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  • 1
    Language: English
    In: Environmental Science and Pollution Research, 2015, Vol.22(21), pp.16384-16392
    Description: Transcriptomics is often used to investigate changes in an organism’s genetic response to environmental contamination. Data noise can mask the effects of contaminants making it difficult to detect responding genes. Because the number of genes which are found differentially expressed in transcriptome data is often very large, algorithms are needed to reduce the number down to a few robust discriminative genes. We present an algorithm for aggregated analysis of transcriptome data which uses multiple fold-change thresholds (threshold screening) and p values from Bayesian generalized linear model in order to assess the robustness of a gene as a potential indicator for the treatments tested. The algorithm provides a robustness indicator (ROBI) as well as a significance profile, which can be used to assess the statistical significance of a given gene for different fold-change thresholds. Using ROBI, eight discriminative genes were identified from an exemplary dataset ( Danio rerio FET treated with chlorpyrifos, methylmercury, and PCB) which could be potential indicators for a given substance. Significance profiles uncovered genetic effects and revealed appropriate fold-change thresholds for single genes or gene clusters. Fold-change threshold screening is a powerful tool for dimensionality reduction and feature selection in transcriptome data, as it effectively reduces the number of detected genes suitable for environmental monitoring. In addition, it is able to unmask patterns in altered genetic expression hidden by data noise and reduces the chance of type II errors, e.g., in environmental screening.
    Keywords: Bioinformatics ; Masked effects ; Danio rerio ; Aggregated analysis ; Ecotoxicogenomics ; Robustness indicator (ROBI) ; Bayesian generalized linear model
    ISSN: 0944-1344
    E-ISSN: 1614-7499
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  • 2
    Language: English
    In: PLoS ONE, 01 January 2014, Vol.9(9), p.e106523
    Description: PURPOSE: Recently, a proof-of-concept study revealed the suitability of transcriptome analyses to obtain and assess changes in the abundance of transcripts in zebrafish (Danio rerio) embryos after exposure to organic sediment extracts. The present...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 3
  • 4
    Language: English
    In: PLoS ONE, 01 January 2014, Vol.9(2), p.e90036
    Description: Automated analysis of multi-dimensional microscopy images has become an integral part of modern research in life science. Most available algorithms that provide sufficient segmentation quality, however, are infeasible for a large amount of data...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 5
    Language: English
    In: PLoS ONE, 01 January 2013, Vol.8(10), p.e75596
    Description: The estuary of the River Elbe between Hamburg and the North Sea (Germany) is a sink for contaminated sediment and suspended particulate matter (SPM). One major concern is the effect of human activities on the hydrodynamics, particularly the intensive dredging activities in this area that may...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 6
  • 7
    Language: English
    In: Environmental Science and Pollution Research, 2012, Vol.19(2), pp.403-415
    Description: Byline: Bernhard Wetterauer (1), Mathias Ricking (3), Jens C. Otte (1,2), Arnold V. Hallare (6,7), Andrew Rastall (4), Lothar Erdinger (4), Jan Schwarzbauer (5), Thomas Braunbeck (1), Henner Hollert (1,6) Keywords: Cytotoxicity; DDA; DDCN; DDMS; DDT metabolites; DDMU; Dioxin-like activity; Dot blot/RNAse protection-assay; EROD; Estrogenic activity; Yeast estrogen screen Abstract: Background, aim, and scope 2,2-bis(chlorophenyl)-1,1,1-trichloroethane (DDT) metabolites, other than those routinely measured [i.e., 2,2-bis(chlorophenyl)-1,1-dichloroethylene (DDE) and 2,2-bis(chlorophenyl)-1,1-dichloroethane (DDD)], have recently been detected in elevated concentrations not only in the surface water of Teltow Canal, Berlin, but also in sediment samples from Elbe tributaries (e.g., Mulde and Havel/Spree). This was paralleled by recent reports that multiple other metabolites could emerge from the degradation of parent DDT by naturally occurring organisms or by interaction with some heavy metals. Nevertheless, only very few data on the biological activities of these metabolites are available to date. The objective of this communication is to evaluate, for the first time, the cytotoxicity, dioxin-like activity, and estrogenicity of the least-studied DDT metabolites. Methods Four DDT metabolites, p,p -2,2-bis(chlorophenyl)-1-chloroethylene (DDMU), p,p -2,2-bis(chlorophenyl)-1-chloroethane (DDMS), p,p -2,2-bis(4-ch1oropheny1)acetonitrile (DDCN), and p,p -2,2-bis(chlorophenyl)acetic acid (DDA), were selected based on their presence in environmental samples in Germany such as in sediments from the Mulde River and Teltow Canal. O,p -DDT was used as reference in all assays. Cytotoxicity was measured by neutral red retention with the permanent cell line RTG-2 of rainbow trout (Oncorhynchus mykiss). Dioxin-like activity was determined using the 7-ethoxyresorufin-O-deetylase assay. The estrogenic potential was tested in a dot blot/RNAse protection-assay with primary hepatocytes from male rainbow trout (O. mykiss) and in a yeast estrogen screen (YES) assay. Results All DDT metabolites tested revealed a clear dose--response relationship for cytotoxicity in RTG-2 cells, but no dioxin-like activities with RTL-W1 cells. The dot blot/RNAse protection-assay demonstrated that the highest non-toxic concentrations of these DDT metabolites (50 uM) had vitellogenin-induction potentials comparable to the positive control (1 nM 17[beta]-estradiol). The estrogenic activities could be ranked as o,p -DDT 〉 p,p -DDMS 〉 p,p -DDMU 〉 p,p -DDCN. In contrast, p,p -DDA showed a moderate anti-estrogenic effect. In the YES assay, besides the reference o,p -DDT, p,p -DDMS and p,p -DDMU displayed dose-dependent estrogenic potentials, whereas p,p -DDCN and p,p -DDA did not show any estrogenic potential. Discussion The reference toxicant o,p -DDT displayed a similar spectrum of estrogenic activities similar to 17[beta]-estradiol, however, with a lower potency. Both p,p -DDMS and p,p -DDMU were also shown to have dose-dependent estrogenic potentials, which were much lower than the reference o,p -DDT, in both the vitellogenin and YES bioassays. Interestingly, p,p -DDA did not show estrogenic activity but rather displayed a tendency towards anti-estrogenic activity by inhibiting the estrogenic effect of 17[beta]-estradiol. The results also showed that the p,p -metabolites DDMU, DDMS, DDCN, and DDA do not show any dioxin-like activities in RTL-W1 cells, thus resembling the major DDT metabolites DDD and DDE. Conclusions All the DDT metabolites tested did not exhibit dioxin-like activities in RTL-W1 cells, but show cytotoxic and estrogenic activities. Based on the results of the in vitro assays used in our study and on the reported concentrations of DDT metabolites in contaminated sediments, such substances could, in the future, pose interference with the normal reproductive and endocrine functions in various organisms exposed to these chemicals. Consequently, there is an urgent need to examine more comprehensively the risk of environmental concentrations of the investigated DDT metabolites using in vivo studies. However, this should be paralleled also by periodic evaluation and monitoring of the current levels of the DDT metabolites in environmental matrices. Recommendations and perspectives Our results clearly point out the need to integrate the potential ecotoxicological risks associated with the "neglected" p,p -DDT metabolites. For instance, these DDT metabolites should be integrated into sediment risk assessment initiatives in contaminated areas. One major challenge would be the identification of baseline data for such risk assessment. Further studies are also warranted to determine possible additive, synergistic, or antagonistic effects that may interfere with the fundamental cytotoxicity and endocrine activities of these metabolites. For a more conclusive assessment of the spectrum of DDT metabolites, additional bioassays are needed to identify potential anti-estrogenic, androgenic, and/or anti-androgenic effects. Author Affiliation: (1) Aquatic Ecology and Toxicology, COS-Center for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, 69120, Heidelberg, Germany (2) Karlsruhe Institute of Technology, Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021, Karlsruhe, Germany (3) Department of Earth Sciences, Free University of Berlin, Malteserstr. 74-100, 12249, Berlin, Germany (4) Hygiene and Medical Microbiology, Department of Hygiene, University of Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany (5) Institute of Geology and Geochemistry of Petroleum and Coal, RWTH Aachen University, Lochnerstr. 4-20, 52056, Aachen, Germany (6) Department of Ecosystem Analysis, Institute for Environmental Research (Biology V), RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany (7) Department of Biology, CAS, University of the Philippines, Padre Faura, Manila, Philippines Article History: Registration Date: 11/07/2011 Received Date: 11/01/2010 Accepted Date: 26/05/2011 Online Date: 27/07/2011 Article note: Responsible editor: Markus Hecker
    Keywords: Cytotoxicity ; DDA ; DDCN ; DDMS ; DDT metabolites ; DDMU ; Dioxin-like activity ; Dot blot/RNAse protection-assay ; EROD ; Estrogenic activity ; Yeast estrogen screen
    ISSN: 0944-1344
    E-ISSN: 1614-7499
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  • 8
    Language: English
    In: PLoS ONE, Sept 4, 2014, Vol.9(9)
    Description: Purpose Recently, a proof-of-concept study revealed the suitability of transcriptome analyses to obtain and assess changes in the abundance of transcripts in zebrafish (Danio rerio) embryos after exposure to organic sediment extracts. The present study investigated changes in the transcript abundance in zebrafish embryos exposed to whole sediment samples and corresponding organic extracts in order to identify the impact of different exposure pathways on sediment toxicity. Materials and Methods Danio rerio embryos were exposed to sublethal concentrations of three sediment samples from the Danube River, Germany. The sediment samples were investigated both as freeze-dried samples and as organic extracts. Silica dust and a process control of the extraction procedure were used as references. After exposure, mRNA was isolated and changes in profiles of gene expression levels were examined by an oligonucleotide microarray. The microarray results were compared with bioassays, chemical analysis of the sediments and profiles of gene expression levels induced by several single substances. Results and Discussion The microarray approach elucidated significant changes in the abundance of transcripts in exposed zebrafish embryos compared to the references. Generally, results could be related to Ah-receptor-mediated effects as confirmed by bioassays and chemical analysis of dioxin-like contaminants, as well as to exposure to stress-inducing compounds. Furthermore, the results indicated that mixtures of chemicals, as present in sediment and extract samples, result in complex changes of gene expression level profiles difficult to compare with profiles induced by single chemical substances. Specifically, patterns of transcript abundances were less influenced by the chemical composition at the sampling site compared t the method of exposure (sediment/extract). This effect might be related to different bioavailability of chemicals. Conclusions The apparent difference between the exposure scenarios is an important aspect that needs to be addressed when conducting analyses of alterations in the expression level of mRNA.
    Keywords: Dioxins – Analysis ; Dioxins – Investigations ; Embryonic Development – Analysis ; Embryonic Development – Investigations ; RNA – Analysis ; RNA – Investigations ; Gene Expression – Analysis ; Gene Expression – Investigations ; Sediments (Geology) – Analysis ; Sediments (Geology) – Investigations
    ISSN: 1932-6203
    Source: Cengage Learning, Inc.
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  • 9
    Language: English
    In: Aquatic Toxicology, 2010, Vol.100(1), pp.38-50
    Description: Endpoints of planar halogenated aromatic hydrocarbon (pHAH) and polycyclic aromatic hydrocarbon (PAH) toxicity are mediated activation of the aryl hydrocarbon receptor (AhR) followed by activation of the so called “AhR-battery” of genes including the cytochrome P450 1 (CYP1) isoforms. The aim of this study was to develop a method to identify CYP1 activity in early life-stages of zebrafish ( ) in order to elucidate the spatio-temporal pattern of basal and induced CYP1 activities. Preliminary experiments with the fish embryo toxicity test (FET) were carried out to determine toxic effect thresholds of the AhR agonist β-naphthoflavone. To assess basal and β-naphthoflavone-induced CYP1 activity during early life-stages of zebrafish, the commonly used 7-ethoxyresorufin- -deethylase (EROD) assay was developed further for use in confocal laser scanning microscopy (CLSM) and spectrometry. Following exposure to selected cytochrome P450 inducers, zebrafish embryos were dechorionated, anaesthetized and inspected under the CLSM. Alternatively, embryos were homogenized, and EROD activity was measured using classical spectrometry . CLSM of CYP-induced fluorescence allowed for the detection of CYP1 enzyme activity down to the cellular level as early as in the gastrulation stage. Basal and induced CYP1 activity was detected at all time points examined from 8 h post-fertilization to early adulthood and showed a highly dynamic spatio-temporal pattern throughout zebrafish development. Basal and induced EROD activity was prominent in tissues of the cardiovascular system, the urinary tract, the digestive system, and parts of the brain as well as in the central portion of the eye and the otic vesicle during distinct stages of development. The differentiation between constitutive and induced spatio-temporal patterns of CYP1 activity even as early as the gastrula stage provide further insights into the endogenous role of CYP1 activity.
    Keywords: Zebrafish ; Development ; Cytochrome P450 ; Erod ; Clsm ; Chemistry ; Ecology
    ISSN: 0166-445X
    E-ISSN: 1879-1514
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  • 10
    In: Toxicological Sciences, 2017, Vol. 159(1), pp.86-93
    Description: Early life stages of zebrafish ( Danio rerio , zf) are gaining attention as an alternative invivo test system for drug discovery, early developmental toxicity screenings and chemical testing in ecotoxicological and toxicological testing strategies. Previous studies have demonstrated transcriptional evidence for xenobiotic metabolizing enzymes (XME) during early zf development. However, elaborate experiments on XME activities during development are incomplete. In this work, the intrinsic activities of representative phase I and II XME were monitored by transformation of putative zf model substrates analyzed using photometry and high pressure liquid chromatography techniques. Six different defined stages of zf development (between 2.5 h postfertilization (hpf) to 120 hpf) were investigated by preparing a subcellular fraction from whole organism homogenates. We demonstrated that zf embryos as early as 2.5 hpf possess intrinsic metabolic activities for esterase, Aldh, Gst, and Cyp1a above the methodological detection limit. The activities of the enzymes Cyp3a and Nat were measurable during later stages in development. Activities represent dynamic patterns during development. The role of XME activities revealed in this work is relevant for the assessing toxicity in this test system and therefore contributes to a valuable characterization of zf embryos as an alternative testing organism in toxicology.
    Keywords: Zebrafish ; Early Life Stages ; Xenobiotic Metabolism ; Enzyme Activity
    ISSN: 1096-6080
    E-ISSN: 1096-0929
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