Karlijn van der Ven

Molecular targets of TBBPA in zebrafish analysed through integration of genomic and proteomic approaches

Tetrabromobisphenol-A (TBBPA) is nowadays one of the most frequently used brominated flame retardants (BFRs) and can be considered as a high production volume chemical. Over the last decade, numerous reports of increasing concentrations of BFRs in the environment and humans have been published. However, the toxicological knowledge on TBBPA, and more specifically its molecular mode of action, is rather fragmentary. In this study two populations of adult zebrafish (Danio rerio) were exposed for 14 days to 0.75 microM and 1.5 microM TBBPA. Subsequently, we employed a combined transcriptomic and proteomic approach to evaluate the molecular effects of TBBPA in zebrafish liver. Oligonucleotide microarrays were used to study the effects on gene expression levels. These results were validated through real-time PCR. The proteome of the liver was analysed by means of differential in-gel electrophoresis (DiGE), an innovative application of traditional 2D-PAGE. Combination of the extracted datasets allowed reassembling of individual molecular responses into a comprehensive overview of affected molecular pathways. Interpretation of the results depicted an interference of thyroid and Vitamin A homeostasis in the exposed zebrafish, TBBPA also elicited responses indicating onset of oxidative stress and general stress responses. Additionally, numerous differentially expressed transcripts could be associated with defence mechanisms or corresponded to metabolizing enzymes. Furthermore, cellular metabolism was clearly affected, illustrated as disturbance of e.g. lipid, carbohydrate, and organic acid metabolic processes. Summarizing, these results enabled us to hypothesize several working mechanisms of TBBPA and demonstrated the potential of a combined genome and proteome approach to generate detailed mechanistic toxicological information.

An integrated transcriptomic and proteomic approach characterizing estrogenic and metabolic effects of 17 α-ethinylestradiol in zebrafish (Danio rerio)

Nowadays there is much concern about the presence of endocrine disrupting compounds (EDCs) in the environment due to their ability to interfere with the endocrine system. In the presented study, adult zebrafish (Danio rerio) were exposed to 30 ng L(-1) 17alpha-ethinylestradiol (EE2) for 4 and 28 days. The underlying molecular mechanisms of EE2 were studied in the zebrafish liver by applying a combined transcriptomics and proteomics approach. In addition, we assessed the added value of such an integrated-omics approach. Oligo microarrays, spotted with 3479 zebrafish-specific oligos, were employed to generate differential gene expression levels. The proteomic responses were evaluated by means of differential in-gel electrophoresis (DiGE), combined with MALDI-tandem mass spectrometry. Assessment of the major biological functions of the differentially expressed transcripts and proteins illustrated that both individual platforms could profile a clear estrogenic interference, next to numerous metabolism-related effects and stress responses. Cross-comparison of both transcriptomics and proteomics datasets displayed limited concordance, though, thorough revision of the results illustrated that transcriptional effects were projected on protein level as downstream effects of affected signalling pathways. Overall, this study demonstrated that a proteomics approach can lift the biological interpretation of microarrays to a higher level, and moreover, opens a window for identification of possible new biomarkers.

miRNA genes and the brain: implications for psychiatric disorders.

MicroRNAs (miRNAs) are a class of nonprotein coding genes with a growing importance in regulatory mechanisms of gene expression related to brain function and plasticity. Considering the relative lack of success of the analysis of variations in candidate protein coding genes and of genome‐wide association studies to identify strong risk factors for common psychiatric disorders (PDs), miRNA genes are of particular interest for the field of psychiatric genetics as deregulation of the rate of transcription or translation of a normal gene may be phenotypically similar to disruption of the gene itself. In this article we review the current knowledge on the contribution of miRNAs in basic mechanisms of brain development and plasticity and their possible involvement in the pathogenesis of several PDs. Because future functional and genomic explorations of brain expressed miRNAs, and other types of noncoding RNAs, may identify additional candidate genes and pathways for common PDs, we believe that implementing additional strategies to further elucidate the role of miRNAs in the etiology of common PDs is of great importance. Hum Mutat 31:1–10, 2010.

Sublethal effects of waterborne uranium exposures on the zebrafish brain:transcriptional responses and alterations of the olfactory bulb ultrastructure

The toxic action modes of uranium (U) in fish are still scarcely known. U is known to modify the acetylcholinesterase activity in the fish brain. To gain further insight into U neurotoxicity in fish, we examined transcriptional responses in the brain of the zebrafish, Danio rerio, exposed to 15 microg L(-1) and 100 microg L(-1) of waterborne U for 3 and 10 days. In parallel, an ultrastructure analysis of the neuropil of the olfactory bulb, an area in the brain of fish sensitive to metal contamination, was performed after 10 days of U exposure. This combined transcriptomic and histological study is the first report performed in the brain and specifically the olfactory bulb of fish exposed to U. We found that 56 transcripts responded to the metal exposure, and the anatomical structure of the olfactory bulb was damaged. The greatest gene response occurred at the lower U concentration and the numbers of responding genes common to any two U exposures were much smaller than those unique to each exposure. These data showed that the intensity of gene response may not correlate positively with toxicant concentrations according to our experimental design. Instead, different patterns of gene expression are expected for each exposure. Gene responses were categorized into eight functional classes, and the transcriptional responses of genes involved in the olfactory system were significantly affected. Collectively, the data suggest that genes in the olfactory region may be ecologically relevant and sensitive transcriptional biomarkers of U waterborne exposure.

Targeted screening and validation of copy number variations.

The accessibility of genome-wide screening technologies considerably facilitated the identification and characterization of copy number variations (CNVs). The increasing amount of available data describing these variants, clearly demonstrates their abundance in the human genome. This observation shows that not only SNPs, but also CNVs and other structural variants strongly contribute to genetic variation. Even though not all structural variants have an obvious phenotypic effect, there is evidence that CNVs influence gene dosage and hence can have profound effects on human disease susceptibility, disease manifestation, and disease severity. Therefore, CNV screening and analysis methodologies, specifically focusing on disease-related CNVs are actively progressing. This chapter specifically describes different techniques currently available for the targeted screening and validation of CNVs. We not only provide an overview of all these CNV analysis methods, but also address their strong and weak points. Methods covered include fluorescence in situ hybridization (FISH), quantitative real-time PCR (qPCR), paralogue ratio test (PRT), molecular copy-number counting (MCC), and multiplex PCR-based approaches, such as multiplex amplifiable probe hybridization (MAPH), multiplex ligation-dependent probe amplification (MLPA), multiplex PCR-based real-time invader assay (mPCR-RETINA), quantitative multiplex PCR of short fluorescent fragments (QMPSF), and multiplex amplicon quantification (MAQ). We end with some general remarks and conclusions, furthermore briefly addressing the future perspectives.

Development of a microarray for Enchytraeus Albidus (oligochaeta): preliminary tool with diverse applications

Standard bioassays allow hazard assessment at the population level, but much remains to be learned about the molecular level response of organisms to stressors. The main aim of this study was the development of a DNA microarray for Enchytraeus albidus, a common soil worm species. Further, this microarray was tested using worms exposed to Cu, phenmedipham, and different soil types. Hybridization onto the developed microarray revealed several genes with homology to known sequences. Genes of interest were confirmed through real-time polymerase chain reaction. It was possible to discriminate between natural and chemical stressors and chemical concentrations. Gene responses were detected under conditions known to have effects in the reproduction of individuals. It was confirmed that the integration of different endpoints improves the assessment process and enhances the understanding of the modes of action of stressors. The chemical stress-induced genes were related to factors such as immune response, stress response, metabolic processes, and/or signal transduction. The present study represents the first step of a gene-level study in the ecologically relevant and standard test species E. albidus. It demonstrates the usefulness of cDNA normalization in the production of cDNA libraries of ecotoxicological standard organisms that are not genome models like E. albidus.

Use of suppression subtractive hybridization PCR for the development of cDNA arrays for the detection of endocrine disruption in carp (Cyprinus carpio)

The potential of a variety of xenobiotic compounds to modulate or disrupt the endocrine system of humans and wildlife is now widely recognized. In the present study, we developed a molecular tool for the evaluation of endocrine disruption in common carp (Cyprinus carpio). Suppression Subtractive Hybridization PCR was applied for the isolation of a relevant gene set, consisting of gender- and hormone-responsive gene fragments. This resulted in 398 different gene fragments that were most related to endocrine functioning. To investigate the applicability of this gene collection for studying endocrine disruption in fish, the gender-related genes were spotted on a cDNA macroarray, and expression profiles were generated for 17beta-estradiol (E2) and cortisol. Therefore, fish were injected with these hormones, and after 24 h and 96 h RNA was extracted and used for macroarray hybridizations. E2 exposure resulted in a total of 35 differentially expressed genes, whereas cortisol only affected 3 genes spotted on the macroarray. These results indicate the discriminating power of the developed array, and its usefulness to describe the toxicological mode of action of endocrine disruptive chemicals.

Abstract 4328: New perspectives on the use of polo-like kinase 1 as a prognostic biomarker in non-small cell lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Polo-like kinase 1 (Plk1) overexpression is observed in various tumors, including non-small cell lung cancer (NSCLC), and is correlated with poor patient prognosis and metastasis, suggesting a role in aggressive tumors. Previous studies reported Plk1 downregulation by P53 upon DNA damage, suggesting that TP53 mutations might have an influence on Plk1 expression. In this study, we determined the Plk1 protein level in NSCLC patients and correlated these results with the TP53 status, presence of hypoxia (carbonic anhydrase 9, CA-9) and apoptosis induction (cleaved caspase 3). Material & methods: Tumor tissues of 84 NSCLC patients and 16 control samples were obtained from the Antwerp University Hospital. Immunohistochemistry was performed using antibodies to Plk1 (208G4, 1/50), CA-9 (EPR4151(2), 1/350) and cleaved caspase 3 (9579S, 1/250). Based on the% positive cells and staining intensity, an overall score of negative, weak, moderate or strong expression was assigned for both the Plk1 and CA-9 protein. For the TP53 mutation analysis, DNA was isolated using the QIAamp® DNA FFPE tissue kit. For each sample, DNA concentration and purity were assessed using NanoDrop measurement. The relation between DNA input and FFPE derived DNA quality was determined using the QC-plex assay from Multiplicom. Samples were considered to be usable when the DNA quality coefficient was higher than 0.2. Determination of the TP53 mutation status was performed using Multiplicoms TP53 MASTRTM Plus test with MID for Illumina Miseq®. Results & discussion: Plk1 and CA-9 positivity was detected in 95% and 83% of all tumor samples, respectively. Of the 16 control samples, all samples stained negative, except for 4/16 and 1/16 samples that showed a low expression for Plk1 and CA-9, respectively. In a next phase, cleaved caspase 3 staining will be scored and both CA-9 and cleaved caspase 3 expression patterns will be correlated to Plk1 expression. Furthermore, results will be correlated with clinicopathological parameters, including incidence age, smoking behavior, tumor differentiation and stage, metastasis and survival. For the TP53 mutation analysis, sufficient DNA with acceptable purity was isolated from 82 patients. A QC plex reaction has already been performed for 20 samples, 12 of them showed an acceptable DQC value and 8 sampled were marked as low DNA quality samples. Nonetheless, the latter could be used for further analysis by increasing the DNA input. At present, 15 samples have been sequenced successfully. Besides polymorphisms, 5 exon mutations (c.536A>G, c.916C>T, c.734G>A, c.578A>C, c.559+1G>T) and 4 intron mutations (c.920-2A>T, c.993+352C>T, c.559+1G>T) were observed. As soon as all samples have been processed, it will be evaluated whether a link between Plk1 overexpression and TP53 mutations can be evidenced. Citation Format: Jolien Van den Bossche, Filip Lardon, Christophe Hermans, Christophe Deben, Vanessa Deschoolmeester, Julie Jacobs, Karlijn van der Ven, Jurgen Del-Favero, Patrick Pauwels, Marc Peeters, An Wouters. New perspectives on the use of polo-like kinase 1 as a prognostic biomarker in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4328. doi:10.1158/1538-7445.AM2015-4328