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

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

Altered Inactivation of Ca2+ Current and Ca2+ Release in Mouse Muscle Fibers Deficient in the DHP receptor γ1 subunit

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit γ1 have previously been studied using coexpression with the cardiac α1C polypeptide in nonmuscle cells and primary-cultured myotubes of γ1-deficient mice. Data from single adult muscle fibers of γ−/− mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from γ+/+ and γ−/− mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of −80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage–dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in γ−/− compared with γ+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the γ1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse

Cytotoxicity, metabolism, and isozyme mapping of the synthetic cannabinoids JWH-200, A-796260, and 5F-EMB-PINACA studied by means of in vitro systems

Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography–high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely