AI for predicting chemical-effect associations at the chemical universe level-deepFPlearn.

Many chemicals are present in our environment, and all living species are exposed to them. However, numerous chemicals pose risks, such as developing severe diseases, if they occur at the wrong time in the wrong place. For the majority of the chemicals, these risks are not known. Chemical risk assessment and subsequent regulation of use require efficient and systematic strategies. Lab-based methods-even if high throughput-are too slow to keep up with the pace of chemical innovation. Existing computational approaches are designed for specific chemical classes or sub-problems but not usable on a large scale. Further, the application range of these approaches is limited by the low amount of available labeled training data. We present the ready-to-use and stand-alone program deepFPlearn that predicts the association between chemical structures and effects on the gene/pathway level using a combined deep learning approach. deepFPlearn uses a deep autoencoder for feature reduction before training a deep feed-forward neural network to predict the target association. We received good prediction qualities and showed that our feature compression preserves relevant chemical structural information. Using a vast chemical inventory (unlabeled data) as input for the autoencoder did not reduce our prediction quality but allowed capturing a much more comprehensive range of chemical structures. We predict meaningful-experimentally verified-associations of chemicals and effects on unseen data. deepFPlearn classifies hundreds of thousands of chemicals in seconds. We provide deepFPlearn as an open-source and flexible tool that can be easily retrained and customized to different application settings at https://github.com/yigbt/deepFPlearn.

Analysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity.

Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.

Prospects and challenges of multi-omics data integration in toxicology.

Exposure of cells or organisms to chemicals can trigger a series of effects at the regulatory pathway level, which involve changes of levels, interactions, and feedback loops of biomolecules of different types. A single-omics technique, e.g., transcriptomics, will detect biomolecules of one type and thus can only capture changes in a small subset of the biological cascade. Therefore, although applying single-omics analyses can lead to the identification of biomarkers for certain exposures, they cannot provide a systemic understanding of toxicity pathways or adverse outcome pathways. Integration of multiple omics data sets promises a substantial improvement in detecting this pathway response to a toxicant, by an increase of information as such and especially by a systemic understanding. Here, we report the findings of a thorough evaluation of the prospects and challenges of multi-omics data integration in toxicological research. We review the availability of such data, discuss options for experimental design, evaluate methods for integration and analysis of multi-omics data, discuss best practices, and identify knowledge gaps. Re-analyzing published data, we demonstrate that multi-omics data integration can considerably improve the confidence in detecting a pathway response. Finally, we argue that more data need to be generated from studies with a multi-omics-focused design, to define which omics layers contribute most to the identification of a pathway response to a toxicant.

Comparison between transcriptomic responses to short-term stress exposures of a common Holarctic and endemic Lake Baikal amphipods.

BACKGROUND: Lake Baikal is one of the oldest freshwater lakes and has constituted a stable environment for millions of years, in stark contrast to small, transient bodies of water in its immediate vicinity. A highly diverse endemic endemic amphipod fauna is found in one, but not the other habitat. We ask here whether differences in stress response can explain the immiscibility barrier between Lake Baikal and non-Baikal faunas. To this end, we conducted exposure experiments to increased temperature and the toxic heavy metal cadmium as stressors. RESULTS: Here we obtained high-quality de novo transcriptome assemblies, covering mutiple conditions, of three amphipod species, and compared their transcriptomic stress responses. Two of these species, Eulimnogammarus verrucosus and E. cyaneus, are endemic to Lake Baikal, while the Holarctic Gammarus lacustris is a potential invader. CONCLUSIONS: Both Baikal species possess intact stress response systems and respond to elevated temperature with relatively similar changes in their expression profiles. G. lacustris reacts less strongly to the same stressors, possibly because its transcriptome is already perturbed by acclimation conditions.

Can Environmentally Relevant Neuroactive Chemicals Specifically Be Detected with the Locomotor Response Test in Zebrafish Embryos?

Chemicals considered as neuroactive (such as certain pesticides, pharmaceuticals, and industrial chemicals) are among the largest groups of bioactive substances recently detected in European rivers. However, the determination of nervous-system-specific effects has been limited using in vitro tests or conventional end points including lethality. Thus, neurobehavioral tests using in vivo models (e.g., zebrafish embryo) have been proposed as complementary approaches. To investigate the specificity and sensitivity of a light-dark transition locomotor response (LMR) test in 4 to 5 days post fertilization zebrafish with respect to different modes of action (MoAs), we analyzed a set of 18 environmentally relevant compounds with various anticipated MoAs. We found that exposure-induced behavioral alterations were reproducible and dependent on concentration and time. Comparative and quantitative analyses of the obtained locomotor patterns revealed that behavioral effects were not restricted to compounds primarily known to target the nervous system. A clear distinction of MoAs based on locomotor patterns was not possible for most compounds. Furthermore, chemicals with an anticipated same MoA did not necessarily provoke similar behavioral phenotypes. Finally, we determined an increased sensitivity (≥10-fold) compared to observed mortality in the LMR assay for five of eight neuroactive chemicals as opposed to non-neuroactive compounds.

Screening of Pesticide and Biocide Patterns As Risk Drivers in Sediments of Major European River Mouths: Ubiquitous or River Basin-Specific Contamination?

Pesticides and biocides (PaB) are ubiquitously present in aquatic ecosystems due to their widespread application and have been detected in rivers at concentrations that may cause distress to aquatic life. Many of these compounds accumulate in sediments acting as long-term source for aquatic ecosystems. However, data on sediment contamination with current-use PaB in Europe are scarce. Thus, in this study, we elucidated PaB patterns and associated risks in sediments of seven major European rivers focusing on their last stretch as an integrative sink of particles transported by these rivers. Sediments were extracted with pressurized liquid extraction (PLE) using a broad-spectrum method recovering many compound classes with a wide range of physicochemical properties. Altogether 126 compounds were analyzed and 81 of them were detected with LC-HRMS and GC-NCI-MS/MS at least in one of the sediments. The highest number of compounds was detected (59) in River Elbe sediments close to Cuxhaven with outstanding concentrations ranging from 0.8 to 1691 mg/g organic carbon. Multivariate analysis identified a cluster with 3 ubiquitous compounds (cyhalothrin, carbendazim, fenpropimorph) and three clusters of chemicals with higher variability within and between rivers. Risk assessment indicates an acute toxic risk to benthic crustaceans at all investigated sites with the pyrethroids tefluthrin and cyfluthrin together with the fungicide carbendazim as the main drivers. Risks to algae were driven at most sites almost exclusively by photosynthesis inhibitors with estuary-specific herbicide mixtures, while in the rivers Po and Gironde cell division inhibitors played an important role at some sites. Mixtures of specific concern have been defined and suggested for integration in future monitoring programs.

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Curated mode-of-action data and effect concentrations for chemicals relevant for the aquatic environment.

Chemicals in the aquatic environment can be harmful to organisms and ecosystems. Knowledge on effect concentrations as well as on mechanisms and modes of interaction with biological molecules and signaling pathways is necessary to perform chemical risk assessment and identify toxic compounds. To this end, we developed criteria and a pipeline for harvesting and summarizing effect concentrations from the US ECOTOX database for the three aquatic species groups algae, crustaceans, and fish and researched the modes of action of more than 3,300 environmentally relevant chemicals in literature and databases. We provide a curated dataset ready to be used for risk assessment based on monitoring data and the first comprehensive collection and categorization of modes of action of environmental chemicals. Authorities, regulators, and scientists can use this data for the grouping of chemicals, the establishment of meaningful assessment groups, and the development of in vitro and in silico approaches for chemical testing and assessment.

Investigation of Peroxisome Proliferator-Activated Receptor Genes as Requirements for Visual Startle Response Hyperactivity in Larval Zebrafish Exposed to Structurally Similar Per- and Polyfluoroalkyl Substances (PFAS).

BACKGROUND: Per- and polyfluoroalkyl Substances (PFAS) are synthetic chemicals widely detected in humans and the environment. Exposure to perfluorooctanesulfonic acid (PFOS) or perfluorohexanesulfonic acid (PFHxS) was previously shown to cause dark-phase hyperactivity in larval zebrafish. OBJECTIVES: The objective of this study was to elucidate the mechanism by which PFOS or PFHxS exposure caused hyperactivity in larval zebrafish. METHODS: Swimming behavior was assessed in 5-d postfertilization (dpf) larvae following developmental (1-4 dpf) or acute (5 dpf) exposure to 0.43-7.86µM PFOS, 7.87-120µM PFHxS, or 0.4% dimethyl sulfoxide (DMSO). After developmental exposure and chemical washout at 4 dpf, behavior was also assessed at 5-8 dpf. RNA sequencing was used to identify differences in global gene expression to perform transcriptomic benchmark concentration-response (BMCT) modeling, and predict upstream regulators in PFOS- or PFHxS-exposed larvae. CRISPR/Cas9-based gene editing was used to knockdown peroxisome proliferator-activated receptors (ppars) pparaa/ab, pparda/db, or pparg at day 0. Knockdown crispants were exposed to 7.86µM PFOS or 0.4% DMSO from 1-4 dpf and behavior was assessed at 5 dpf. Coexposure with the ppard antagonist GSK3787 and PFOS was also performed. RESULTS: Transient dark-phase hyperactivity occurred following developmental or acute exposure to PFOS or PFHxS, relative to the DMSO control. In contrast, visual startle response (VSR) hyperactivity only occurred following developmental exposure and was irreversible up to 8 dpf. Similar global transcriptomic profiles, BMCT estimates, and enriched functions were observed in PFOS- and PFHxS-exposed larvae, and ppars were identified as putative upstream regulators. Knockdown of pparda/db, but not pparaa/ab or pparg, blunted PFOS-dependent VSR hyperactivity to control levels. This finding was confirmed via antagonism of ppard in PFOS-exposed larvae. DISCUSSION: This work identifies a novel adverse outcome pathway for VSR hyperactivity in larval zebrafish. We demonstrate that developmental, but not acute, exposure to PFOS triggered persistent VSR hyperactivity that required ppard function. https://doi.org/10.1289/EHP13667.

A data-derived reference mixture representative of European wastewater treatment plant effluents to complement mixture assessment.

Aquatic environments are polluted with a multitude of organic micropollutants, which challenges risk assessment due the complexity and diversity of pollutant mixtures. The recognition that certain source-specific background pollution occurs ubiquitously in the aquatic environment might be one way forward to approach mixture risk assessment. To investigate this hypothesis, we prepared one typical and representative WWTP effluent mixture of organic micropollutants (EWERBmix) comprised of 81 compounds selected according to their high frequency of occurrence and toxic potential. Toxicological relevant effects of this reference mixture were measured in eight organism- and cell-based bioassays and compared with predicted mixture effects, which were calculated based on effect data of single chemicals retrieved from literature or different databases, and via quantitative structure-activity relationships (QSARs). The results show that the EWERBmix supports the identification of substances which should be considered in future monitoring efforts. It provides measures to estimate wastewater background concentrations in rivers under consideration of respective dilution factors, and to assess the extent of mixture risks to be expected from European WWTP effluents. The EWERBmix presents a reasonable proxy for regulatory authorities to develop and implement assessment approaches and regulatory measures to address mixture risks. The highlighted data gaps should be considered for prioritization of effect testing of most prevalent and relevant individual organic micropollutants of WWTP effluent background pollution. The here provided approach and EWERBmix are available for authorities and scientists for further investigations. The approach presented can furthermore serve as a roadmap guiding the development of archetypic background mixtures for other sources, geographical settings and chemical compounds, e.g. inorganic pollutants.

A metabolomics approach to reveal the mechanism of developmental toxicity in zebrafish embryos exposed to 6-propyl-2-thiouracil.

A crucial component of a substance registration and regulation is the evaluation of human prenatal developmental toxicity. Current toxicological tests are based on mammalian models, but these are costly, time consuming and may pose ethical concerns. The zebrafish embryo has evolved as a promising alternative model to study developmental toxicity. However, the implementation of the zebrafish embryotoxicity test is challenged by lacking information on the relevance of observed morphological alterations in fish for human developmental toxicity. Elucidating the mechanism of toxicity could help to overcome this limitation. Through LC-MS/MS and GC-MS metabolomics, we investigated whether changes to the endogenous metabolites can indicate pathways associated with developmental toxicity. To this aim, zebrafish embryos were exposed to different concentrations of 6-propyl-2-thiouracil (PTU), a compound known to induce developmental toxicity. The reproducibility and the concentration-dependence of the metabolome response and its association with morphological alterations were studied. Major morphological findings were reduced eye size, and other craniofacial anomalies; major metabolic changes included increased tyrosine, pipecolic acid and lysophosphatidylcholine levels, decreased methionine levels, and disturbance of the 'Phenylalanine, tyrosine and tryptophan biosynthesis' pathway. This pathway, and the changes in tyrosine and pipecolic acid levels could be linked to the mode of action of PTU, i.e., inhibition of thyroid peroxidase (TPO). The other findings suggested neurodevelopmental impairments. This proof-of-concept study demonstrated that metabolite changes in zebrafish embryos are robust and provide mechanistic information associated with the mode of action of PTU.

New approach methodologies in human regulatory toxicology - Not if, but how and when!

The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of "New Approach Methodologies" (NAMs). While this term does not define the age or the state of readiness of the innovation, it covers a wide range of methods, including quantitative structure-activity relationship (QSAR) predictions, high-throughput screening (HTS) bioassays, omics applications, cell cultures, organoids, microphysiological systems (MPS), machine learning models and artificial intelligence (AI). In addition to promising faster and more efficient toxicity testing, NAMs have the potential to fundamentally transform today's regulatory work by allowing more human-relevant decision-making in terms of both hazard and exposure assessment. Yet, several obstacles hamper a broader application of NAMs in current regulatory risk assessment. Constraints in addressing repeated-dose toxicity, with particular reference to the chronic toxicity, and hesitance from relevant stakeholders, are major challenges for the implementation of NAMs in a broader context. Moreover, issues regarding predictivity, reproducibility and quantification need to be addressed and regulatory and legislative frameworks need to be adapted to NAMs. The conceptual perspective presented here has its focus on hazard assessment and is grounded on the main findings and conclusions from a symposium and workshop held in Berlin in November 2021. It intends to provide further insights into how NAMs can be gradually integrated into chemical risk assessment aimed at protection of human health, until eventually the current paradigm is replaced by an animal-free "Next Generation Risk Assessment" (NGRA).

Mixture toxicity revisited from a toxicogenomic perspective.

The advent of new genomic techniques has raised expectations that central questions of mixture toxicology such as for mechanisms of low dose interactions can now be answered. This review provides an overview on experimental studies from the past decade that address diagnostic and/or mechanistic questions regarding the combined effects of chemical mixtures using toxicogenomic techniques. From 2002 to 2011, 41 studies were published with a focus on mixture toxicity assessment. Primarily multiplexed quantification of gene transcripts was performed, though metabolomic and proteomic analysis of joint exposures have also been undertaken. It is now standard to explicitly state criteria for selecting concentrations and provide insight into data transformation and statistical treatment with respect to minimizing sources of undue variability. Bioinformatic analysis of toxicogenomic data, by contrast, is still a field with diverse and rapidly evolving tools. The reported combined effect assessments are discussed in the light of established toxicological dose-response and mixture toxicity models. Receptor-based assays seem to be the most advanced toward establishing quantitative relationships between exposure and biological responses. Often transcriptomic responses are discussed based on the presence or absence of signals, where the interpretation may remain ambiguous due to methodological problems. The majority of mixture studies design their studies to compare the recorded mixture outcome against responses for individual components only. This stands in stark contrast to our existing understanding of joint biological activity at the levels of chemical target interactions and apical combined effects. By joining established mixture effect models with toxicokinetic and -dynamic thinking, we suggest a conceptual framework that may help to overcome the current limitation of providing mainly anecdotal evidence on mixture effects. To achieve this we suggest (i) to design studies to establish quantitative relationships between dose and time dependency of responses and (ii) to adopt mixture toxicity models. Moreover, (iii) utilization of novel bioinformatic tools and (iv) stress response concepts could be productive to translate multiple responses into hypotheses on the relationships between general stress and specific toxicity reactions of organisms.

A risk based assessment approach for chemical mixtures from wastewater treatment plant effluents.

In this study, 56 effluent samples from 52 European wastewater treatment plants (WWTPs) were investigated for the occurrence of 499 emerging chemicals (ECs) and their associated potential risks to the environment. The two main objectives were (i) to extend our knowledge on chemicals occurring in treated wastewater, and (ii) to identify and prioritize compounds of concern based on three different risk assessment approaches for the identification of consensus mixture risk drivers of concern. Approaches include (i) PNEC and EQS-based regulatory risk quotients (RQs), (ii) species sensitivity distribution (SSD)-based hazard units (HUs) and (iii) toxic units (TUs) for three biological quality elements (BQEs) algae, crustacean, and fish. For this purpose, solid-phase extracts were analysed with wide-scope chemical target screening via liquid chromatography high-resolution mass spectrometry (LC-HRMS), resulting in 366 detected compounds, with concentrations ranging from < 1 ng/L to > 100 µg/L. The detected chemicals were categorized with respect to critical information relevant for risk assessment and management prioritization including: (1) frequency of occurrence, (2) measured concentrations, (3) use groups, (4) persistence & bioaccumulation, and (5) modes of action. A comprehensive assessment using RQ, HU and TU indicated exceedance of risk thresholds for the majority of effluents with RQ being the most sensitive metric. In total, 299 out of the 366 compounds were identified as mixture risk contributors in one of the approaches, while 32 chemicals were established as consensus mixture risk contributors of high concern, including a high percentage (66%) of pesticides and biocides. For samples which have passed an advanced treatment using ozonation or activated carbon (AC), consistently much lower risks were estimated.

Disentangling multiple chemical and non-chemical stressors in a lotic ecosystem using a longitudinal approach.

Meeting ecological and water quality standards in lotic ecosystems is often failed due to multiple stressors. However, disentangling stressor effects and identifying relevant stressor-effect-relationships in complex environmental settings remain major challenges. By combining state-of-the-art methods from ecotoxicology and aquatic ecosystem analysis, we aimed here to disentangle the effects of multiple chemical and non-chemical stressors along a longitudinal land use gradient in a third-order river in Germany. We distinguished and evaluated four dominant stressor categories along this gradient: (1) Hydromorphological alterations: Flow diversity and substrate diversity correlated with the EU-Water Framework Directive based indicators for the quality element macroinvertebrates, which deteriorated at the transition from near-natural reference sites to urban sites. (2) Elevated nutrient levels and eutrophication: Low to moderate nutrient concentrations together with complete canopy cover at the reference sites correlated with low densities of benthic algae (biofilms). We found no more systematic relation of algal density with nutrient concentrations at the downstream sites, suggesting that limiting concentrations are exceeded already at moderate nutrient concentrations and reduced shading by riparian vegetation. (3) Elevated organic matter levels: Wastewater treatment plants (WWTP) and stormwater drainage systems were the primary sources of bioavailable dissolved organic carbon. Consequently, planktonic bacterial production and especially extracellular enzyme activity increased downstream of those effluents showing local peaks. (4) Micropollutants and toxicity-related stress: WWTPs were the predominant source of toxic stress, resulting in a rapid increase of the toxicity for invertebrates and algae with only one order of magnitude below the acute toxic levels. This toxicity correlates negatively with the contribution of invertebrate species being sensitive towards pesticides (SPEARpesticides index), probably contributing to the loss of biodiversity recorded in response to WWTP effluents. Our longitudinal approach highlights the potential of coordinated community efforts in supplementing established monitoring methods to tackle the complex phenomenon of multiple stress.

Tungsten carbide cobalt nanoparticles exert hypoxia-like effects on the gene expression level in human keratinocytes.

BACKGROUND: Tungsten carbide (WC) and tungsten carbide cobalt (WC-Co) nanoparticles are of occupational health relevance because of the increasing usage in hard metal industries. Earlier studies showed an enhanced toxic potential for WC-Co compared to WC or cobalt ions alone. Therefore, we investigated the impact of these particles, compared to cobalt ions applied as CoCl(2), on the global gene expression level in human keratinocytes (HaCaT) in vitro. RESULTS: WC nanoparticles exerted very little effects on the transcriptomic level after 3 hours and 3 days of exposure. In contrast, WC-Co nanoparticles caused significant transcriptional changes that were similar to those provoked by CoCl(2). However, CoCl(2) exerted even more pronounced changes in the transcription patterns. Gene set enrichment analyses revealed that the differentially expressed genes were related to hypoxia response, carbohydrate metabolism, endocrine pathways, and targets of several transcription factors. The role of the transcription factor HIF1 (hypoxia inducible factor 1) is particularly highlighted and aspects of downstream events as well as the role of other transcription factors related to cobalt toxicity are considered. CONCLUSIONS: This study provides extensive data useful for the understanding of nanoparticle and cobalt toxicity. It shows that WC nanoparticles caused low transcriptional responses while WC-Co nanoparticles are able to exert responses similar to that of free cobalt ions, particularly the induction of hypoxia-like effects via interactions with HIF1alpha in human keratinocytes. However, the enhanced toxicity of WC-Co particles compared to CoCl(2) could not be explained by differences in gene transcription.

A Self-Organizing Map of the Fathead Minnow Liver Transcriptome to Identify Consistent Toxicogenomic Patterns across Chemical Fingerprints.

Lack of consistent findings in different experimental settings remains a major challenge in toxicogenomics. The present study investigated whether consistency between findings of different microarray experiments can be improved when the analysis is based on a common reference frame ("toxicogenomic universe"), which can be generated using the machine learning algorithm of the self-organizing map (SOM). This algorithm arranges and clusters genes on a 2-dimensional grid according to their similarity in expression across all considered data. In the present study, 19 data sets, comprising of 54 different adult fathead minnow liver exposure experiments, were retrieved from Gene Expression Omnibus and used to train a SOM. The resulting toxicogenomic universe aggregates 58 872 probes to 2500 nodes and was used to project, visualize, and compare the fingerprints of these 54 different experiments. For example, we could identify a common pattern, with 14% of significantly regulated nodes in common, in the data sets of an interlaboratory study of ethinylestradiol exposures. Consistency could be improved compared with the 5% total overlap in regulated genes reported before. Furthermore, we could determine a specific and consistent estrogen-related pattern of differentially expressed nodes and clusters in the toxicogenomic universe by applying additional clustering steps and comparing all obtained fingerprints. Our study shows that the SOM-based approach is useful for generating comparable toxicogenomic fingerprints and improving consistency between results of different experiments. Environ Toxicol Chem 2020;39:526-537. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Transcriptome-level effects of the model organic pollutant phenanthrene and its solvent acetone in three amphipod species.

Polyaromatic hydrocarbons (PAH) are common pollutants of water ecosystems originating from incineration processes and contamination with mineral oil. Water solubility of PAHs is generally low; for toxicity tests with aquatic organisms, they are therefore usually dissolved in organic solvents. Here we examined the effects of a typical model PAH, phenanthrene, and a solvent, acetone, on amphipods as relevant aquatic invertebrate models. Two of these species, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, are common endemics of the oligotrophic and pristine Lake Baikal, while one, Gammarus lacustris, is widespread throughout the Holarctic and inhabits smaller and more eutrophic water bodies in the Baikal area. Neither solvent nor phenanthrene caused mortality at the applied concentrations, but both substances affected gene expression in all species. Differential gene expression was more profound in the species from Lake Baikal than in the Holarctic species. Moreover, in one of the Baikal species, E. cyaneus, we found that many known components of the cellular xenobiotic detoxification system reacted to the treatments. Finally, we detected a negative relationship between changes in transcript abundances in response to the solvent and phenanthrene. This mixture effect, weaker than the impact by a single mixture component, needs further exploration.

Environmental risk or benefit? Comprehensive risk assessment of groundwater treated with nano Fe0-based Carbo-Iron®.

Groundwater is essential for the provision of drinking water in many areas around the world. The performance of the groundwater-bearing aquifer relies on the ecosystem services provided by groundwater-related organisms. Therefore, if remediation of contaminated groundwater is necessary, the remediation method has to be carefully selected to avoid risk-risk trade-offs that might impact these ecosystems. In the present study, the environmental risk of the in situ remediation agent Carbo-Iron was performed. Carbo-Iron® is a composite of zero valent nano-iron and active carbon. Existing ecotoxicity data were complemented by studies with Daphnia magna (Crustacea), Scenedesmus vacuolatus (Algae), Chironomus riparius (Insecta) and nitrifying soil microorganisms. The predicted no effect concentration of 0.1 mg/L was derived from acute and chronic ecotoxicity studies. It was compared to measured and modelled environmental concentrations of Carbo-Iron applied in a groundwater contaminated with chlorohydrocarbons in a field study and risk ratios were derived. A comprehensive assessment approach was developed further based on existing strategies and used to identify changes of the environmental risk due to the remediation of the contaminated site with Carbo-Iron. With the data used in the present study, the total environmental risk decreased by approximately 50% in the heavily contaminated zones after the application of Carbo-Iron. Thus, based on the results of the present study, the benefit of remediation with Carbo-Iron seems to outweigh its negative effects on the environment.

Combined effects of environmental xeno-estrogens within multi-component mixtures: Comparison of in vitro human- and zebrafish-based estrogenicity bioassays.

Some recent studies showed that in vitro bioassays based on fish or human estrogen receptor (ER) activation may have distinct responses to environmental samples, highlighting the need to better understand bioassay-specific ER response to environmental mixtures. For this purpose, we investigated a 12-compound mixture in two mixture ratios (M1 and M2) on zebrafish (zf) liver cells stably expressing zfERα (ZELHα cells) or zfERß2 (ZELHß2 cells) and on human ER-reporter gene (MELN) cells. The mixture included the well-known ER ligands bisphenol A (BPA) and genistein (GEN), and other compounds representatives of a freshwater background contamination. In this context, the study aimed at assessing the robustness of concentration addition (CA) model and the potential confounding influence of other chemicals by testing subgroups of ER activators, ER inhibitors or ER activators and inhibitors combined. Individual chemical testing showed a higher prevalence of ER inhibitors in zebrafish than human cells (e.g. propiconazole), and some chemicals inhibited zfER but activated hER response (e.g. benzo(a)pyrene, triphenylphosphate). The estrogenic activity of M1 and M2 was well predicted by CA in MELN cells, whereas it was significantly lower than predicted in ZELHß2 cells, contrasting with the additive effects observed for BPA and GEN binary mixtures. When testing the subgroups of ER activators and inhibitors combined, the deviation from additivity in ZELHß2 cells was caused by zebrafish-specific inhibiting chemicals. This study provides novel information on the ability of environmental pollutants to interfere with zfER signalling and shows that non-estrogenic chemicals can influence the response to a mixture of xeno-estrogens in a bioassay-specific manner.