Tools for studying the metabolism of new psychoactive substances for toxicological screening purposes – A comparative study using pooled human liver S9, HepaRG cells, and zebrafish larvae

Richter, Lilian H.J.; Herrmann, Jennifer; Andreas, Anastasia; Park, Yu Mi; Wagmann, Lea; Flockerzi, Veit; Müller, Rolf; Meyer, Markus R.

doi:10.1016/j.toxlet.2019.01.010

Cited by 41 publications

(64 citation statements)

References 28 publications

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“…Following the study of Richter et al (2019a), zebrafish maintenance and all experiments with larvae were performed according to internal protocols based on standard methods (Westerfield 2007). Zebrafish larvae were raised at 28 °C in Danieau's medium consisting of 17 mM NaCl, 2 mM KCl, 0.12 mM MgSO 4 , 1.8 mM Ca(NO 3 ) 2 , 1.5 mM HEPES, and 1.2 µM methylene blue.…”

Section: Maximum-tolerated Concentration (Mtc) Studies In Zebrafish Lmentioning

confidence: 99%

“…In compliance with an already described procedure (Richter et al 2019a), 4F-Cy-BAP or Fu-BAP were administered to the zebrafish larvae (4 dpf) via medium. One well of a 6-well plate contained 10 zebrafish larvae and 2 mL of Danieau's medium spiked with the compound (100 µM 4F-Cy-BAP or 80 µM Fu-BAP final concentrations, respectively).…”

Section: In Vivo Identification Of Metabolitesmentioning

confidence: 99%

“…Although in vitro metabolism studies may have some advantages, e.g. cost-effectivity or feasibility, there is often a discrepancy between the metabolites identified in vitro to those in human (Richter et al 2019a). As no human samples after intake of 4F-Cy-BAP or Fu-BAP were available, an additional in vivo assay should confirm possible main targets for toxicological screenings.…”

Section: Comparison Of In Vitro and In Vivo Metabolitesmentioning

confidence: 99%

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Toxicokinetics and toxicodynamics of the fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine and furanoyl-1-benzyl-4-anilinopiperidine

et al. 2020

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The two fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine (4F-Cy-BAP) and furanoyl-1-benzyl-4-anilinopiperidine (Fu-BAP) have recently been seized as new psychoactive substances (NPS) on the drugs of abuse market. As their toxicokinetic and toxicodynamic characteristics are completely unknown, this study focused on elucidating their in vitro metabolic stability in pooled human liver S9 fraction (pHLS9), their qualitative in vitro (pHLS9), and in vivo (zebrafish larvae) metabolism, and their in vitro isozyme mapping using recombinant expressed isoenzymes. Their maximum-tolerated concentration (MTC) in zebrafish larvae was studied from 0.01 to 100 µM. Their µ-opioid receptor (MOR) activity was analyzed in engineered human embryonic kidney (HEK) 293 T cells. In total, seven phase I and one phase II metabolites of 4F-Cy-BAP and 15 phase I and four phase II metabolites of Fu-BAP were tentatively identified by means of liquid chromatography high-resolution tandem mass spectrometry, with the majority detected in zebrafish larvae. N-Dealkylation, N-deacylation, hydroxylation, and N-oxidation were the most abundant metabolic reactions and the corresponding metabolites are expected to be promising analytical targets for toxicological analysis. Isozyme mapping revealed the main involvement of CYP3A4 in the phase I metabolism of 4F-Cy-BAP and in terms of Fu-BAP additionally CYP2D6. Therefore, drug-drug interactions by CYP3A4 inhibition may cause elevated drug levels and unwanted adverse effects. MTC experiments revealed malformations and changes in the behavior of larvae after exposure to 100 µM Fu-BAP. Both substances were only able to produce a weak activation of MOR and although toxic effects based on MOR activation seem unlikely, activity at other receptors cannot be excluded. Keywords In vitro and in vivo metabolism • Metabolic stability • LC-HRMS/MS • Zebrafish larvae • In vitro µ-opioid receptor activity Electronic supplementary material The online version of this article (

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Section: Maximum-tolerated Concentration (Mtc) Studies In Zebrafish Lmentioning

confidence: 99%

Section: In Vivo Identification Of Metabolitesmentioning

confidence: 99%

Section: Comparison Of In Vitro and In Vivo Metabolitesmentioning

confidence: 99%

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Toxicokinetics and toxicodynamics of the fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine and furanoyl-1-benzyl-4-anilinopiperidine

et al. 2020

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“…The zebra sh has been widely used in developmental biology, toxicology, genetics, and pharmacology because the sh is small, easy to cultivate and manage, and has a short reproductive cycle; in vitro fertilization and development are possible; and the embryos are easily observed and treated. 33,34 The components of PM2.5 are complex and enter the cardiovascular system through the lung-capillary barrier. Currently, the toxicity screening research and application of drugs resulting from this research primarily focus on neurotoxicity, cardiotoxicity, embryonic developmental toxicity, and so on.…”

Section: Discussionmentioning

confidence: 99%

Walnut protein isolates attenuate particulate matter-induced lung and cardiac injury in mice and zebra fish

Zhang

Liu²,

Fan

et al. 2019

RSC Adv.

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“…Incubations using HepaRG cells were performed according to a previous publication (Richter et al 2019). For each experiment, cells were maintained in an incubator (Binder, Tuttlingen, Germany) at 37 °C with 95% air humidity and 5% CO 2 atmosphere.…”

Section: Incubations Using Heparg Cellsmentioning

confidence: 99%

Toxicometabolomics of the new psychoactive substances α-PBP and α-PEP studied in HepaRG cell incubates by means of untargeted metabolomics revealed unexpected amino acid adducts

et al. 2020

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Toxicometabolomics, essentially applying metabolomics to toxicology of endogenous compounds such as drugs of abuse or new psychoactive substances (NPS), can be investigated by using different in vitro models and dedicated metabolomics techniques to enhance the number of relevant findings. The present study aimed to study the toxicometabolomics of the two NPS α-pyrrolidinobutiophenone (1-phenyl-2-(pyrrolidin-1-yl)butan-1-one, α-PBP) and α-pyrrolidinoheptaphenone (1-phenyl-2-(pyrrolidin-1-yl)heptan-1-one, α-PEP, PV8) in HepaRG cell line incubates. Evaluation was performed using reversed-phase and normal-phase liquid chromatography coupled with high-resolution mass spectrometry in positive and negative ionization mode, respectively, to analyze cells and cell media. Statistical evaluation was performed using one-way ANOVA, principal component discriminant function analysis, as well as hierarchical clustering. In general, the analysis of cells did not mainly reveal any features, but the parent compounds of the drugs of abuse. For α-PBP an increase in N-methylnicotinamide was found, which may indicate hepatotoxic potential of the substance. After analysis of cell media, significant features led to the identification of several metabolites of both compounds. Amino acid adducts with glycine and alanine were found, and these have not been described in any study before and are likely to appear in vivo. Additionally, significant changes in the metabolism of cholesterol were revealed after incubation with α-PEP. In summary, the application of metabolomics techniques after HepaRG cells exposure to NPS did not lead to an increased number of identified drug metabolites compared to previously published studies, but gave a wider perspective on the physiological effect of the investigated compounds on human liver cells.

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Tools for studying the metabolism of new psychoactive substances for toxicological screening purposes – A comparative study using pooled human liver S9, HepaRG cells, and zebrafish larvae

Cited by 41 publications

References 28 publications

Toxicokinetics and toxicodynamics of the fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine and furanoyl-1-benzyl-4-anilinopiperidine

Toxicokinetics and toxicodynamics of the fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine and furanoyl-1-benzyl-4-anilinopiperidine

Walnut protein isolates attenuate particulate matter-induced lung and cardiac injury in mice and zebra fish

Toxicometabolomics of the new psychoactive substances α-PBP and α-PEP studied in HepaRG cell incubates by means of untargeted metabolomics revealed unexpected amino acid adducts

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