EDA-EMERGE: an FP7 initial training network to equip the next generation of young scientists with the skills to address the complexity of environmental contamination with emerging pollutants

The initial training network consortium novel tools in effect-directed analysis to support the identification and monitoring of emerging toxicants on a European scale (EDA-EMERGE) was formed in response to the seventh EU framework program call to train a new generation of young scientists (13 PhD fellows and 1 postdoctoral fellow) in the interdisciplinary techniques required to meet the major challenges in the monitoring, assessment, and management of toxic pollution in European river basins. This 4-year project is of particular relevance considering the multidisciplinary analytical chemistry and biology skills required to investigate the enormous complexity of contamination, effects, and cause-effect relationships. By integrating innovative mode-of-action-based biodiagnostic tools including in vitro and in vivo tests, transgenic organisms, and ‘omics’ techniques with powerful fractionation and cutting edge, analytical, and computational structure elucidation tools, powerful new EDA approaches are being developed for the identification of toxicants in European surface and drinking waters. Innovative method development by young researchers at major European universities, research centers, and private companies has been closely interlinked with a joint European demonstration program, higher-tier EDA, and specialized training courses and secondments. Using a simplified protocol based on existing EDA tools, EDA-EMERGE fellows are also being trained to organize and run international and interdisciplinary sampling and monitoring campaigns within selected European river basin sites. Strong networking between academia, the private sector, and leading regulators in the field of river basin management and pollution management ensures the relevance of the research for practice and excellent employment opportunities for the fellows. Additionally, an internationally composed advisory board has been tasked to introduce new perspectives on monitoring, assessment, and management of emerging pollutants within and outside of Europe. The combination of cutting edge science with specialized training in complimentary soft skills is being offered with a strong emphasis on commercial exploitation and media competence which further enhances the employability of the fellows in research, academia, and beyond.publishedVersio

Activated STAT3 Is a Novel Regulator of the XRCC1 Promoter and Selectively Increases XRCC1 Protein Levels in Triple Negative Breast Cancer

Base Excision Repair (BER) addresses base lesions and abasic sites induced by exogenous and endogenous stressors. X-ray cross complementing group 1 (XRCC1) functions as a scaffold protein in BER and single-strand break repair (SSBR), facilitating and coordinating repair through its interaction with a host of critical repair proteins. Alterations of XRCC1 protein and gene expression levels are observed in many cancers, including colorectal, ovarian, and breast cancer. While increases in the expression level of XRCC1 are reported, the transcription factors responsible for this up-regulation are not known. In this study, we identify the signal transducer and activator of transcription 3 (STAT3) as a novel regulator of XRCC1 through chromatin immunoprecipitation. Activation of STAT3 through phosphorylation at Y705 by cytokine (IL-6) signaling increases the expression of XRCC1 and the occupancy of STAT3 within the XRCC1 promoter. In triple negative breast cancer, the constitutive activation of STAT3 upregulates XRCC1 gene and protein expression levels. Increased expression of XRCC1 is associated with aggressiveness and resistance to DNA damaging chemotherapeutics. Thus, we propose that activated STAT3 regulates XRCC1 under stress and growth conditions, but constitutive activation in cancers results in dysregulation of XRCC1 and subsequently BER and SSBR

Dihydronicotinamide riboside promotes cell-specific cytotoxicity by tipping the balance between metabolic regulation and oxidative stress.

Nicotinamide adenine dinucleotide (NAD+), the essential cofactor derived from vitamin B3, is both a coenzyme in redox enzymatic processes and substrate in non-redox events; processes that are intimately implicated in all essential bioenergetics. A decrease in intracellular NAD+ levels is known to cause multiple metabolic complications and age-related disorders. One NAD+ precursor is dihydronicotinamide riboside (NRH), which increases NAD+ levels more potently in both cultured cells and mice than current supplementation strategies with nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) or vitamin B3 (nicotinamide and niacin). However, the consequences of extreme boosts in NAD+ levels are not fully understood. Here, we demonstrate the cell-specific effects of acute NRH exposure in mammalian cells. Hepatocellular carcinoma (HepG3) cells show dose-dependent cytotoxicity when supplemented with 100-1000 μM NRH. Cytotoxicity was not observed in human embryonic kidney (HEK293T) cells over the same dose range of NRH. PUMA and BAX mediate the cell-specific cytotoxicity of NRH in HepG3. When supplementing HepG3 with 100 μM NRH, a significant increase in ROS was observed concurrent with changes in the NAD(P)H and GSH/GSSG pools. NRH altered mitochondrial membrane potential, increased mitochondrial superoxide formation, and induced mitochondrial DNA damage in those cells. NRH also caused metabolic dysregulation, altering mitochondrial respiration. Altogether, we demonstrated the detrimental consequences of an extreme boost of the total NAD (NAD+ + NADH) pool through NRH supplementation in HepG3. The cell-specific effects are likely mediated through the different metabolic fate of NRH in these cells, which warrants further study in other systemic models

Use of zebrafish cell lines to assess estrogenic compounds in environmental samples

Aquatic endocrine disrupting chemicals (EDCs) encompass a great diversity of chemicals that affect exposed organisms through different molecular mechanisms, including their ability to bind to estrogen receptors (ERs) and to modulate the transcription of target genes. Till date, most of in vitro assays used to assess xeno-estrogens have been established using human ER in either yeast or mammalian cell lines. However, cross-species differences due to cellular context (metabolic capacities, transcription) or different receptor sequences may lead to significant differences between-assay in estrogenic potency of chemicals and eventually influence the response of complex environmental mixtures. We recently established an in vitro luciferase assay based on stable expression of zebrafish ER subtypes (zfERa, zfERß1, zfERß2) in the ZFL zebrafish liver cell line (Cosnefroy et al 2012). Luciferase induction by 17ß-estradiol (E2) in zfERa and zfERß2 cell lines results in EC 50 s of 0.2 and 0.03 nM, respectively. In the present study, we have implemented this assay to screen and characterize the individual effect of different chemical contaminants, including bisphenols, zearalenone, alkylphenols, parabens and phthalates. Comparison with the response in the human cell based MELN assay revealed differences in both sensitivity (i.e. EC50) and receptor binding potency (i.e. full versus partial agonists). In addition, the ability of the zebrafish in vitro assays to detect active compounds in complex mixtures will be tested by screening organic extracts of river sediment, effluents and surface waters. These results will be compared with those obtained with the MELN assay which will provide new information on fish specific effects of environmental xenoestrogens present in aquatic ecosystems

Toward identification of fish specific estrogens through effect-directed analysis based on zebrafish in vitro and in vivo reporter gene assays

Estrogens are present in all vertebrates, from telesots to mammals, which can bind to and activate estrogen receptors (ERs) through a conserved mechanism. However, existence of cross-species differences as regards ER subtypes and the binding affinity of (xeno)estrogens on ERs, as well as the cellular context may lead to differential ER activation by environmental contaminants when assessing hazard on aquatic organisms. For this purpose, we previously developed, characterized and demonstrated the functionality of stable zebrafish (zf)-ER subtypes (zfERa, zfERb1, zfERb2) in the zebrafish liver (ZFL) cell line and transgenic cyp19a1b-GFP zf embryosto quantify estrogenic activity in complex mixtures. In the present study, we used zebrafish (zf)-based in vitro and in vivo bioassays to assess estrogenic activity in surface waters by investigating 20 French river sites using polar organic chemical integrative sampling (POCIS). Simultaneously, the human MELN bioassay (MCF-7 cells-based, expressing hERa) was used to study cross-species differences. POCIS-based bio-monitoring provide significant information on zf-based in vitro bioassays as zfERs and hERa assays significantly differed at some sites, identifying zf-specific estrogenic activities in surface waters. Higher numbers of POCIS extracts were found to be more active on zfERbs than on zfERa, which is in line with the better sensitivity of zfERbs to steroid estrogens. The responsiveness of zfERb2 to environmental samples is important as this ER subtype is present in fish species but not in humans. In complementary to in vitro assays, the estrogenic activities at the organism level were observed using zf embryo assay at sites that were highly active on in vitro bioassays. These in vivo results are relevant for hazard assessment as they reveal that detected active compounds target radial glial cells expressing brain aromatase which are known to be involved in neurogenesis. In addition, combining these zf-based tools within an EDA approach, we were able to isolate fractions that were active only on zfERb2 subtype, hence reinforcing the hypothesis of species-specificity for, yet unknown, ER ligands. Further investigation using mass spectrometry techniques is foreseen to identify fish-specific active compounds which will allow improving bioassay-based environmental risk assessment towards aquatic species

Dihydroxyacetone suppresses mTOR nutrient signaling and induces mitochondrial stress in liver cells

Dihydroxyacetone (DHA) is the active ingredient in sunless tanning products and a combustion product from e-juices in electronic cigarettes (e-cigarettes). DHA is rapidly absorbed in cells and tissues and incorporated into several metabolic pathways through its conversion to dihydroxyacetone phosphate (DHAP). Previous studies have shown DHA induces cell cycle arrest, reactive oxygen species, and mitochondrial dysfunction, though the extent of these effects is highly cell-type specific. Here, we investigate DHA exposure effects in the metabolically active, HepG3 (C3A) cell line. Metabolic and mitochondrial changes were evaluated by characterizing the effects of DHA in metabolic pathways and nutrient-sensing mechanisms through mTOR-specific signaling. We also examined cytotoxicity and investigated the cell death mechanism induced by DHA exposure in HepG3 cells. Millimolar doses of DHA were cytotoxic and suppressed glycolysis and oxidative phosphorylation pathways. Nutrient sensing through mTOR was altered at both short and long time points. Increased mitochondrial reactive oxygen species (ROS) and mitochondrial-specific injury induced cell cycle arrest and cell death through a non-classical apoptotic mechanism. Despite its carbohydrate nature, millimolar doses of DHA are toxic to liver cells and may pose a significant health risk when higher concentrations are absorbed through e-cigarettes or spray tanning

Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay

Endocrine disrupting chemicals (EDCs) act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As the most commonly used estrogen screening assays are either yeast- or human-cell based systems, the question of their (eco)toxicological relevance when assessing risks for aquatic species can be raised. The present study addresses the use of zebrafish (zf) derived reporter gene assays, both in vitro (i.e. zf liver cell lines stably expressing zfERα, zfERβ1 and zfERβ2 subtypes) and in vivo (i.e. transgenic cyp19a1b-GFP zf embryos), to assess estrogenic contaminants in river waters. By investigating 20 French river sites using passive sampling, high frequencies of in vitro zfER-mediated activities in water extracts were measured. Among the different in vitro assays, zfERβ2 assay was the most sensitive and responsive one, enabling the detection of active compounds at all investigated sites. In addition, comparison with a conventional human-based in vitro assay highlighted sites that were able to active zfERs but not human ER, suggesting the occurrence of zf-specific ER ligands. Furthermore, a significant in vivo estrogenic activity was detected at the most active sites in vitro, with a good accordance between estradiol equivalent (E2-EQ) concentrations derived from both in vitro and in vivo assays. Overall, this study shows the relevance and usefulness of such novel zebrafish-based assays as screening tools to monitor estrogenic activities in complex mixtures such as water extracts. It also supports their preferred use compared to human-based assays to assess the potential risks caused by endocrine disruptive chemicals for aquatic species such as fish

Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases

An integrative approach combining passive sampling, bioassays, and effect‐directed analysis to assess the impact of wastewater effluent

Wastewater treatment plant (WWTP) effluents are major sources of endocrine-disrupting chemicals (EDCs) and other chemicals of toxicological concern for the aquatic environment. In the present study, we used an integrated strategy combining passive sampling (Chemcatcher®), developmental toxicity, and mechanism-based in vitro and in vivo bioassays to monitor the impacts of a WWTP on a river. In vitro screening revealed the WWTP effluent as a source of estrogen, glucocorticoid, and aryl hydrocarbon (AhR) receptor-mediated activities impacting the downstream river site where significant activities were also measured, albeit to a lesser extent than in the effluent. Effect-directed analysis of the effluent successfully identified the presence of potent estrogens (estrone, 17α-ethinylestradiol, and 17β-estradiol) and glucocorticoids (clobetasol propionate and fluticasone propionate) as the major contributors to the observed in vitro activities, even though other unidentified active chemicals were likely present. The impact of the WWTP was also assessed using zebrafish embryo assays, highlighting its ability to induce estrogenic response through up-regulation of the aromatase promoter-dependent reporter gene in the transgenic (cyp19a1b–green fluorescent protein [GFP]) zebrafish assay and to generate teratogenic effects at nonlethal concentrations in the zebrafish embryo toxicity test. The present study argues for the use of such an integrated approach, combining passive sampling, bioassays, and effect-directed analysis, to comprehensively identify endocrine active compounds and associated hazards of WTTP effluents