Publications by authors named "Pascale Berckmans"

6 Publications

  • Page 1 of 1

Comparison of extracellular vesicle isolation and storage methods using high-sensitivity flow cytometry.

PLoS One 2021 4;16(2):e0245835. Epub 2021 Feb 4.

Health Unit, Flemish Institute for Technological Research, Mol, Belgium.

Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0245835PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7861365PMC
July 2021

Repeatability and Reproducibility of the RTgill-W1 Cell Line Assay for Predicting Fish Acute Toxicity.

Toxicol Sci 2019 06;169(2):353-364

Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, 8600 Dübendorf, Switzerland.

Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout (Oncorhynchus mykiss) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC50) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings.
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http://dx.doi.org/10.1093/toxsci/kfz057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542334PMC
June 2019

Phenotypic and biomarker evaluation of zebrafish larvae as an alternative model to predict mammalian hepatotoxicity.

J Appl Toxicol 2016 09 4;36(9):1194-206. Epub 2016 Mar 4.

VITO NV, Applied Bio & Molecular Systems, Boeretang 200, B-2400, Mol, Belgium.

Zebrafish phenotypic assays have shown promise to assess human hepatotoxicity, though scoring of liver morphology remains subjective and difficult to standardize. Liver toxicity in zebrafish larvae at 5 days was assessed using gene expression as the biomarker approach, complementary to phenotypic analysis and analytical data on compound uptake. This approach aimed to contribute to improved hepatotoxicity prediction, with the goal of identifying biomarker(s) as a step towards the development of transgenic models for prioritization. Morphological effects of hepatotoxic compounds (acetaminophen, amiodarone, coumarin, methapyrilene and myclobutanil) and saccharin as the negative control were assessed after exposure in zebrafish larvae. The hepatotoxic compounds induced the expected zebrafish liver degeneration or changes in size, whereas saccharin did not have any phenotypic adverse effect. Analytical methods based on liquid chromatography-mass spectrometry were optimized to measure stability of selected compounds in exposure medium and internal concentration in larvae. All compounds were stable, except amiodarone for which precipitation was observed. There was a wide variation between the levels of compound in the zebrafish larvae with a higher uptake of amiodarone, methapyrilene and myclobutanil. Detection of hepatocyte markers (CP, CYP3A65, GC and TF) was accomplished by in situ hybridization of larvae to coumarin and myclobutanil and confirmed by real-time reverse transcription-quantitative polymerase chain reaction. Experiments showed decreased expression of all markers. Next, other liver-specific biomarkers (i.e. FABP10a and NR1H4) and apoptosis (i.e. CASP-3 A and TP53) or cytochrome P450-related (CYP2K19) and oxidoreductase activity-related (ZGC163022) genes, were screened. Links between basic mechanisms of liver injury and results of biomarker responses are described. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/jat.3288DOI Listing
September 2016

Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds.

Reprod Toxicol 2010 Aug 10;30(1):18-24. Epub 2010 May 10.

BioDetection Systems BV, Science Park 406, Amsterdam, The Netherlands.

To date there are no validated methods available to test androgenicity or antiandrogenicity in vitro. A problem with testing androgenicity using reporter genes is the possibility by other steroid receptors than androgen receptors to activate the same reporter gene, thereby lowering selectivity. To avoid this we have established a robust and very selective method, the AR CALUX reporter gene assay, to test androgenic and antiandrogenic activity of compounds in vitro. This assay uses a human U2-OS cell line stably transfected with the human androgen receptor and an androgen receptor responsive reporter gene. We optimized protocols to be used in combination with AR CALUX cells and carried out an in house prevalidation. In addition we successfully transferred this assay to another laboratory, leading to comparable test results with a panel of androgen receptor agonists and antagonists. The assay was able to readily rank a range of chemicals on the basis of their EC50 values. The CALUX assay was found to be selective for androgens and seemed not influenced by signaling through other steroid receptors.
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http://dx.doi.org/10.1016/j.reprotox.2010.04.012DOI Listing
August 2010

Optimization and prevalidation of the in vitro ERalpha CALUX method to test estrogenic and antiestrogenic activity of compounds.

Reprod Toxicol 2010 Aug 8;30(1):73-80. Epub 2010 May 8.

BioDetection Systems BV, Science Park 406, Amsterdam, The Netherlands.

Estrogenicity of chemicals has received significant attention and is linked to endocrine-disrupting activities. However, there is a paucity of validated methods to assess estrogenicity in vitro. We have established a robust method to test estrogenic and antiestrogenic activity of compounds in vitro, as an alternative to using animal models such as the uterotrophic assay. To this end we optimized protocols to be used in combination with CALUX reporter gene assays and carried out an in house prevalidation, followed by two rounds of tests to establish transferability. Problems in the initial test with transferability were solved by isolation of a novel cell clone of the ERalpha CALUX line with greatly improved stability and luciferase levels. This cell line proved to be a very suitable and reliable predictor of estrogenicity of chemicals and was able to readily rank a range of chemicals on the basis of their EC50 values.
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http://dx.doi.org/10.1016/j.reprotox.2010.04.007DOI Listing
August 2010

Comparison of different androgen bioassays in the screening for environmental (anti)androgenic activity.

Environ Toxicol Chem 2005 Oct;24(10):2646-56

Division of Biochemistry, Faculty of Medicine, Campus Gasthuisberg, K.U.Leuven, Herestraat 49, B-3000 Leuven, Belgium.

Endocrine disruptors pose a growing threat to human and wildlife health. Validated test systems are required to study the mechanisms by which chemicals may interfere with the endocrine system. In order to identify compounds with (anti)androgenic activity, we used several in vitro bioassays, based on different androgen receptor (AR) functions, including AR transactivation and interaction between the amino-terminal domain and the ligand-binding domain. The AR activity assay, based on activation of the transcription of an androgen-responsive reporter gene in the presence of androgen, proved to excel in terms of high fold induction range and low minimal detection limit. The EC50 value, defined as the concentration that leads to half the maximal response and reflecting the potency of the synthetic androgen R1881 (methyltrienolone), was found to be 250 pM, consistent with the high affinity of this ligand to the human AR. A number of environmental samples were tested in the bioassay. The bioassay also could be used to detect antiandrogenic activity, because known AR-antagonists were able to inhibit R1881-mediated transactivation.
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http://dx.doi.org/10.1897/05-126r.1DOI Listing
October 2005
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