Matthias Cuykx

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

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.

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

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.

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

HighlightsNine in vitro metabolites of cloniprazepam identified with LC‐QTOF‐MS after incubation with human liver microsomes.Five metabolites proposed as potential analytical targets for cloniprazepam consumption.Clonazepam appeared to be the most prominent metabolite of cloniprazepam. Abstract 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.

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

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.

Optimisation of in vitro sample preparation for LC-MS metabolomics applications on HepaRG cell cultures

Untargeted metabolomics studies are designed to observe as many metabolites as possible. A reliable protocol should be fast, efficient, unspecific and it should introduce as little variance as possible. Liquid–liquid extraction (LLE) is often applied during sample preparation, since it leads to a polar (aqueous) and a non-polar (solvent) fraction. In the present study, we have applied for the first time pH buffers and other additives during the LLE to prevent degradation of the metabolites and to improve the precision of the method. The use of chamber slides in comparison to well plates improved the washing and quenching step and reduced the average quench time. Extraction efficiency and usability improved as well. The stabilisation of the pH of the LLE-solvent and the addition of anti-oxidants and chelators improved the precision of the method. Optimal results were observed at buffer strength of 10 mM at pH 4.4. Ascorbic acid (0.5 mM) and butyl-hydroxytoluene (1 mM) in combination with EDTA (1 mM) prevented oxidation and degradation.

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

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.

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

In this pilot study three fractions of particulate matter (PM0.25, PM2.5-0.25, and PM10-2.5) 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 childrens 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 PM10-2.5 reaching the alveoli was minor, while PM2.5-0.25 and PM0.25 apportion for most of the PM mass retained in the lungs. Consequently, PM2.5-0.25 and PM0.25 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 (LC5) were estimated. Afterwards, two LC-MS metabolomic assays were conducted: one applying LC5, and another applying real dose. A lower estimated LC5 value was obtained for PM0.25 than PM2.5-0.25 (8.08 and 73.7 ng/mL respectively). The number of altered features after LC5 exposure was similar for both fractions (39 and 38 for PM0.25 and PM2.5-0.25 respectively), while after real dose exposure these numbers differed (10 and 5 for PM0.25 and PM2.5-0.25 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.