. In this study, zebrafish embryos were exposed to PFOS, F-53B, and OBS to evaluate their bioconcentration and acute metabolic consequences. Per- and polyfluoroalkyl substances (PFASs) accumulated in larvae in the order of F-53B > PFOS > OBS, with the bioconcentration factors ranging from 20 to 357. Exposure to F-53B and PFOS, but not OBS, increased energy expenditure, and reduced feed intake in a concentration-dependent manner and the expression of genes involved in metabolic pathways at the transcriptional and translational levels. Molecular docking revealed that the binding affinities of PFASs to glucokinase were decreased in the following order: F-53B > PFOS > OBS. Finally, the results of Point of Departure (PoD) indicate that metabolic end points at the molecular and organismal level are most sensitive to F-53B followed by PFOS and OBS. Collectively, F-53B has the highest bioconcentration potential and the strongest metabolism-disrupting effects, followed by PFOS and OBS. Our findings have important implications for the assessment of early developmental metabolic effects of PFOS alternatives F-53B and OBS in wildlife and humans.

Morphometric signatures of exposure to endocrine disrupting chemicals in zebrafish eleutheroembryos.

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.

Unravelling the mechanisms of PFOS toxicity by combining morphological and transcriptomic analyses in zebrafish embryos.

Exposure to PFOS (perfluorooctanesulfonate) has been related to toxic effects on lipid metabolism, immunological response, and different endocrine systems. We present here a transcriptomic analysis of zebrafish embryos exposed to different concentrations of PFOS (0.03-1.0 mg/L) from 48 to 120 hpf. No major survival or morphological alterations (swimming bladder inflation, kyphosis, eye separation and size…) were observed below the 1.0 mg/L mark. Conversely, we observed significant increase in transcripts related to lipid transport and metabolism even at the lowest used concentration. In addition, we observed a general decrease on transcripts related to natural immunity and defense again infections, which adds to the recent concerns about PFOS as immunotoxicant, particularly in humans. Derived PoD (Point of Departure) values for transcriptional changes (0.011 mg/L) were about 200-fold lower than the corresponding PoD values for morphometric effects (2.53 mg/L), and close to levels observed in human blood serum or bird eggs. Our data suggest that currently applicable tolerable levels of PFOS in commercial goods should be re-evaluated, taking into account its potential effects on lipid metabolism and the immune system.

Developmental fluoxetine exposure in zebrafish reduces offspring basal cortisol concentration via life stage-dependent maternal transmission.

Fluoxetine (FLX) is a pharmaceutical used to treat affective disorders in humans, but as environmental contaminant also affects inadvertently exposed fish in urban watersheds. In humans and fish, acute FLX treatment and exposure are linked to endocrine disruption, including effects on the reproductive and stress axes. Using the zebrafish model, we build on the recent finding that developmental FLX exposure reduced cortisol production across generations, to determine possible parental and/or life-stage-dependent (age and/or breeding experience) contributions to this phenotype. Specifically, we combined control and developmentally FLX-exposed animals of both sexes (F0) into four distinct breeding groups mated at 5 and 9 months, and measured offspring (F1) basal cortisol at 12 dpf. Basal cortisol was lower in F1 descended from developmentally FLX-exposed F0 females bred at 5, but not 9 months, revealing a maternal, life-stage dependent effect. To investigate potential molecular contributions to this phenotype, we profiled maternally deposited transcripts involved in endocrine stress axis development and regulation, epigenetic (de novo DNA methyltransferases) and post-transcriptional (miRNA pathway components and specific miRNAs) regulation of gene expression in unfertilized eggs. Maternal FLX exposure resulted in decreased transcript abundance of glucocorticoid receptor, dnmt3 paralogues and miRNA pathway components in eggs collected at 5 months, and increased transcript abundance of miRNA pathway components at 9 months. Specific miRNAs predicted to target stress axis transcripts decreased (miR-740) or increased (miR-26, miR-30d, miR-92a, miR-103) in eggs collected from FLX females at 5 months. Increased abundance of miRNA-30d and miRNA-92a persisted in eggs collected from FLX females at 9 months. Clustering and principal component analyses of egg transcript profiles separated eggs collected from FLX-females at 5 months from other groups, suggesting that oocyte molecular signatures, and miRNAs in particular, may serve as predictive tools for the offspring phenotype of reduced basal cortisol in response to maternal FLX exposure.

Dose-dependent transcriptomic responses of zebrafish eleutheroembryos to Bisphenol A.

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-1, 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 the exposed animals, well below the macroscopic LOEC, but related to at least some of the observed morphological alterations, particularly in eye size and yolk sac resorption. Our data supports the idea that the physiological effects of BPA cannot be only explained by its rather weak interaction with the estrogen receptor, and that multivariate analyses are required to analyze the effects of toxicants like BPA, which interact with different cellular targets producing complex phenotypes.