Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay.

The continuous emergence of new psychoactive substances (NPS) attracted a great deal of attention within recent years. Lately, the two hallucinogenic NPS 1cP-LSD and 4-AcO-DET have appeared on the global market. Knowledge about their metabolism to identify potential metabolic targets for analysis and their cytotoxic properties is lacking. The aim of this work was thus to study their in vitro and in vivo metabolism in pooled human liver S9 fraction (pHLS9) and in zebrafish larvae (ZL) by means of liquid chromatography-high-resolution tandem mass spectrometry. Monooxygenases involved in the initial metabolic steps were elucidated using recombinant human isozymes. Investigations on their cytotoxicity were performed on the human hepatoma cell line HepG2 using a multiparametric, fluorescence-based high-content screening assay. This included measurement of CYP-enzyme mediated effects by means of the unspecific CYP inhibitor 1-aminbenzotriazole (ABT). Several phase I metabolites of both compounds and two phase II metabolites of 4-AcO-DET were produced in vitro and in vivo. After microinjection of 1cP-LSD into the caudal vein of ZL, three out of seven metabolites formed in pHLS9 were also detected in ZL. Twelve 4-AcO-DET metabolites were identified in ZL after exposure via immersion bath and five of them were found in pHLS9 incubations. Notably, unique metabolites of 4-AcO-DET were only produced by ZL, whereas 1cP-LSD specific metabolites were found both in ZL and in pHLS9. No toxic effects were observed for 1cP-LSD and 4-AcO-DET in HepG2 cells, however, two parameters were altered in incubations containing 4-AcO-DET together with ABT compared with incubations without ABT but in concentrations far above expected in vivo concentration. Further investigations should be done with other hepatic cell lines expressing higher levels of CYP enzymes.

A Comparison of In Vitro Metabolic Clearance of Various Regulatory Fish Species Using Hepatic S9 Fractions.

Bioaccumulation predictions can be substantially improved by combining in vitro metabolic rate measurements derived from rainbow trout hepatocytes and/or hepatic S9 fractions with quantitative structure-activity relationship (QSAR) modeling approaches. Compared with in vivo testing guidelines Organisation for Economic Co-operation and Development (OECD) 305 and Office of Chemical Safety and Pollution Prevention (OCSPP; an office of the US Environmental Protection Agency) 850.1730, the recently adopted OECD test guidelines 319A and 319B are in vitro approaches that have the potential to provide a time- and cost-efficient, humane solution, reducing animal use while addressing uncertainties in bioaccumulation across species. The present study compares the hepatic clearance of the S9 subcellular fraction of rainbow trout, bluegill, common carp, fathead minnow, and largemouth bass, discerning potential differences in metabolism between different warm- and cold-water species. With refinements to the in vitro metabolic S9 assay for high-throughput analysis, we measured in vitro clearance rates of seven chemicals crossing multiple classes of chemistry and modes of action. We confirmed that data from rainbow trout liver S9 fraction metabolic rates can be utilized to predict rainbow trout bioconcentration factors using an in vitro to in vivo extrapolation model, as intended in the OECD 319B applicability domain per the bioaccumulation prediction. Also, we determined that OECD 319B can be applied to other species, modified according to their habitat, adaptations to feeding behavior, and environmental conditions (e.g., temperature). Once toxicokinetics for each species is better understood and appropriate models are developed, this method can be an excellent tool to determine hepatic clearance and potential bioaccumulation across species. The present study could be leveraged prior to or in place of initiating in vivo bioconcentration studies, thus optimizing selection of appropriate fish species. Environ Toxicol Chem 2024;43:1390-1405. © 2024 SETAC.

Comparison of the in vitro assays to investigate the hepatic metabolism of seven pesticides in Cyprinus carpio and Oncorhynchus mykiss.

Liver S9 fractions from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were incubated with seven pesticides (fenamidone, fenoxaprop-p-ethyl, penflufen, spirotetramat, tebuconazole, tembotrione and trifloxystrobin) and the metabolic pathways of the applied chemicals were determined by HPLC-high-resolution mass spectrometry. Five of the seven pesticides (fenamidone, penflufen, spirotetramat, trifloxystrobin and fenoxaprop-p-ethyl) revealed a higher metabolic capacity of rainbow trout liver fractions compared to carp liver fractions. The other two pesticides (tebuconazole and tembotrione) showed a similar and marginal biotransformation for liver S9 fractions of both species. Furthermore, four compounds (penflufen, spirotetramat, tembotrione and tebuconazole) were incubated with cryo-preserved hepatocytes of rainbow trout showing additional conjugated metabolites compared to liver S9 fractions. The incubations were performed with concentrations of 1 and 10 µM for experiments with liver S9 fractions and 5 µM with hepatocytes for up to 120 (liver S9 fractions) or 240 min (hepatocytes). A set of positive controls was used to confirm the metabolic capability of the in vitro systems. The comparison of the in vitro results from hepatocyte assays of penflufen and tebuconazole with the data from corresponding in vivo studies performed according to OECD (Organisation for Economic Co-operation and Development) guideline 305 exhibited a similar metabolic behavior for these pesticides and emphasizes the reliability of the in vitro assays. Besides investigation of the metabolism of plant protection products for research purposes, inter-species comparison by in vitro assays and the use of PBTK modelling approaches will allow improved environmental and dietary risk assessments.

In vitro and in vivo metabolic stability of various fragrance materials and insect repellent in rainbow trout (Oncorhynchus mykiss).

Commercial fragrances consist of several thousand natural and synthetic substances formulated in complex combinations. These ingredients are frequently blended at very low concentrations but they are typically lipophilic and a few of them (e.g., synthetic musks) have been detected in aquatic systems, albeit at low concentrations. Few fragrances have guideline in vivo data on bioaccumulation, so in silico modeling has been widely used to estimate bioconcentration factors (BCFs). This study used in vitro metabolism assays with trout S9 cell fractions and cryopreserved hepatocytes to improve estimates of fish BCFs and to test published methods for extrapolating in vitro metabolic rate data to whole fish and corresponding BCFs. These estimates for several chemicals were compared with new in vivo bioconcentration measurements and previously published data on fragrances and the insect repellent, DEET. In total, 17 (20 including isomers) fragrance chemicals (abalyn, amberwood, amboryl acetate, bisabolene, cedroxide, coniferan, elemol, givescone, maritima, precyclemone B, polysantol, sandela, sanjinol, santalex, timberol and vernaldehyde) and DEET were metabolized at various rates. Only three materials tested did not appear to undergo enzymatic degradation (caryophyllene oxide, galaxolide and ketone patchouli). Even relatively slow rates of metabolism had a large influence on bioconcentration estimates. This work adds valuable information to the evolving body of work supporting the use of in vitro determinations of hepatic clearance to improve modeled predictions of bioaccumulation. It can also be used directly to help prioritize testing of potential bioaccumulative chemicals or serve as a more economical method for screening these chemicals.

Toxicokinetics and analytical toxicology of the abused opioid U-48800 - in vitro metabolism, metabolic stability, isozyme mapping, and plasma protein binding.

Due to the risk of new synthetic opioids (NSOs) for human health, the knowledge of their toxicokinetic characteristics is important for clinical and forensic toxicology. U-48800 is an NSO structurally non-related to classical opioids such as morphine or fentanyl and offered for abuse. As toxicokinetic data of U-48800 is not currently available, the aims of this study were to identify the in vitro metabolites of U-48800 in pooled human liver S9 fraction (pS9), to map the isozymes involved in the initial metabolic steps, and to determine further toxicokinetic data such as metabolic stability, including the in vitro half-life (t1/2 ), and the intrinsic (CLint ) and hepatic clearance (CLh ). Furthermore, drug detectability studies in rat urine should be done using hyphenated mass spectrometry. In total, 13 phase I metabolites and one phase II metabolite were identified. N-Dealkylation, hydroxylation, and their combinations were the predominant metabolic reactions. The isozymes CYP2C19 and CYP3A4 were mainly involved in these initial steps. CYP2C19 poor metabolizers may suffer from an increased U-48800 toxicity. The in vitro t1/2 and CLint could be rated as moderate, compared to structural related compounds. After administration of an assumed consumer dose to rats, the unchanged parent compound was found only in very low abundance but three metabolites were detected additionally. Due to species differences, metabolites found in rats might be different from those in humans. However, phase I metabolites found in rat urine, the parent compound, and additionally the N-demethyl metabolite should be used as main targets in toxicological urine screening approaches.

In vitro biotransformation of pharmaceuticals and pesticides by trout liver S9 in the presence and absence of carbamazepine.

The aim of the present study was to develop (i) a technique for identifying metabolites of organic contaminants by using an in vitro system of trout S9 and liquid chromatography-high-resolution mass spectrometry-based identification method and (ii) to apply this technique to identify the interactive potential of carbamazepine on the formation rate of other metabolites. The pharmaceuticals carbamazepine and propranolol and the pesticides azoxystrobin, diazinon, and fipronil were selected as test contaminants. As a result, a total of ten metabolites were identified for the five parent substances, six of which were confirmed using reference standards. Metabolic reactions included hydroxylation, epoxidation, S-oxidation, and dealkylation. The metabolic transformation rate ranged from 0.2 to 3.5 pmol/mg protein/min/µmol substrate. In the binary exposure experiment with increasing carbamazepine concentration, the formation rates of diazinon and fipronil metabolites (MDI2 and MFP2, respectively) increased, while formation of metabolites of propranolol and azoxystrobin (MPR1, MPR2, MPR3, and MAZ1) slowed down. Meanwhile, S9 pre-exposed to carbamazepine produced diazoxon, a toxic metabolite of diazinon, and pyrimidinol, a less toxic metabolite, more rapidly. These results suggest that carbamazepine, a perennial environmental pollutant, might modulate the toxicity of other substances such as diazinon but further in vivo studies are needed.

Identification of metabolic pathways related to the bisphenol A-induced adipogenesis in differentiated murine adipocytes by using RNA-sequencing.

We evaluated the effect of bisphenol A and its metabolites on the 3T3-L1 cells, in terms of glucose and lipid metabolism. We also aimed to obtain the information on the genome-wide expression changes in the 3T3-L1 cells treated with Bisphenol A by using RNA-seq, which involves whole-transcriptome sequencing. Differentially Expressed Genes (DEGs) collected from RNA-seq can be used to produce a complete picture of related metabolism pathways. The KEGG pathway was extracted based on the DEGs. Bisphenol A significantly increased the mRNA level of Sterol regulatory element binding transcription factor 1 (Srebf1) and CCAAT/enhancer binding protein alpha (Cebpa). Lipoprotein lipase (Lpl) was also significantly influenced by bisphenol A and its metabolites. Acetyl-Coenzyme A carboxylase beta (Acacb) and Fatty acid synthase (Fasn) mRNA levels were elevated by bisphenol A and its metabolites. The insulin signaling pathway, neurotrophin signaling pathway, and endometrial cancer-related pathway were focused by the functional enrichment analyses, and the pathways were well coincided with recent previous reports. DEGs collected from RNA-seq were confirmed as a reliable evidence in the exposure to the chemicals such as bisphenol A. Collecting pieces of the puzzles obtained from the RNA-seq will help us to produce a complete picture of the metabolic pathway for such chemicals.

Comparative Study on Transformation of Tecomin by Using Liver Microsomes and Liver S9 QIAO Shan-shan, / 中国中医药信息杂志

Objective To compare the difference of transformation profile and transformation rate of tecomin by using two in vitro liver metabolism models. Methods Liver microsomes and liver S9 fraction models were employed to transform tecomin. HPLC was used to determine the contents of tecomin and its metabolites at the detecting wavelength of 254 nm. The gradient elution (0–6 min, 5％–40％ A; 6–9 min, 40％–50％ A; 9–11 min, 50％–5％ A) was carried out by using mobile phase of acetonitrile (A) - 1％ acetic acid (B) at a flow rate of 1 mL/min. Results Both models could transform tecomin into veratric acid; however, the metabolites obtained with liver S9 were more than those obtained with liver microsomes, and the transformation rate of the former was higher than that of the latter. Conclusion The liver S9 fraction can more efficiently transform esters than liver microsomes.

Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism.

BACKGROUND: Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. METHODS: Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification. RESULTS: The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R²>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases. CONCLUSIONS: Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin, a pore-forming peptide.

1. An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency. 2. Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. 3. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. 4. These results clearly demonstrate the advantages of using alamethicin for the removal of latency in UGT activity studies with trout and may have broad implications for the study of UGTs in other fish species.

Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism

BACKGROUND: Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. METHODS: Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification. RESULTS: The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R 2>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases. CONCLUSIONS: Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

Metabolism of xenobiotics by aldehyde oxidase.

Aldehyde oxidase (AO) is a cytosolic molybdoflavoprotein whose contribution to the metabolism and clearance of xenobiotics-containing heterocyclic rings has attracted increased interest in recent years. This unit details methods for identification and confirmation of AO as a metabolic pathway as well as a method for estimating clearance of compounds that are AO substrates.

Liver S9 Fraction-Derived Metabolites of Curcumin Analogue UBS109.

To address the shortcomings of the natural product curcumin, many groups have created analogues that share similar structural features while displaying superior properties, particularly in anticancer drug discovery. Relatively unexplored have been the mechanisms by which such compounds are metabolized. A comprehensive in vitro study of a curcumin analogue (UBS109) in liver S9 fractions from five different species is presented. Further, we examine the cell-based bioactivity of the major metabolites. In spite of the fact that UBS109 reduces tumor growth in mice, it is quickly metabolized in vitro and 94% protein bound in mouse plasma. The primary monounsaturated metabolite is only modestly bioactive against MDA-MB-231 breast cancer cells. These observations suggest that while the α,ß-unsaturated ketone common to curcumin analogues is important for bioactivity, protein binding and tissue distribution may serve to protect UBS109 from full metabolism in vivo while allowing it to exert a pharmacological effect by means of slow drug release.

Study on sulfation metabolism of scutellarein in FVB/NCrIVr mice / 中国药学杂志

OBJECTIVE: To investigate the characteristics of sulfation of scutellarein in FVB/NCrIVr (FVB) mice. METHODS: FVB mouse intestinal perfusion model and incubation system with FVB mouse liver S9 fractions were adapted to conduct the study. HPLC-MS/MS and HPLC-UV were used to identify and quantify scutellarein and its metabolites in the samples. RESULTS: One sulfation metabolite and one glucuronidation metabolite were detected in the small intestinal perfusate. There was no significant difference between the excretion rates of sulfation metabolite and glucuronidation metabolite in small intestinal perfusate (P=0.435), while only sulfation metabolite of scutellarein could be detected in colon perfusate, scutellarein, sulfation metabolite and glucuronidation metabolite of scutellarein could all be detected in biliary samples, indicating an entero-hepatic circulation of scutellarein. In the liver S9 fractions, sulfation rate at 20 μmol·L-1 was sig nificantly higher than those at 10 and 40 μmol·L-1 (P<0.05). CONCLUSION: Sulfation was found to be the most important metabolism route in the intestinal disposition of scutellarein. There is probably a substrate inhibition effect in the sulfation of scutellarein in liver S9 fractions.