Jens C. Otte

Relative differences in aryl hydrocarbon receptor-mediated response for 18 polybrominated and mixed halogenated dibenzo-P-dioxins and -furans in cell lines from four different species†

As a consequence of ubiquitous use of brominated organic chemicals, there is a concern for persistent or increasing environmental levels of polybrominated dibenzo-p-dioxins/furans (PBDD/Fs) and mixed polychlorinated and polybrominated dibenzo-p-dioxins/furans (PXDD/Fs). Hence, there is a need to broaden the toxicological and environmental knowledge about these compounds, as a basis for risk assessment. In the study presented here, the relative potencies (REPs) for 18 PBDD/F and PXDD/ F congeners were determined in four dioxin-specific bioassays from different species: dioxin receptor chemically activated luciferase expression assay (DR-CALUX, rat hepatoma cells), TV101L (human hepatoma cells), and GPC.2D (guinea pig adenoma cells), as well as ethoxyresorufin-O-deethylase induction in the fish cell line RTL-W1 (rainbow trout liver cells). The bioassay specific REP factors presented here enable the assessment of the contribution from PBDD/Fs and PXDD/Fs to total 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQs: toxic equivalents), using bioassay analysis. The PBDD/Fs were found to be equally potent as their chlorinated analogues in the three mammalian assays, whereas the PXDD/Fs showed relatively higher potencies. Of special concern were the 2,3,7,8-substituted penta- and tetrahalogenated congeners, for which mean REPs were > or =1. The 2-B-1,3,7,8-CDD (2-bromo-1,3,7,8-tetrachlorodibenzo-p-dioxin) was up to three times more potent than TCDD in individual experiments (on weight basis). The RTL-W1 was less sensitive to the tested compounds with overall 10-fold lower REPs than the mammalian cell lines. Although the REP factors exhibited species-specific differences, overall resembling rank orders of dioxin-like potency were obtained.

Fast Segmentation of Stained Nuclei in Terabyte-Scale, Time Resolved 3D Microscopy Image Stacks

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 due to their high complexity. In this contribution we present a fast parallelized segmentation method that is especially suited for the extraction of stained nuclei from microscopy images, e.g., of developing zebrafish embryos. The idea is to transform the input image based on gradient and normal directions in the proximity of detected seed points such that it can be handled by straightforward global thresholding like Otsu’s method. We evaluate the quality of the obtained segmentation results on a set of real and simulated benchmark images in 2D and 3D and show the algorithm’s superior performance compared to other state-of-the-art algorithms. We achieve an up to ten-fold decrease in processing times, allowing us to process large data sets while still providing reasonable segmentation results.

A combined DNA-microarray and mechanism-specific toxicity approach with zebrafish embryos to investigate the pollution of river sediments

The zebrafish embryo has repeatedly proved to be a useful model for the analysis of effects by environmental toxicants. This proof-of-concept study was performed to investigate if an approach combining mechanism-specific bioassays with microarray techniques can obtain more in-depth insights into the ecotoxicity of complex pollutant mixtures as present, e.g., in sediment extracts. For this end, altered gene expression was compared to data from established bioassays as well as to results from chemical analysis. Mechanism-specific biotests indicated a defined hazard potential of the sediment extracts, and microarray analysis revealed several classes of significantly regulated genes which could be related to the hazard potential. Results indicate that potential classes of contaminants can be assigned to sediment extracts by both classical biomarker genes and corresponding expression profile analyses of known substances. However, it is difficult to distinguish between specific responses and more universal detoxification of the organism.

Contribution of Priority PAHs and POPs to Ah Receptor-Mediated Activities in Sediment Samples from the River Elbe Estuary, Germany

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 result in remobilization of sediment-bound pollutants. The aim of this study was to identify pollutants contributing to the toxicological risk associated with re-suspension of sediments in the Elbe Estuary by use of an effect-directed analysis that combines chemical and biological analyses in with specific fractionation techniques. Sediments were collected from sites along the Elbe Estuary and a site from a small harbor basin of the Elbe Estuary that is known to be polluted. The sixteen priority EPA-PAHs were quantified in organic extracts of sediments. In addition, dioxin equivalents of sediments were investigated by use of the 7-ethoxyresorufin O-deethylase assay with RTL-W1 cells and the Ah receptor-mediated luciferase transactivation assay with H4IIE-luc cells. Quantification of the 16 priority PAHs revealed that sediments were moderately contaminated at all of the sites in the Elbe River Estuary (<0.02–0.906 µg/g dw). Sediments contained relatively small concentrations of dioxin equivalents (Bio-TEQ) with concentrations ranging from 15.5 to 322 pg/g dw, which were significantly correlated with dioxin equivalents calculated based on toxicity reference values and concentrations of PAH. The concentration of Bio-TEQ at the reference site exceeded 200,000 pg/g dw. In a potency balance the 16 PAHs explained between 47 and 118% of the Bio-TEQ in the luciferase assay, which can be explained by the constant input of PAHs bound to SPM from the upper course of the Elbe River into its estuary. Successful identification of a significant portion of dioxin-like activity to priority PAHs in complex environmental samples such as sediments has rarely been reported.

An ensemble-averaged, cell density-based digital model of zebrafish embryo development derived from light-sheet microscopy data with single-cell resolution

A new era in developmental biology has been ushered in by recent advances in the quantitative imaging of all-cell morphogenesis in living organisms. Here we have developed a light-sheet fluorescence microscopy-based framework with single-cell resolution for identification and characterization of subtle phenotypical changes of millimeter-sized organisms. Such a comparative study requires analyses of entire ensembles to be able to distinguish sample-to-sample variations from definitive phenotypical changes. We present a kinetic digital model of zebrafish embryos up to 16 h of development. The model is based on the precise overlay and averaging of data taken on multiple individuals and describes the cell density and its migration direction at every point in time. Quantitative metrics for multi-sample comparative studies have been introduced to analyze developmental variations within the ensemble. The digital model may serve as a canvas on which the behavior of cellular subpopulations can be studied. As an example, we have investigated cellular rearrangements during germ layer formation at the onset of gastrulation. A comparison of the one-eyed pinhead (oep) mutant with the digital model of the wild-type embryo reveals its abnormal development at the onset of gastrulation, many hours before changes are obvious to the eye.

Generating semi-synthetic validation benchmarks for embryomics

Systematic validation is an essential part of algorithm development. The enormous dataset sizes and the complexity observed in many recent time-resolved 3D fluorescence microscopy imaging experiments, however, prohibit a comprehensive manual ground truth generation. Moreover, existing simulated benchmarks in this field are often too simple or too specialized to sufficiently validate the observed image analysis problems. We present a new semi-synthetic approach to generate realistic 3D+t benchmarks that combines challenging cellular movement dynamics of real embryos with simulated fluorescent nuclei and artificial image distortions including various parametrizable options like cell numbers, acquisition deficiencies or multiview simulations. We successfully applied the approach to simulate the development of a zebrafish embryo with thousands of cells over 14 hours of its early existence.

Impacts of different exposure scenarios on transcript abundances in Danio rerio embryos when investigating the toxicological burden of riverine sediments.

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.

LAMBDA -- The LSDF Execution Framework for Data Intensive Applications

To cope with the growing requirements of data intensive scientific experiments, models and simulations the Large Scale Data Facility (LSDF) at KIT aims to support many scientific disciplines. The LSDF is a distributed storage facility at Exabyte scale providing storage, archives, data bases and meta data repositories. Apart from data storage many scientific communities need to perform data processing operations as well. For this purpose the LSDF Execution Framework for Data Intensive Applications (LAMBDA) was developed to allow asynchronous high-performance data processing next to the LSDF. However, it is not restricted to the LSDF or any special feature only available at the LSDF. The main goal of LAMBDA is to simplify large scale data processing for scientific users by reducing complexity, responsibility and error-proneness. The description of an execution is realized as part of LAMBDA administration in the background via meta data that can be obtained from arbitrary sources. Thus, the scientific user has only to select which applications he wants to apply to his data.

Data-intensive analysis for scientific experiments at the Large Scale Data Facility

The Large Scale Data Facility (LSDF) was conceived and launched at the Karlsruhe Institute of Technology (KIT) end of 2009 to address the growing need for value-added storage services for data intensive experiments. The LSDF main focus is to support scientific experiments producing large data sets reaching into the petabyte range with adequate storage, support and value added services for data management, processing and preservation. In this work we describe the approach taken to perform data analysis in LSDF, as well as for data management of the scientific datasets.

Data management and analysis at the Large Scale Data Facility

The Large Scale Data Facility (LSDF) was started at the Karlsruhe Institute of Technology (KIT) end of 2009 to address the growing need for value-added storage services for its data intensive experiments. The main focus of the project is to provide scientific communities producing data collections in the tera — to petabyte range with the necessary hardware infrastructure as well as with adequate value-added services and support for the data management, processing, and preservation. In this work we describe the projects infrastructure and services design, as well as its meta data handling. Both community specific meta data schemes, a meta data repository, an application programming interface and a graphical tool for accessing the resources were developed to further support the processing workflows of the partner scientific communities. The analysis workflow of high throughput microscopy images for studying biomedical processes is described in detail.