Publications by authors named "Matthias Cuykx"

17 Publications

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Optimization of a liquid chromatography-ion mobility-high resolution mass spectrometry platform for untargeted lipidomics and application to HepaRG cell extracts.

Talanta 2021 Dec 17;235:122808. Epub 2021 Aug 17.

University of Antwerp, Toxicological Centre, Universiteitsplein 1, 2610, Antwerp, Belgium. Electronic address:

Analytical methods to evaluate the lipidome of biological samples need to provide high data quality to ensure comprehensive profiling and reliable structural elucidation. In this perspective, liquid chromatography-high resolution mass spectrometry (LC-HRMS) is the state-of-the-art technique for lipidomic analysis of biological samples. There are thousands of lipids in most biological samples, and therefore separation methods before introduction to the mass spectrometer is key for relative quantitation and identification. Chromatographic methods differ across laboratories, without any consensus on the best methodologies. Therefore, we designed an experiment to determine the optimal LC methodology, and assessed the value of ion mobility for an additional dimension of separation. To apply an untargeted method for hypothesis generation focused on lipidomics, LC-HRMS parameters were optimized based on the measurement of 50 panel lipids covering key human metabolic pathways. Reversed-phase liquid chromatography columns were compared based on a quality scoring system considering the signal-to-noise ratio, peak shape, and retention factor. Furthermore, drift tube ion mobility spectrometry (DTIMS) was implemented to increase peak capacity and confidence during annotation by providing collision cross section (CCS) values for the analytes under investigation. However, hyphenating DTIMS to LC-HRMS may result in a reduced sensitivity due to impaired duty cycles. To increase the signal intensity, a Box-Behnken design (BBD) was used to optimize four key factors, i.e. drift entrance voltage, drift exit voltage, rear funnel entrance, and rear funnel exit voltages. Application of a maximized desirability function provided voltages for the above-mentioned parameters resulting in higher signal intensity compared to each combination of parameters used during the BBD. In addition, the influence of single pulse and Hadamard 4-bit multiplexed modes on signal intensity was explored and different trap filling and release times of ions were evaluated. The optimized LC-DTIM-HRMS platform was applied to extracts from HepaRG cells and resulted in 3912 high-quality features (<30% median relative standard deviation; n = 6, t = 24 h). From these features, 436 lipid species could be annotated (i.e., matching based on accurate mass <5 ppm, isotopic pattern, in-silico MS/MS fragmentation, and in-silico CCS database matching <3%). The application of LC-DTIM-HRMS for untargeted analysis workflows is growing and the platform optimization, as described here, can be used to guide the method development and CCS database comparison for high confidence lipid annotation.
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http://dx.doi.org/10.1016/j.talanta.2021.122808DOI Listing
December 2021

Serological response in health care workers after a single dose of SARS-CoV-2 vaccine using six automated SARS-CoV-2 antibody assays.

Diagn Microbiol Infect Dis 2021 Oct 10;101(2):115486. Epub 2021 Jul 10.

University Hospital Antwerp, Laboratory Medicine, Edegem, Belgium. Electronic address:

Spike (S)- and nucleocapsid (N)-specific serological assay responses were determined before and/or after first dose SARS-CoV-2 vaccination in 22 individuals. S-specific assays quantified antibodies after vaccination with significant higher levels in participants with a previous infection. Be cautious combining N-/S-specific assay results, potentially differentiating post-infection/vaccination immunization as assay-specific N-antibody waning was observed.
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http://dx.doi.org/10.1016/j.diagmicrobio.2021.115486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8272050PMC
October 2021

An exploratory approach for an oriented development of an untargeted hydrophilic interaction liquid chromatography-mass spectrometry platform for polar metabolites in biological matrices.

J Chromatogr A 2021 Jan 15;1637:461807. Epub 2020 Dec 15.

Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium. Electronic address:

The analysis of polar metabolites based on liquid chromatography-mass spectrometry (LC-MS) methods should take into consideration the complexity of interactions in LC columns to be able to cover a broad range of metabolites of key biological pathways. Therefore, in this study, different chromatographic columns were tested for polar metabolites including reversed-phase and hydrophilic interaction liquid chromatography (HILIC) columns. Based on a column screening, two new generations of zwitterionic HILIC columns were selected for further evaluation. A tree-based method optimization was applied to investigate the chromatographic factors affecting the retention mechanisms of polar metabolites with zwitterionic stationary phases. The results were evaluated based on a scoring system which was applied for more than 80 polar metabolites with a high coverage of key human metabolic pathways. The final optimized methods showed high complementarity to analyze a wide range of metabolic classes including amino acids, small peptides, sugars, amino sugars, phosphorylated sugars, organic acids, nucleobases, nucleosides, nucleotides and acylcarnitines. Optimized methods were applied to analyze different biological matrices, including human urine, plasma and liver cell extracts using an untargeted approach. The number of high-quality features (< 30% median relative standard deviation) ranged from 3,755 for urine to 5,402 for the intracellular metabolome of liver cells, showing the potential of the methods for untargeted purposes.
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http://dx.doi.org/10.1016/j.chroma.2020.461807DOI Listing
January 2021

Occurrence of Selected Organic Contaminants in Edible Insects and Assessment of Their Chemical Safety.

Environ Health Perspect 2019 12 31;127(12):127009. Epub 2019 Dec 31.

Toxicological Center, University of Antwerp, Wilrijk, Belgium.

Background: Feeding the continuously growing world population is challenging, and edible insects offer a sustainable alternative to conventional sources of animal proteins. As with any food source, the potential presence of hazardous organic chemicals, such as persistent organic pollutants (POPs), plasticizers and flame retardants (FRs), must be investigated to guarantee consumer chemical safety.

Objectives: Here, we have investigated the contamination levels of several classes of organic compounds in edible insects. To evaluate their chemical safety, a dietary exposure risk assessment was then performed by combining the measured chemical contamination with the most recent food consumption data from local surveys.

Methods: Insect samples, belonging to six orders (Orthoptera, Coleoptera, Lepidoptera, Hemiptera, Odonata, Hymenoptera) were purchased from five European and three Asian countries. POPs and halogenated FRs were analyzed by gas chromatography-mass spectrometry (GC/MS) and organophosphorus FRs and plasticizers were quantified by liquid chromatography-MS/MS, according to validated protocols.

Results: The overall levels of chemical contamination varied greatly among the insect orders and country of purchase, but they were generally low and comparable with other commonly consumed animal products.

Discussion: Here we show that, besides the activities during rearing, the industrial post-harvesting handling and addition of ingredients are supplementary factors influencing the chemical load of the final insect food-product. The total estimated dietary intakes of the considered classes of compounds through insect consumption are comparable with those generally assessed in common food of animal origin worldwide and, when compared with existing reference dose values, suggest that the risk of adverse health effects from exposure to the targeted organic compounds via insect consumption is unlikely. https://doi.org/10.1289/EHP5782.
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http://dx.doi.org/10.1289/EHP5782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957287PMC
December 2019

Exposure of HepaRG Cells to Sodium Saccharin Underpins the Importance of Including Non-Hepatotoxic Compounds When Investigating Toxicological Modes of Action Using Metabolomics.

Metabolites 2019 Nov 4;9(11). Epub 2019 Nov 4.

Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.

Metabolites represent the most downstream information of the cellular organisation. Hence, metabolomics experiments are extremely valuable to unravel the endogenous pathways involved in a toxicological mode of action. However, every external stimulus can introduce alterations in the cell homeostasis, thereby obscuring the involved endogenous pathways, biasing the interpretation of the results. Here we report on sodium saccharin, which is considered to be not hepatotoxic and therefore can serve as a reference compound to detect metabolic alterations that are not related to liver toxicity. Exposure of HepaRG cells to high levels of sodium saccharin (>10 mM) induced cell death, probably due to an increase in the osmotic pressure. Yet, a low number ( = 15) of significantly altered metabolites were also observed in the lipidome, including a slight decrease in phospholipids and an increase in triacylglycerols, upon daily exposure to 5 mM sodium saccharin for 72 h. The observation that a non-hepatotoxic compound can affect the metabolome underpins the importance of correct experimental design and data interpretation when investigating toxicological modes of action via metabolomics.
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http://dx.doi.org/10.3390/metabo9110265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918164PMC
November 2019

Untargeted liquid chromatography-mass spectrometry metabolomics to assess drug-induced cholestatic features in HepaRG® cells.

Toxicol Appl Pharmacol 2019 09 16;379:114666. Epub 2019 Jul 16.

Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

Cholestasis is a liver disease associated with retention of bile in the liver, which leads to local hepatic inflammation and severe liver damage. In order to investigate the mode of action of drug-induced cholestasis, in vitro models have shown to be able to recapitulate important elements of this disease. In this study, we applied untargeted metabolomics to investigate the metabolic perturbances in HepaRG® cells exposed for 24 h and 72 h to bosentan, a cholestatic reference toxicant. Intracellular profiles were extracted and analysed with liquid chromatography and accurate-mass spectrometry. Metabolites of interest were selected using partial least-squares discriminant analysis and random forest classifier models. The observed metabolic patterns associated with cholestasis in vitro were complex. Acute (24 h) exposure revealed metabolites related to apoptosis, such as ceramide and triglyceride accumulation, in combination with phosphatidylethanolamine, choline and carnitine depletion. Metabolomic alterations during exposure to lower dosages and a prolonged exposure (72 h) included carnitine upregulation and changes in the polyamine metabolism. These metabolites were linked to changes in phospholipid metabolism, mitochondrial pathways and energy homeostasis. The metabolic changes confirmed the mitotoxic effects of bosentan and revealed the potential involvement of phospholipid metabolism as part of the mode of action of drug-induced cholestasis.
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http://dx.doi.org/10.1016/j.taap.2019.114666DOI Listing
September 2019

Using Expert Driven Machine Learning to Enhance Dynamic Metabolomics Data Analysis.

Metabolites 2019 Mar 20;9(3). Epub 2019 Mar 20.

Adrem Data Lab, Department of Mathematics and Computer Science, University of Antwerp, 2000 Antwerp, Belgium.

Data analysis for metabolomics is undergoing rapid progress thanks to the proliferation of novel tools and the standardization of existing workflows. As untargeted metabolomics datasets and experiments continue to increase in size and complexity, standardized workflows are often not sufficiently sophisticated. In addition, the ground truth for untargeted metabolomics experiments is intrinsically unknown and the performance of tools is difficult to evaluate. Here, the problem of dynamic multi-class metabolomics experiments was investigated using a simulated dataset with a known ground truth. This simulated dataset was used to evaluate the performance of tinderesting, a new and intuitive tool based on gathering expert knowledge to be used in machine learning. The results were compared to EDGE, a statistical method for time series data. This paper presents three novel outcomes. The first is a way to simulate dynamic metabolomics data with a known ground truth based on ordinary differential equations. This method is made available through the MetaboLouise R package. Second, the EDGE tool, originally developed for genomics data analysis, is highly performant in analyzing dynamic case vs. control metabolomics data. Third, the tinderesting method is introduced to analyse more complex dynamic metabolomics experiments. This tool consists of a Shiny app for collecting expert knowledge, which in turn is used to train a machine learning model to emulate the decision process of the expert. This approach does not replace traditional data analysis workflows for metabolomics, but can provide additional information, improved performance or easier interpretation of results. The advantage is that the tool is agnostic to the complexity of the experiment, and thus is easier to use in advanced setups. All code for the presented analysis, MetaboLouise and tinderesting are freely available.
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http://dx.doi.org/10.3390/metabo9030054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468718PMC
March 2019

Targeted and non-target screening of persistent organic pollutants and organophosphorus flame retardants in leachate and sediment from landfill sites in Gauteng Province, South Africa.

Sci Total Environ 2019 Feb 28;653:1231-1239. Epub 2018 Oct 28.

Department of Environmental, Water and Earth Sciences, Tshwane University of Technology, Tshwane, South Africa. Electronic address:

In the present study, target analysis and a non-target screening method were employed to investigate the degree of contamination of landfill sediment and leachate in Gauteng Province, South Africa. Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), organochlorinated pesticides (OCPs) and organophosphorus flame retardants (OPFRs) were extracted from sediment and leachate samples using solid-liquid extraction and liquid-liquid extraction methods, respectively; and analysed by target analysis using gas chromatography mass spectrometry (GC-MS). Targeted PBDEs were all detected in sediment samples. However, PCBs, CB 52 and CB 101 were below the quantification limit (LOQ) in the sediment samples. The concentrations of OCPs measured ranged from 0.09 ng/g dw (α-HCH) to 5.29 ng/g dw (pp-DDE) in Marie Louis, and from 0.14 ng/g dw (α-HCH) to 11.3 ng/g dw (γ-HCH) in Hatherly, with an average mass fraction of 1.0 (±2.0, SD) ng/g dw in Marie Louis and 2.0 (±3.0, SD) ng/g dw in Hatherly. Among the OPFRs, TnPP, TPTP, T35DMPP and TBPP were below the LOQ in both leachate and sediment samples. Overall, high concentrations of TDCIPP and TCIPP were obtained in both media, ranging from 226 to 14,500 ng/L and 52.0-13,800 ng/L and from 19.6-741 ng/g dw and 32.8-1240 ng/g dw in leachate and sediment, respectively. The high concentrations of certain OPFRs suggest that these may have replaced both PCBs and PBDEs in consumer products currently imported into South Africa. Common compounds identified by non-target screening with high-resolution mass spectrometry of leachate were diethyl [2‑(1, 3‑dioxolan‑2‑yl) ethyl] malonate (CHO) and 4‑(benzyloxy) cyclohexanone (CHO). These compounds are possibly associated with plasticizers used in the production of plastic and plastic coatings.
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http://dx.doi.org/10.1016/j.scitotenv.2018.10.356DOI Listing
February 2019

Mass spectrometric identification of in vitro-generated metabolites of two emerging organophosphate flame retardants: V6 and BDP.

Chemosphere 2018 Dec 3;212:1047-1057. Epub 2018 Sep 3.

Toxicological Centre, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium. Electronic address:

The aim of the present study was to investigate the in vitro metabolism of two emerging organophosphate flame retardants, namely tetrekis(2-chlorethyl)dichloroisopentyldiphosphate (V6) and bisphenol-A bis-diphenyl phosphate (BDP) in human liver microsomes (HLMs), HLM S9 fractions and in human serum. In particular, the role of cytochrome P450 (CYPs) enzymes and/or paraoxonases (PONs) in the formation of V6 and BDP phase I metabolites was studied. Mono-, di-hydroxylated and hydrolytic phase I metabolites of V6 were mainly formed by CYPs in HLMs, while hydrolytic and O-dealkylated phase I metabolites of BDP were generated by PONs mainly in serum experiments. Limited number of glucuronidated and sulfated phase II metabolites were also identified for the two chemicals. The activity of seven recombinant CYPs (rCYPs) including rCYP1A2, rCYP2B6, rCYP2C9, rCYP2C19, rCYP2D6, rCYP2E1 and rCYP3A4 in the in vitro phase I metabolism of V6 and BDP was investigated. The formation of V6 metabolites was catalyzed by several enzymes, especially rCYP1A2 that was responsible for the exclusive formation of two metabolites, one primary (M1) and its secondary metabolite (M9). For BDP, only one phase I metabolite (MM1) was catalyzed by the seven rCYPs. Collectively, these results indicate that CYPs have a predominant role in the metabolism of V6, while PONs have a predominant role in BDP in vitro metabolism. These results are a starting point for future studies involving the study of the toxicity, bioaccumulation and in vivo biomonitoring of V6 and BDP.
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http://dx.doi.org/10.1016/j.chemosphere.2018.08.142DOI Listing
December 2018

Occurrence of organophosphorus flame retardants and plasticizers in wild insects from a former e-waste recycling site in the Guangdong province, South China.

Sci Total Environ 2019 Feb 6;650(Pt 1):709-712. Epub 2018 Sep 6.

Toxicological Center, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

Due to the fast growth of the electronic industry, a large quantity of electronic waste (e-waste) is generated worldwide and then often inappropriately dismantled and disposed of. In a pilot study, the occurrence of organophosphorus flame retardants and plasticizers (PFRs) was investigated for the first time in several wild insect species collected from a former e-waste recycling site in the Guangdong province, South China. TEHP was the most abundant PFR (average concentration of 5.8 ng/g ww), followed by TPHP (2.5 ng/g ww), TCIPP (2.2 ng/g ww), TCEP (0.8 ng/g ww), EHDPHP and TCP (both 0.1 ng/g ww). Dragonfly nymphs were the most contaminated insects, with total PFR concentrations of 68 ng/g ww, followed by moth adults (26 ng/g ww) and terrestrial stinkbug (17 ng/g ww). The different contamination patterns observed in the analyzed insects could be explained by their different habitats and feeding habits. This study shows that e-waste recycling areas can be an important local source of contamination with PFRs, mainly caused by inadequate recycling activities.
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http://dx.doi.org/10.1016/j.scitotenv.2018.09.067DOI Listing
February 2019

In vitro assessment of hepatotoxicity by metabolomics: a review.

Arch Toxicol 2018 10 25;92(10):3007-3029. Epub 2018 Aug 25.

Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Belgium.

Omics technologies, and in particular metabolomics, have received an increasing attention during the assessment of hepatotoxicity in vitro. However, at present, a consensus on good metabolomics practices has yet to be reached. Therefore, in this review, a range of experimental approaches, applied methodologies, and data processing workflows are compared and critically evaluated. Experimental designs among the studies are similar, reporting the use of primary hepatocytes or hepatic cell lines as the most frequently used cell sources. Experiments are usually conducted in short time-frames (< 48 h) at sub-toxic dosages. Applied sample preparations are protein precipitation or Bligh-and-Dyer extraction. Most analytical platforms rely on chromatographic separations with mass spectrometric detection using high-resolution instruments. Untargeted metabolomics was typically used to allow the simultaneous detection of several classes of the metabolome, including endogenous metabolites that are not initially linked to toxicity. This non-biased detection platform is a valuable tool for generating hypothesis-based mechanistic research. The most frequently reported metabolites that are altered under toxicological impulses are alanine, lactate, and proline, which are often correlated. Other unspecific biomarkers of hepatotoxicity in vitro are the down-regulation of choline, glutathione, and 3-phospho-glycerate. Disruptions on the Krebs cycle are associated with increased glutamate, tryptophan, and valine. Phospholipid alterations are described in steatosis, lipo-apoptosis, and oxidative stress. Although there is a growing trend towards quality control, data analysis procedures do often not follow good contemporary metabolomics practices, which include feature filtering, false-discovery rate correction, and reporting the confidence of metabolite annotation. The currently annotated biomarkers can be used to identify hepatotoxicity in general and provide, to a certain extent, a tool for mechanistic distinction.
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http://dx.doi.org/10.1007/s00204-018-2286-9DOI Listing
October 2018

In-vitro metabolomics to evaluate toxicity of particulate matter under environmentally realistic conditions.

Chemosphere 2018 Oct 10;209:137-146. Epub 2018 Jun 10.

Universitat Rovira i Virgili, Chemical Engineering Department, Environmental Analysis and Management Group, Av. Països Catalans 26, 43007, Tarragona, Spain. Electronic address:

In this pilot study three fractions of particulate matter (PM, PM, and PM) were collected in three environments (classroom, home, and outdoors) in a village located nearby an industrial complex. Time-activity pattern of 20 students attending the classroom was obtained, and the dose of particles reaching the children's lungs under actual environmental conditions (i.e. real dose) was calculated via dosimetry model. The highest PM concentrations were reached in the classroom. Simulations showed that heavy intensity outdoor activities played a major role in PM deposition, especially in the upper part of the respiratory tract. The mass of PM reaching the alveoli was minor, while PM and PM apportion for most of the PM mass retained in the lungs. Consequently, PM and PM were the only fractions used in two subsequent toxicity assays onto alveolar cells (A549). First, a cytotoxicity dose-response assay was performed, and doses corresponding to 5% mortality (LC) were estimated. Afterwards, two LC-MS metabolomic assays were conducted: one applying LC, and another applying real dose. A lower estimated LC value was obtained for PM than PM (8.08 and 73.7 ng/mL respectively). The number of altered features after LC exposure was similar for both fractions (39 and 38 for PM and PM respectively), while after real dose exposure these numbers differed (10 and 5 for PM and PM respectively). The most metabolic changes were related to membrane and lung surfactant lipids. This study highlights the capacity of PM to alter metabolic profile of lung cells at conventional environmental levels.
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http://dx.doi.org/10.1016/j.chemosphere.2018.06.065DOI Listing
October 2018

In vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

J Pharm Biomed Anal 2018 May 21;153:158-167. Epub 2018 Feb 21.

Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium.

Designer benzodiazepines have recently emerged as a class of new psychoactive substances. These substances are used in recreational settings and as alternatives to prescription benzodiazepines as self-medication for patients suffering from anxiety or other mental disorders. Due to the limited information available on the metabolic fate of these new substances, it is challenging to reliably detect their usage in bioanalytical (e.g. clinical and forensic) settings. The objective of this study was to investigate the in vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam and identify potential biomarkers for its detection in human biological fluids. Cloniprazepam was incubated with human liver microsomes and cytosolic fractions to generate both Phase I and II metabolites. The extracts were analysed using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Identification of the metabolites was performed using two complementary workflows, including a suspect screening based on in silico predictions and a non-targeted screening. A total of nine metabolites were identified, eight Phase I metabolites and one Phase II metabolite, of which five were specific for cloniprazepam. Clonazepam was the major metabolite of cloniprazepam. Hydroxy-cloniprazepam, dihydroxy-cloniprazepam, 3-keto-cloniprazepam, 7-amino-cloniprazepam, hydroxy-clonazepam, 7-amino-clonazepam and 3-hydroxy-7-amino-clonazepam were formed through oxidation, hydroxylation, and/or reduction of the nitro-group. Glucuronidated hydroxy-cloniprazepam was the only Phase II metabolite detected. Five metabolites were specific for cloniprazepam. This study provided a set of human in vitro biotransformation products which can assist specific detection of cloniprazepam consumption in future studies.
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http://dx.doi.org/10.1016/j.jpba.2018.02.032DOI Listing
May 2018

Metabolomics profiling of steatosis progression in HepaRG cells using sodium valproate.

Toxicol Lett 2018 Apr 31;286:22-30. Epub 2018 Jan 31.

Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

Non-alcoholic Fatty Liver Disease (NAFLD) is a frequently encountered Drug-Induced Liver Injury (DILI). Although this stage of the disease is reversible, it can lead to irreversible damage provoked by non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Therefore, the assessment of NAFLD is a paramount objective in toxicological screenings of new drug candidates. In this study, a metabolomic fingerprint of NAFLD induced in HepaRG cells at four dosing schemes by a reference toxicant, sodium valproate (NaVPA), was obtained using liquid-liquid extraction followed by liquid chromatography and accurate mass-mass spectrometry (LC-AM/MS). The combination of a strict design of experiment with a robust detection method, applied on sodium valproate, validated the possibilities of untargeted metabolomics in hepatic toxicological research. Distinctive patterns between exposed and control cells were consistently observed, multivariate analyses selected up to 200 features of interest, revealing hallmark NAFLD-biomarkers, such as diacylglycerol and triglyceride accumulation and carnitine deficiency. Initial toxic responses show increased levels of S-adenosylmethionine and mono-acetylspermidine in combination with only a moderate increase in triglycerides. New specific markers of toxicity have been observed, such as spermidines, creatine, and acetylcholine. The described design of experiment provides a valuable metabolomics platform for mechanistic research of toxicological hazards and identified new markers for steatotic progression.
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http://dx.doi.org/10.1016/j.toxlet.2017.12.015DOI Listing
April 2018

Tailored liquid chromatography-mass spectrometry analysis improves the coverage of the intracellular metabolome of HepaRG cells.

J Chromatogr A 2017 Mar 23;1487:168-178. Epub 2017 Jan 23.

Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk-Antwerp, Belgium. Electronic address:

Metabolomics protocols are often combined with Liquid Chromatography-Mass Spectrometry (LC-MS) using mostly reversed phase chromatography coupled to accurate mass spectrometry, e.g. quadrupole time-of-flight (QTOF) mass spectrometers to measure as many metabolites as possible. In this study, we optimised the LC-MS separation of cell extracts after fractionation in polar and non-polar fractions. Both phases were analysed separately in a tailored approach in four different runs (two for the non-polar and two for the polar-fraction), each of them specifically adapted to improve the separation of the metabolites present in the extract. This approach improves the coverage of a broad range of the metabolome of the HepaRG cells and the separation of intra-class metabolites. The non-polar fraction was analysed using a C18-column with end-capping, mobile phase compositions were specifically adapted for each ionisation mode using different co-solvents and buffers. The polar extracts were analysed with a mixed mode Hydrophilic Interaction Liquid Chromatography (HILIC) system. Acidic metabolites from glycolysis and the Krebs cycle, together with phosphorylated compounds, were best detected with a method using ion pairing (IP) with tributylamine and separation on a phenyl-hexyl column. Accurate mass detection was performed with the QTOF in MS-mode only using an extended dynamic range to improve the quality of the dataset. Parameters with the greatest impact on the detection were the balance between mass accuracy and linear range, the fragmentor voltage, the capillary voltage, the nozzle voltage, and the nebuliser pressure. By using a tailored approach for the intracellular HepaRG metabolome, consisting of three different LC techniques, over 2200 metabolites can be measured with a high precision and acceptable linear range. The developed method is suited for qualitative untargeted LC-MS metabolomics studies.
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http://dx.doi.org/10.1016/j.chroma.2017.01.050DOI Listing
March 2017

Evaluation of hazardous chemicals in edible insects and insect-based food intended for human consumption.

Food Chem Toxicol 2017 Feb 19;100:70-79. Epub 2016 Dec 19.

Toxicological Center, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

Due to the rapid increase in world population, the waste of food and resources, and non-sustainable food production practices, the use of alternative food sources is currently strongly promoted. In this perspective, insects may represent a valuable alternative to main animal food sources due to their nutritional value and sustainable production. However, edible insects may be perceived as an unappealing food source and are indeed rarely consumed in developed countries. The food safety of edible insects can thus contribute to the process of acceptance of insects as an alternative food source, changing the perception of developed countries regarding entomophagy. In the present study, the levels of organic contaminants (i.e. flame retardants, PCBs, DDT, dioxin compounds, pesticides) and metals (As, Cd, Co, Cr, Cu, Ni, Pb, Sn, Zn) were investigated in composite samples of several species of edible insects (greater wax moth, migratory locust, mealworm beetle, buffalo worm) and four insect-based food items currently commercialized in Belgium. The organic chemical mass fractions were relatively low (PCBs: 27-2065 pg/g ww; OCPs: 46-368 pg/g ww; BFRs: up to 36 pg/g ww; PFRs 783-23800 pg/g ww; dioxin compounds: up to 0.25 pg WHO-TEQ/g ww) and were generally lower than those measured in common animal products. The untargeted screening analysis revealed the presence of vinyltoluene, tributylphosphate (present in 75% of the samples), and pirimiphos-methyl (identified in 50% of the samples). The levels of Cu and Zn in insects were similar to those measured in meat and fish in other studies, whereas As, Co, Cr, Pb, Sn levels were relatively low in all samples (<0.03 mg/kg ww). Our results support the possibility to consume these insect species with no additional hazards in comparison to the more commonly consumed animal products.
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http://dx.doi.org/10.1016/j.fct.2016.12.006DOI Listing
February 2017

Metabolomics analysis of the toxicity pathways of triphenyl phosphate in HepaRG cells and comparison to oxidative stress mechanisms caused by acetaminophen.

Toxicol In Vitro 2015 Dec 28;29(8):2045-54. Epub 2015 Aug 28.

Department of In Vitro Toxicology and Dermato-cosmetology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium.

Since the publication of REACH guidelines, the need for in vitro tools for toxicity testing has increased. We present here the development of a hepatotoxicity testing tool using human HepaRG cell cultures and metabolomics. HepaRG cells were exposed to either 4mM acetaminophen (APAP) as reference toxicant for oxidative stress or 50 μM triphenyl phosphate (TPHP) as toxicant with unknown toxicity pathways (TPs). After 72 h exposure, cells were subjected to quenching and liquid-liquid extraction which resulted in a polar and an apolar fraction. Analysis of fractions was performed by ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-QTOF-MS). Significantly up or down regulated metabolites were selected by univariate statistics prior to identification. In order to obtain robust and specific TP biomarkers, the experiment was also repeated using a different culture medium composition to assess which metabolites show consistent changes. Potential biomarkers belonging to different TPs were found for APAP and TPHP. For APAP, the biomarkers were related to a decrease in unsaturated phospholipids, and for TPHP to an accumulation of phosphoglycerolipids and increase of palmitoyl lysophosphatidylcholine. This first proof-of-concept opens new perspectives for the analysis of other (reference) toxicants with different TPs and it can be used to expand the in vitro tool for hepatotoxicity screening of various compounds.
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http://dx.doi.org/10.1016/j.tiv.2015.08.012DOI Listing
December 2015
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