Multiomics Point of Departure (moPOD) Modeling Supports an Adverse Outcome Pathway Network for Ionizing Radiation.

While adverse biological effects of acute high-dose ionizing radiation have been extensively investigated, knowledge on chronic low-dose effects is scarce. The aims of the present study were to identify hazards of low-dose ionizing radiation to Daphnia magna using multiomics dose-response modeling and to demonstrate the use of omics data to support an adverse outcome pathway (AOP) network development for ionizing radiation. Neonatal D. magna were exposed to γ radiation for 8 days. Transcriptomic analysis was performed after 4 and 8 days of exposure, whereas metabolomics and confirmative bioassays to support the omics analyses were conducted after 8 days of exposure. Benchmark doses (BMDs, 10% benchmark response) as points of departure (PODs) were estimated for both dose-responsive genes/metabolites and the enriched KEGG pathways. Relevant pathways derived using the BMD modeling and additional functional end points measured by the bioassays were overlaid with a previously published AOP network. The results showed that several molecular pathways were highly relevant to the known modes of action of γ radiation, including oxidative stress, DNA damage, mitochondrial dysfunction, protein degradation, and apoptosis. The functional assays showed increased oxidative stress and decreased mitochondrial membrane potential and ATP pool. Ranking of PODs at the pathway and functional levels showed that oxidative damage related functions had relatively low PODs, followed by DNA damage, energy metabolism, and apoptosis. These were supportive of causal events in the proposed AOP network. This approach yielded promising results and can potentially provide additional empirical evidence to support further AOP development for ionizing radiation.

Application of AOPs to assist regulatory assessment of chemical risks - Case studies, needs and recommendations.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

A Bayesian Approach to Incorporating Spatiotemporal Variation and Uncertainty Limits into Modeling of Predicted Environmental Concentrations from Chemical Monitoring Campaigns.

Environmental monitoring studies provide key information to assess ecosystem health. Results of chemical monitoring campaigns can be used to identify the exposure scenarios of regulatory concern. In environmental risk assessment (ERA), measured concentrations of chemicals can be used to model predicted environmental concentrations (PECs). As the PEC is, by definition, a predicted variable, it is highly dependent on the underlying modeling approach from which it is derived. We demonstrate the use of Bayesian distributional regression models to derive PECs by incorporating spatiotemporal conditional variances, and limits of quantification (LOQ) and detection (LOD) as de facto data censoring. Model accuracies increase when incorporating spatiotemporal conditional variances, and the inclusion of LOQ and LOD results in potentially more robust PEC distributions. The methodology is flexible, credibly quantifies uncertainty, and can be adjusted to different scientific and regulatory needs. Posterior sampling allows to express PECs as distributions, which makes this modeling procedure directly compatible with other Bayesian ERA approaches. We recommend the use of Bayesian modeling approaches with chemical monitoring data to make realistic and robust PEC estimations and encourage the scientific debate about the benefits and challenges of Bayesian methodologies in the context of ERA.

Effects of artificial ultraviolet B radiation on the macrophyte Lemna minor: a conceptual study for toxicity pathway characterization.

MAIN CONCLUSION: UVB radiation caused irradiance-dependent and target-specific responses in non-UVB acclimated Lemna minor. Conceptual toxicity pathways were developed to propose causal relationships between UVB-mediated effects at multiple levels of biological organisation. Macrophytes inhabit waterways around the world and are used in hydroponics or aquaponics for different purposes such as feed and wastewater treatment and are thus exposed to elevated levels of UVB from natural and artificial sources. Although high UVB levels are harmful to macrophytes, mechanistic understanding of irradiance-dependent effects and associated modes of action in non-UVB acclimated plants still remains low. The present study was conducted to characterise the irradiance-dependent mechanisms of UVB leading to growth inhibition in Lemna minor as an aquatic macrophyte model. The L. minor were continuously exposed to UVB (0.008-4.2 W m-2) and constant UVA (4 W m-2) and photosynthetically active radiation, PAR (80 µmol m-2 s-1) for 7 days. A suite of bioassays was deployed to assess effects on oxidative stress, photosynthesis, DNA damage, and transcription of antioxidant biosynthesis, DNA repair, programmed cell death, pigment metabolism and respiration. The results showed that UVB triggered both irradiance-dependent and target-specific effects at multiple levels of biological organization, whereas exposure to UVA alone did not cause any effects. Inhibition of photosystem II and induction of carotenoids were observed at 0.23 W m-2, whereas growth inhibition, excessive reactive oxygen species, lipid peroxidation, cyclobutane pyrimidine dimer formation, mitochondrial membrane potential reduction and chlorophyll depletion were observed at 0.5-1 W m-2. Relationships between responses at different levels of biological organization were used to establish a putative network of toxicity pathways to improve our understanding of UVB effects in aquatic macrophytes under continuous UVB exposures. Additional studies under natural illuminations were proposed to assess whether these putative toxicity pathways may also be relevant for more ecologically relevant exposure scenarios.

De Novo Development of a Quantitative Adverse Outcome Pathway (qAOP) Network for Ultraviolet B (UVB) Radiation Using Targeted Laboratory Tests and Automated Data Mining.

Ultraviolet B (UVB) radiation is a natural nonchemical stressor posing potential hazards to organisms such as planktonic crustaceans. The present study was conducted to revisit the lethal effects of UVB on crustaceans, generate new experimental evidence to fill in knowledge gaps, and develop novel quantitative adverse outcome pathways (qAOPs) for UVB. A combination of laboratory and computational approaches was deployed to achieve the goals. For targeted laboratory tests, Daphnia magna was used as a prototype and exposed to a gradient of artificial UVB. Targeted bioassays were used to quantify the effects of UVB at multiple levels of biological organization. A toxicity pathway network was assembled based on the new experimental evidence and previously published data extracted using a novel computational tool, the NIVA Risk Assessment Database (NIVA RAdb). A network of AOPs was developed, and weight of evidence was assessed based on a combination of the current and existing data. In addition, quantitative key event relationships in the AOPs were developed by fitting the D. magna data to predefined models. A complete workflow for assembly and evaluation of qAOPs has been presented, which may serve as a good example for future de novo qAOP development for chemical and nonchemical stressors.

Epigenetic, transcriptional and phenotypic responses in Daphnia magna exposed to low-level ionizing radiation.

Ionizing radiation is known to induce oxidative stress and DNA damage as well as epigenetic effects in aquatic organisms. Epigenetic changes can be part of the adaptive responses to protect organisms from radiation-induced damage, or act as drivers of toxicity pathways leading to adverse effects. To investigate the potential roles of epigenetic mechanisms in low-dose ionizing radiation-induced stress responses, an ecologically relevant crustacean, adult Daphnia magna were chronically exposed to low and medium level external 60Co gamma radiation ranging from 0.4, 1, 4, 10, and 40 mGy/h for seven days. Biological effects at the molecular (global DNA methylation, histone modification, gene expression), cellular (reactive oxygen species formation), tissue/organ (ovary, gut and epidermal histology) and organismal (fecundity) levels were investigated using a suite of effect assessment tools. The results showed an increase in global DNA methylation associated with loci-specific alterations of histone H3K9 methylation and acetylation, and downregulation of genes involved in DNA methylation, one-carbon metabolism, antioxidant defense, DNA repair, apoptosis, calcium signaling and endocrine regulation of development and reproduction. Temporal changes of reactive oxygen species (ROS) formation were also observed with an apparent transition from ROS suppression to induction from 2 to 7 days after gamma exposure. The cumulative fecundity, however, was not significantly changed by the gamma exposure. On the basis of the new experimental evidence and existing knowledge, a hypothetical model was proposed to provide in-depth mechanistic understanding of the roles of epigenetic mechanisms in low dose ionizing radiation induced stress responses in D. magna.

No evidence of a protective or cumulative negative effect of UV-B on growth inhibition induced by gamma radiation in Scots pine (Pinus sylvestris) seedlings.

Exposure to ambient UV-B radiation may prime protective responses towards various stressors in plants, though information about interactive effects of UV-B and gamma radiation is scarce. Here, we aimed to test whether UV-B exposure could prime acclimatisation mechanisms contributing to tolerance to low-moderate gamma radiation levels in Scots pine seedlings, and concurrently whether simultaneous UV-B and gamma exposure may have an additive adverse effect on seedlings that had previously not encountered either of these stressors. Responses to simultaneous UV-B (0.35 W m-2) and gamma radiation (10.2-125 mGy h-1) for 6 days with or without UV-B pre-exposure (0.35 W m-2, 4 days) were studied across various levels of organisation, as compared to effects of either radiation type. In contrast to UV-B, and regardless of UV-B presence, gamma radiation at ≥42.9 mGy h-1 caused increased formation of reactive oxygen species and reduced shoot length, and reduced root length at 125 mGy h-1. In all experiments there was a gamma dose rate-dependent increase in DNA damage at ≥10.8 mGy h-1, generally with additional UV-B-induced damage. Gamma-induced growth inhibition and gamma- and UV-B-induced DNA damage were still visible 44 days post-irradiation, even at 20.7 mGy h-1, probably due to genomic instability, but this was reversed after 8 months. In conclusion, there was no evidence of a protective effect of UV-B on gamma-induced growth inhibition and DNA damage in Scots pine, and no additive adverse effect of gamma and UV-B radiation on growth in spite of the additional UV-B-induced DNA damage.

Deciphering the Combined Effects of Environmental Stressors on Gene Transcription: A Conceptual Approach.

The use of classical mixture toxicity models to predict the combined effects of environmental stressors based on toxicogenomics (OMICS) data is still in its infancy. Although several studies have made attempts to implement mixture modeling in OMICS analysis to understand the low-dose interactions of stressors, it is not clear how interactions occur at the molecular level and how results generated from such approaches can be better used to inform future studies and cumulative hazard assessment of multiple stressors. The present work was therefore conducted to propose a conceptual approach for combined effect assessment using global gene expression data, as illustrated by a case study on assessment of combined effects of gamma radiation and depleted uranium (DU) on Atlantic salmon ( Salmo salar). Implementation of the independent action (IA) model in reanalysis of a previously published microarray gene expression dataset was performed to describe gene expression patterns of combined effects and identify key gene sets and pathways that were relevant for understanding the interactive effects of these stressors. By using this approach, 3120 differentially expressed genes (DEGs) were found to display additive effects, whereas 279 (273 synergistic, 6 antagonistic) were found to deviate from additivity. Functional analysis further revealed that multiple toxicity pathways, such as oxidative stress responses, cell cycle regulation, lipid metabolism, and immune responses were enriched by DEGs showing synergistic gene expression. A key toxicity pathway of DNA damage leading to enhanced tumorigenesis signaling is highlighted and discussed in detail as an example of how to take advantage of the approach. Furthermore, a conceptual workflow describing the integration of combined effect modeling, OMICS analysis, and bioinformatics is proposed. The present study presents a conceptual framework for utilizing OMICS data in combined effect assessment and may provide novel strategies for dealing with data analysis and interpretation of molecular responses of multiple stressors.

Ecdysone Receptor Agonism Leading to Lethal Molting Disruption in Arthropods: Review and Adverse Outcome Pathway Development.

Molting is critical for growth, development, reproduction, and survival in arthropods. Complex neuroendocrine pathways are involved in the regulation of molting and may potentially become targets of environmental endocrine disrupting chemicals (EDCs). Based on several known ED mechanisms, a wide range of pesticides has been developed to combat unwanted organisms in food production activities such as agriculture and aquaculture. Meanwhile, these chemicals may also pose hazards to nontarget species by causing molting defects, and thus potentially affecting the health of the ecosystems. The present review summarizes the available knowledge on molting-related endocrine regulation and chemically mediated disruption in arthropods (with special focus on insects and crustaceans), to identify research gaps and develop a mechanistic model for assessing environmental hazards of these compounds. Based on the review, multiple targets of EDCs in the molting processes were identified and the link between mode of action (MoA) and adverse effects characterized to inform future studies. An adverse outcome pathway (AOP) describing ecdysone receptor agonism leading to incomplete ecdysis associated mortality was developed according to the OECD guideline and subjected to weight of evidence considerations by evolved Bradford Hill Criteria. This review proposes the first invertebrate ED AOP and may serve as a knowledge foundation for future environmental studies and AOP development.

Characterizing combined effects of antiestrogenic chemicals on vitellogenin production in rainbow trout (Oncorhynchus mykiss) hepatocytes.

Fish are exposed to a complex mixture of endocrine disrupting compounds (EDC), some of which display antiestrogenic activity leading to suppression of estrogen receptor (ER)- mediated reproductive processes. Although the main mode of action (MoA) of these antiestrogens is to directly interfere with natural ligand binding of the ER, several other MoA have been proposed. The aim of the present study was to characterize single and combined antiestrogenic effects of the aryl hydrocarbon receptor (AhR)-agonist ß-naphthoflavone (BNF) and ER-antagonist 4-hydroxytamoxifen (OHT) on vitellogenin (Vtg) protein using primary rainbow trout (Oncorhynchus mykiss) hepatocytes. Supporting transcriptional analysis of ER-responsive genes (estrogen receptor-α (er-α), vitellogenin-1 (vtg-1), eggshell zona radiata protein (zrp)) and AhR-mediated genes (aryl hydrocarbon receptor-2ß, cytochrome p450-1a (cyp1a)) was performed by qPCR to characterize the antiestrogenic influence on ER- and AhR-mediated responses. Data demonstrated that both BNF and OHT significantly reduced 17ß-estradiol (E2)-induced Vtg protein expression in a concentration responsive manner, whereas exposure to a mixture of these produced an additive antiestrogenic effect. The results observed at the protein level were further supported by transcriptional analysis of ER-responsive genes (er-α, vtg-1, zrp), where only E2-induced vtg-1 gene expression was significantly decreased by OHT and the mixture of OHT and BNF. E2-induced er-α and zrp gene expression was not markedly altered. The significant reduction of E2-induced vtg-1 gene expression by OHT suggested that the antiestrogenic effect of this compound may be associated with ER signaling pathway. Specific genes involved in putative AhR-ER cross-talk were also investigated, however none were directly associated with the compound anti-estrogenic MoA. Although the MoA of the single compounds and mixture were not completely characterized, the present study enhanced our knowledge of the combined toxicity mediated by antiestrogens acting through different MoA.

Release of chitobiase as an indicator of potential molting disruption in juvenile Daphnia magna exposed to the ecdysone receptor agonist 20-hydroxyecdysone.

During arthropod molting, the old exoskeleton is degraded and recycled by the molting fluid. Chitobiase, a major chitinolytic enzyme in the molting fluid, has been widely used as a biomarker to indicate endocrine disruption of molting in arthropods under environmental stress. Although release of chitobiase was extensively studied in organisms exposed to molting-inhibiting chemicals, enzymic association with molting and response of the molting hormone receptor, ecdysone receptor (EcR), is not well understood. The present study was therefore conducted to identify potential linkages between release of chitobiase, molting frequency, and EcR activation in a freshwater crustacean Daphnia magna after short-term (96 hr) exposure to endogenous molting hormone 20-hydroxyecdysone (20E). A suite of bioassays was used for this purpose, including the chitobiase activity, molting frequency, viability, and in vitro EcR activation. Effect concentrations were compared between different assays analyzed. Results showed that exposure to 20E reduced chitobiase release and molting frequency in a concentration-dependent manner. Exposure to as low as 250 nM 20E significantly decreased release of chitobiase after 72 hr exposure, whereas adverse effects on molting frequency and incomplete molting-associated mortality required higher 20E exposure concentrations. The EcR reporter assay further demonstrated that as low as 100 nM 20E may activate EcR in vitro. Data suggest that release of chitobiase may be employed as a sensitive indicator of potential molting disruption in crustaceans after exposure to EcR agonists such as 20E.

Characterizing cytotoxic and estrogenic activity of Arctic char tissue extracts in primary Arctic char hepatocytes.

Contaminants from various anthropogenic activities are detected in the Arctic due to long-range atmospheric transport, ocean currents, and living organisms such as migrating fish or seabirds. Although levels of persistent organic pollutants (POPs) in Arctic fish are generally low, local hot spots of contamination were found in freshwater systems such as Lake Ellasjøen at Bjørnøya (Bear Island, Norway). Higher concentrations of organic halogenated compounds (OHC), and higher levels of cytochrome P450 and DNA-double strand breaks were reported in Arctic char (Salvelinus alpinus) from this lake compared to fish from other lakes on Bjørnøya. Although several of the measured contaminants are potential endocrine disrupters, few studies have investigated potential endocrine disruptive effects of the contaminant cocktail in this fish population. The aim of this study was to compare acutely toxic and estrogenic potency of the cocktail of pollutants as evidenced by cytotoxic and/or estrogenic effects in vitro using extracts of Arctic char livers from contaminated Lake Ellasjøen with those from less contaminated Lake Laksvatn at Bjørnøya. This was performed by in situ sampling and contaminant extraction from liver tissue, followed by chemical analysis and in vitro testing of the following contaminated tissue extracts: F1-nonpolar OHC, F2-polar pesticides and metabolites of OHC, and F3-polar OHC. Contaminant levels were highest in extracts from Ellasjøen fish. The F2 and F3 extracts from Lake Laksvatn and Lake Ellasjøen fish reduced in vitro cell viability at a concentration ratio of 0.03-1 relative to tissue concentration in Arctic char. Only the F3 liver extract from Ellasjøen fish increased in vitro vitellogenin protein expression. Although compounds such as estrogenic OH-PCBs were quantified in Ellasjøen F3 extracts, it remains to be determined which compounds were inducing estrogenic effects.

Mortality and transcriptional effects of inorganic mercury in the marine copepod Calanus finmarchicus.

Inorganic mercury (Hg) is highly toxic to organisms including crustaceans and displays multiple toxic modes of action (MoA). The main aim of this investigation was to assess the acute and sublethal toxicity mediated by mercury chloride (HgCl2) in the marine copepod Calanus finmarchicus. A combination of short-term static studies to determine acute toxicity and a transcriptional investigation to characterize the sublethal MoA of HgCl2 were conducted with an in-house continuous culture of C. finmarchicus. Transcriptional changes were determined by a custom 6.6 k C. finmarchicus Agilent oligonucleotide microarray and quantitative RT-PCR analysis. Data demonstrate that HgCl2 produced a concentration- and time-dependent reduction in survival (NOEC48 h = 6.9 µg/L [Hg2+] and LC50 of 279, 73, 48, and 34 µg/L [Hg2+] after 24, 48, 72, and 96 h, respectively) and that exposure to sublethal concentrations of HgCl2 (5 µg/L [Hg2+]) induced differential expression of 98 features (probes) on the microarray. Gene ontology (GO) and toxicological pathway analyses suggested that the main MOA were (1) uncoupling of mitochondrial oxidative phosphorylation (OXPHOS) and ATP production, (2) oxidative stress and macromolecular damage, (3) inactivation of cellular enzymes, (4) induction of cellular apoptosis and autophagocytosis, (5) over-excitation of glutamate receptors (neurotoxicity), (6) disruption of calcium homeostasis and signaling, and (7) modulation of nuclear receptor activity involved in vitamin D receptor signaling. Quantitative RT-PCR analysis verified that oligoarray performed reliably in terms of specificity and response, thus demonstrating that Hg2+ exerts multiple potential MoA in C. finmarchicus.

Mixture toxicity of five biocides with dissimilar modes of action on the growth and photosystem II efficiency of Chlamydomonas reinhardtii.

Biocides are extensively used and universally distributed. Some are highly toxic to algae, including antifoulants, herbicides, and fungicides. The inhibition of algal growth is an important regulatory endpoint for toxicity assessment of single compounds. However, in the aquatic environment, mixtures of compounds with unknown toxicities and mode of action (MoA) co-exist, making single toxicity assessment inadequate to ensure protection of the aquatic environment. This study aimed to characterize the combined toxicity of five environmentally relevant biocides-aclonifen, bifenox, dichlofluanid, metribuzin, and triclosan-with different MoA on growth and photosystem (PS) II efficiency of Chlamydomonas reinhardtii. For growth inhibition, herbicides bifenox and metribuzin were the most toxic, whereas triclosan was least. Only aclonifen and metribuzin exerted a significant effect on PSII, which was also correlated with reduced algal growth. The combined effect of the five biocides on growth inhibition was predominantly additive and presumed to act by independent MoA with potential antagonism observed only at low concentrations and at shorter duration of exposure. The binary mixture of metribuzin and aclonifen exhibited additive effects on diminished PSII efficiency, and effects were apparently induced by an independent MoA. Potential synergy of this mixture on growth inhibition was identified at the highest concentrations. Growth inhibition was found to be a more valuable endpoint for regulatory studies than PSII inhibition due to its environmental relevance, integration of multiple MoA and sensitivity.

Whole-Organism Transcriptomic Analysis Provides Mechanistic Insight into the Acute Toxicity of Emamectin Benzoate in Daphnia magna.

Emamectin benzoate (EMB) is an antisea lice chemical widely used in the aquaculture that may also unintentionally affect nontarget crustaceans in the environment. Although the adverse effects of this compound are well documented in various species, the full modes of action (MoAs) are still not well characterized. The current study was therefore conducted to characterize the MoAs of EMB and link perturbations of key toxicological pathways to adverse effects in the model freshwater crustacean Daphnia magna. Effects on molting and survival were determined after 48 h exposure to EMB, whereas global transcriptional changes and the ecdysone receptor (EcR) binding potency was determined to characterize the MoA. The results showed that the molting frequency and survival of D. magna decreased in a concentration-dependent manner, and the observed changes could not be attributed to direct interactions with the EcR. Major MoAs such as activation of glutamate-gated chloride channels and gamma-aminobutyric acid signaling, disruption of neuroendocrine regulation of molting, perturbation of energy homeostasis, suppression of DNA repair and induction of programmed cell death were observed by transcriptional analysis and successfully linked to the adverse effects. This study has demonstrated that acute exposure to intermediate and high pM levels of EMB may pose hazards to nontarget crustaceans in the aquatic environment.

Individual and molecular level effects of produced water contaminants on nauplii and adult females of Calanus finmarchicus.

In the Barents Sea region new petroleum fields are discovered yearly and extraction of petroleum products is expected to increase in the upcoming years. Despite enhanced technology and stricter governmental legislation, establishment of the petroleum industry in the Barents Sea may potentially introduce a new source of contamination to the area, as some discharges of produced water will be allowed. Whether the presence of produced water poses a risk to the Arctic marine life remains to be investigated. The aim of this study was to examine effects of exposure to several compounds found in produced water-a mixture of selected organic compounds (APW), radium-226 ((226)Ra), barium (Ba), and a scale inhibitor-on the copepod species Calanus finmarchicus. Experiments were performed using exposure concentrations at realistic levels based on those detected in the vicinity of known discharge points. The influence of lethal and sublethal effects on early life stages was determined and significantly lower survival in the APW exposure groups was found. In the Ba treatment the life stage development did not proceed to the same advanced stages as observed in the control (filtered sea water). The scale inhibitor and (226)Ra treatments showed no significant difference from control. In addition, adult females were exposed to APW, (226)Ra, and a mixture of the two. Both individual-level effects (egg production and feeding) and molecular-level effects (gene expression) were assessed. On the individual level endpoints, only treatments including APW produced an effect compared to control. However, on the molecular level the possibility that also (226)Ra induced toxicologically relevant effects cannot be ruled out.

Automated high-throughput in vitro screening of the acetylcholine esterase inhibiting potential of environmental samples, mixtures and single compounds.

A high-throughput and automated assay for testing the presence of acetylcholine esterase (AChE) inhibiting compounds was developed, validated and applied to screen different types of environmental samples. Automation involved using the assay in 96-well plates and adapting it for the use with an automated workstation. Validation was performed by comparing the results of the automated assay with that of a previously validated and standardised assay for two known AChE inhibitors (paraoxon and dichlorvos). The results show that the assay provides similar concentration-response curves (CRCs) when run according to the manual and automated protocol. Automation of the assay resulted in a reduction in assay run time as well as in intra- and inter-assay variations. High-quality CRCs were obtained for both of the model AChE inhibitors (dichlorvos IC50=120µM and paraoxon IC50=0.56µM) when tested alone. The effect of co-exposure of an equipotent binary mixture of the two chemicals were consistent with predictions of additivity and best described by the concentration addition model for combined toxicity. Extracts of different environmental samples (landfill leachate, wastewater treatment plant effluent, and road tunnel construction run-off) were then screened for AChE inhibiting activity using the automated bioassay, with only landfill leachate shown to contain potential AChE inhibitors. Potential uses and limitations of the assay were discussed based on the present results.

Profiling of the embryonic Atlantic halibut (Hippoglossus hippoglossus L.) transcriptome reveals maternal transcripts as potential markers of embryo quality.

BACKGROUND: Commercial Atlantic halibut (Hippoglossus hippoglossus) farming is restricted by variable oocyte quality, slow growth, and early maturation of male fish. Maternally transferred components regulate early developmental processes; therefore, they have an effect on the future viability of the embryo. Using a newly developed Agilent 10 k custom-made oligonucleotide array, we profiled components of the transcriptome involved in immune defence as well as germline and muscle development during early developmental stages: 8-cell embryos (8CS), germ ring stage (GR), 10-somite stage (10SS), and hatched embryos (HT). In addition, we identified differentially expressed transcripts in low (≤9 ± 3% hatching) and high (≥86 ± 3°% hatching) quality eggs at 8CS to identify potential maternal markers for embryo quality. RESULTS: Out of 2066 differentially expressed transcripts, 160 were identified as maternal transcripts being specifically expressed at 8CS only. Twenty transcripts were differentially expressed in 8-cell embryos between low and high quality egg groups. Several immune-related transcripts were identified as promising molecular markers of hatching success including interferon regulatory factor 7 and mhc class 2A chain. Differential expression was positively validated with quantitative real-time PCR. CONCLUSIONS: We have demonstrated maternal transfer of innate and adaptive immune system transcripts into Atlantic halibut embryos and their relation with future embryo developmental potential. We identified several transcripts as potential molecular markers of embryo quality. The developed microarray represents a useful resource for improving the commercial production of Atlantic halibut.

Hepatic transcriptomic profiling reveals early toxicological mechanisms of uranium in Atlantic salmon (Salmo salar).

BACKGROUND: Uranium (U) is a naturally occurring radionuclide that has been found in the aquatic environment due to anthropogenic activities. Exposure to U may pose risk to aquatic organisms due to its radiological and chemical toxicity. The present study aimed to characterize the chemical toxicity of U in Atlantic salmon (Salmo salar) using depleted uranium (DU) as a test model. The fish were exposed to three environmentally relevant concentrations of DU (0.25, 0.5 and 1.0 mg U/L) for 48 h. Hepatic transcriptional responses were studied using microarrays in combination with quantitative real-time reverse transcription polymerase chain reaction (qPCR). Plasma variables and chromosomal damages were also studied to link transcriptional responses to potential physiological changes at higher levels. RESULTS: The microarray gene expression analysis identified 847, 891 and 766 differentially expressed genes (DEGs) in the liver of salmon after 48 h exposure to 0.25, 0.5 and 1.0 mg/L DU, respectively. These DEGs were associated with known gene ontology functions such as generation of precursor metabolites and energy, carbohydrate metabolic process and cellular homeostasis. The salmon DEGs were then mapped to mammalian orthologs and subjected to protein-protein network and pathway analysis. The results showed that various toxicity pathways involved in mitochondrial functions, oxidative stress, nuclear receptor signaling, organ damage were commonly affected by all DU concentrations. Eight genes representative of several key pathways were further verified using qPCR No significant formation of micronuclei in the red blood cells or alterations of plasma stress variables were identified. CONCLUSION: The current study suggested that the mitochondrion may be a key target of U chemical toxicity in salmon. The induction of oxidative stress and uncoupling of oxidative phosphorylation may be two potential modes of action (MoA) of DU. These MoAs may subsequently lead to downstream events such as apoptosis, DNA repair, hypoxia signaling and immune response. The early toxicological mechanisms of U chemical toxicity in salmon has for the first time been systematically profiled. However, no other physiological changes were observed. Future efforts to link transcriptional responses to adverse effects have been outlined as important for understanding of potential risk to aquatic organisms.

Aryl hydrocarbon receptor agonists in European herring gull (Larus argentatus) eggs from Norway.

Herring gull eggs from two locations in Norway, an island situated in the north (Musvær, 69.88° N, 18.55° E) and an island in the southeast (Reiaren, 59.15° N, 10.46° E) of the country, were analyzed for the presence of aryl hydrocarbon receptor (AhR) agonists. AhR agonist activity was determined using the dioxin-responsive chemically activated luciferase expression (DR-CALUX) assay to calculate the toxic equivalent quotient (TEQ)CALUX. TEQCALUX ranged from 16 to 401 pg TEQ/g lipid in the samples from the north (n = 11) and between 6 and 360 pg TEQ/g lipid (n = 12) in the southeastern samples. The large variance between the individual samples is postulated to be due to different feeding habits of individual birds. The levels of AhR agonists detected might lead to adverse effects for the developing embryo or to a significant increase of contaminant load for human consumers of eggs.