Metabolites In Hepatocytes Research Articles

Chaiqin chengqi decoction treatment mitigates hypertriglyceridemia-associated acute pancreatitis by modulating liver-mediated glycerophospholipid metabolism

BackgroundThe incidence of hypertriglyceridemia-associated acute pancreatitis (HTG-AP) is increasing globally and more so in China. The characteristics of liver-mediated metabolites and related key enzymes are rarely reported in HTG-AP. Chaiqin chengqi decoction (CQCQD) has been shown to protect against AP including HTG-AP in both patients and rodent models, but the underlying mechanisms in HTG-AP remain unexplored. PurposeTo assess the characteristics of liver-mediated metabolism and the therapeutic mechanisms of CQCQD in HTG-AP. MethodsMale human apolipoprotein C3 transgenic (hApoC3-Tg; leading to HTG) mice or wild-type littermates received 7 intraperitoneal injections of cerulein (100 μg/kg) to establish HTG-AP and CER-AP, respectively. In HTG-AP, some mice received CQCQD (5.5 g/kg) gavage at 1, 5 or 9 h after disease induction. AP severity and related liver injury were determined by serological and histological parameters; and underlying mechanisms were identified by lipidomics and molecular biology. Molecular docking was used to identify key interactions between CQCQD compounds and metabolic enzymes, and subsequently validated in vitro in hepatocytes. ResultsHTG-AP was associated with increased disease severity indices including augmented liver injury compared to CER-AP. CQCQD treatment reduced severity and liver injury of HTG-AP. Glycerophospholipid (GPL) metabolism was the most disturbed pathway in HTG-AP in comparison to HTG alone. In HTG-AP, the mRNA level of GPL enzymes involved in phosphocholine (PC) and phosphatidylethanolamine (PE) synthesis (Pcyt1a, Pcyt2, Pemt, and Lpcat) were markedly upregulated in the liver. Of the GPL metabolites, lysophosphatidylethanolamine LPE(16:0) in serum of HTG-AP was significantly elevated and positively correlated with the pancreas histopathology score (r = 0.65). In vitro, supernatant from Pcyt2-overexpressing hepatocytes co-incubated with LPE(16:0) or phospholipase A2 (a PC- and PE-hydrolyzing enzyme) alone induced pancreatic acinar cell death. CQCQD treatment downregulated PCYT1a and PCYT2 enzyme levels in the liver. Hesperidin and narirutin were identified top two CQCQD compounds with highest affinity docking to PCYT1a and PCYT2. Both hesperidin and narirutin reduced the level of some GPL metabolites in hepatocytes. ConclusionLiver-mediated GPL metabolism is excessively activated in HTG-AP with serum LPE(16:0) level correlating with disease severity. CQCQD reduces HTG-AP severity partially via modulating key enzymes in GPL metabolism pathway.

A breakthrough trial of an artificial liver without systemic heparinization in hyperbilirubinemia beagle models.

The development of wearable artificial livers was restricted to device miniaturization and bleeding risk with water-soluble anticoagulants. Herein, a double-deck column filled with solid anticoagulant microspheres and Kevlar porous microspheres (KPMs, bilirubin adsorbents) was connected with the principle machine of wearable artificial liver (approximately 9 kg) to treat hyperbilirubinemia beagles for the first time. With the initial normal dose of heparin, the double-deck column could afford 3 h hemoperfusion in whole blood without thrombus formation. The removal efficiency of the double-deck column for total bilirubin (TBIL) was 31.4%. Interestingly, the excessive amounts of hepatocyte metabolites were also decreased by approximately 25%. The “anticoagulant + column” realized safe and effective whole blood hemoperfusion without the plasma separation system and heparin pump; however, the proposed principle machine of wearable artificial liver and “anticoagulant + column” cannot completely replace the bio-liver now. The intelligence of the device and the versatility of the adsorbent need to be improved; moreover, advanced experimental techniques need to be developed to validate the survival rates in animals. Overall, this study is a meaningful trial for the development of wearable artificial livers in the future.

Biodistribution and pharmacokinetic profile of berberine and its metabolites in hepatocytes

BackgroundBerberine has been shown in clinical studies to have many health benefits, including anti-inflammatory and antioxidant properties, along with gut-flora balancing properties. However, its clinical efficacy is hindered by its low oral bioavailability and rapid metabolism. PurposeThis study aims to identify the berberine metabolites' forms and characterize their biodistribution patterns in and out of HepG2 cells. MethodsThe qualitative analysis of metabolites of berberine in HepG2 cells was performed using the LC/MSn-IT-TOF method. Subsequent cellular pharmacokinetics characterization of intracellular and extracellular berberine and its metabolites was performed by LC-MS/MS analysis. ResultsBerberine's metabolites of phase I metabolism were demethyleneberberine, jatrorrhizine, columbamine, berberrubine, etc., while its phase II metabolites were sulfate and glucuronide conjugates of phase I metabolites. Among the phase I metabolites of berberine, jatrorrhizine+columbamine accounted for over two-thirds of the total, followed by demethyleneberberine, which accounted for about a quarter. The intracellular demethyleneberberine is 25.14 times more enriched than extracellular demethyleneberberine. On the other hand, jatrorrhizine+columbamine and berberrubine were primarily distributed extracellularly, and their extracellular concentrations were 7.13 times and 15.61 times of their intracellular concentrations, respectively. Berberine metabolites produced in phase II metabolism are predominantly sulfate conjugates. ConclusionOur results show that demethyleneberberine is highly concentrated intracellularly in HepG2, possibly because it is an essential metabolite of berberine that likely contributes to berberine's efficacy. In light of our findings, berberine's poor plasma concentration-effectiveness characteristics have been partially explained.

New insight into the molecular mechanism of protein cross-linking induced by cis-2-butene-1,4-dial, the metabolite of furan: Formation of 2-substituted pyrrole cross-links involving the cysteine and lysine residues

Furan is an environmental pollutant also present in heat-treated food. The compound is toxic and carcinogenic to rats, and classified as a possible human carcinogen. Mechanisms laying behind the furan carcinogenicity remain unclear, however scientific data indicate the involvement of bioactivation catalysed by cytochrome P450 2E1. The resulted initial metabolite of furan, cis-2-butene-1,4-dial (BDA) is an extremely reactive conjugated dialdehyde able to damage important cellular components such as glutathione, proteins and nucleic acids. Earlier works showed that BDA induces protein cross-linking leading to the formation of modifications containing substituted pyrrole ring, and that the cysteine and lysine residues are prone to undergo such processes. The resulted cross-linked protein adducts are important for research aimed at exploring biomarkers of furan exposure. Due to furan's high volatility, biomarker-based methods appear to be a reliable approach to measure a level of exposure to this chemical. To date, numerous urinary and hepatocyte metabolites were identified. However, all the metabolites contain 3-substituted pyrrole ring resulted from the 1,4-addition of the cysteine thiol function to BDA followed by the condensation with the lysine free amino group. In this work we provide evidence that also 2-substituted pyrrole cross-links are formed at the physiological pH, when BDA is subjected to the reaction with N-acetylcysteine and Nα-acetyllysine, Nε-acetyllysine or lysine, respectively. The 2-substituted cross-links arise from the initial 1,2-addition of N-acetylcysteine to BDA accomplished by the condensation with lysine or its N-acetylated derivatives. Formation of the 2-substituted pyrroles sheds new light on the reactivity of cis-2-butene-1,4-dial towards amino acids, and contributes to complete characterisation of the compound chemistry. Our studies show that the 2- and 3-substituted cross-links are indistinguishable based on their MS and MS/MS spectra, and that for reliable qualitative and quantitative measurements the use of isotope-substituted synthetic internal standards is necessary. In the light of these facts, our results are important for research into furan exposure biomarkers.

Identification of the metabolites of methylophiopogonanone A by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry.

Methylophiopogonanone A (MOA) is a naturally occurring homoisoflavonoid from the Chinese herb Ophiopogon japonicus, which has been demonstrated to attenuate myocardial apoptosis. However, the metabolism of MOA remains unknown. The goal of the present work was to investigate the in vitro metabolism of MOA using liver microsomes and hepatocytes. The metabolites were generated by incubating MOA with rat, monkey and human liver microsomes or hepatocytes. The resulting samples were analyzed by using a quadrupole-orbitrap high-resolution mass spectrometer. The metabolites were identified through the measurements of the exact mass, elemental composition and product ions. A total of 15 metabolites were detected and identified. Among these metabolites, M7 (demethylenation) was the most abundant metabolite in liver microsomes, while M6 (hydroxylation) was the predominant metabolite in hepatocytes, and glucuronidation metabolites (M9 and M10) were also the main metabolites in hepatocytes. The metabolic pathways of MOA included hydroxylation, demethylenation, glucuronidation, methylation, sulfation and glutathione conjugation. This study for the first time provides valuable data on the metabolites of MOA, which will be of great importance for a better understanding of its disposition and to predict human pharmacokinetics.

Generation of functional liver organoids on combining hepatocytes and cholangiocytes with hepatobiliary connections ex vivo

In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. Although liver organoids have been reported, it is not clear whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report the generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors and cholangiocytes. Hepatocytes form the bile canalicular network and secrete metabolites into the canaliculi, which are then transported into the biliary tubular structure. Hepatocytes in HBTO acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we establish functional liver tissue incorporating a bile drainage system ex vivo. HBTO enable us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections.

Metabolic profiling of senkyunolide A and identification of its metabolites in hepatocytes by ultra-high-performance liquid chromatography combined with diode-array detector and high-resolution mass spectrometry.

Senkyunolide A is one of the bioactive constituents originally isolated from Ligusticum chuanxiong Hort. To better understand the action of this constituent, it is necessary to study the metabolic profiles in different species. For the metabolic stability study, senkyunolide A at a concentration of 0.5 μM was individually incubated with hepatocytes of mouse, rat, dog, monkey and human at 37°C for 2 h. For metabolite profiling and identification, senkyunolide A (10 μM) was incubated with hepatocytes and the incubation samples were analyzed by ultra-high-performance liquid chromatography combined with diode-array detector and high-resolution mass spectrometry (UHPLC/DAD-HRMS). The identities of the metabolites were characterized by accurate masses, product ions and retention times. Senkyunolide A was metabolically unstable in hepatocytes. The in vitro half-lives were 136.2, 60.6, 33.65, 55.96 and 138 min in mouse, rat, dog, monkey and human hepatocytes, respectively. Furthermore, a total of 14 metabolites were detected. M1 and M9 were the most abundant metabolites in all species. The metabolic pathways of senkyunolide A involved the following pathways: (1) hydroxylation to form 10- and 11-hydroxysenkyunolide A, which further underwent epoxidation followed by GSH conjugation; (2) epoxidation followed by epoxide hydrolysis or GSH conjugation; and (3) aromatization to form 3-butylphthalide followed by hydroxylation. Hydroxylation, epoxidation, aromatization and GSH conjugation were the main metabolic pathways of senkyunolide A. This study provides an overview of the metabolic profiles of senkyunolide A, which is helpful for a better understanding of the action of this compound.

Combined alcoholic and non-alcoholic steatohepatitis.

SummaryWhile metabolic syndrome and alcohol consumption are the two main causes of chronic liver disease, one of the two conditions is often predominant, with the other acting as a cofactor of morbimortality. It has been shown that obesity and alcohol act synergistically to increase the risk of fibrosis progression, hepatic carcinogenesis and mortality, while genetic polymorphisms can strongly influence disease progression. Based on common pathogenic pathways, there are several potential targets that could be used to treat both diseases; based on the prevalence and incidence of these diseases, new therapies and clinical trials are needed urgently.

In Vivo Metabolites of AB-PINACA in Solid Tissues Obtained from Its Abuser: Comparison with In Vitro Experiment.

In this study, solid tissues such as the lung, liver, kidney and urine were highlighted to profile the AB-PINACA in vivo metabolites in a fatal abuse case, although such metabolite analysis is usually made with urine specimens. We compared the relative peak intensities of in vivo metabolites of AB-PINACA in lung, liver, kidney and urine specimens collected at the autopsy of its abuser with its in vitro metabolites in human hepatocytes. The metabolites of AB-PINACA in tissues were extracted after homogenization. The urine specimen and portions of the extracted metabolites from tissues were firstly hydrolyzed with β-glucuronidase, and the metabolites were extracted. For in vitro experiment, AB-PINACA was incubated with human hepatocytes for 3h to produce its metabolites. The identification of the in vivo and in vitro metabolites was performed using liquid chromatography (LC)-high-resolution Orbitrap-tandem mass spectrometry (MS-MS), and the relative intensities of these metabolites were measured using low resolution LC-quadrupole-ion trap-MS-MS. Thirteen metabolites of AB-PINACA were characterized in vivo in several human specimens and in in vitro human hepatocytes. They were produced by the terminal amide hydrolysis to carboxylic acid, hydroxylation, carbonyl formation and/or glucuronidation. The most detectable metabolite in the hepatocytes, lung or liver was the one produced by the terminal amide hydrolysis, whereas the top metabolite in the kidney or urine was the one produced by hydroxylation or carbonyl formation on the pentyl side chain after the terminal amide hydrolysis, respectively. At least 12 metabolites of AB-PINACA were detected in authentic human lung, liver or kidney specimen from a cadaver. It is concluded that the postmortem metabolite profiling of AB-PINACA can be fulfilled with solid tissues, and the lung and kidney were most recommendable especially when urine specimen is not available.

Metabolism of the benzodiazepines norflurazepam, flurazepam, fludiazepam and cinolazepam by human hepatocytes using high-resolution mass spectrometry and distinguishing their intake in authentic urine samples

Norflurazepam, also a metabolite of other prescription benzodiazepines, appeared on the new psychoactive substances (NPS) drug market recently, complicating the interpretation of NPS findings. The aims of the study were to tentatively identify potential metabolites of norflurazepam and structural analogues (flurazepam, fludiazepam and cinolazepam) produced by hepatocytes and in authentic human samples and to discuss the possibility to differentiate drug consumption. Each drug (5 µmol/L) was incubated with pooled human hepatocytes, and metabolites were identified using liquid chromatography–high-resolution mass spectrometry (LC–HRMS). Similarly, urine with/without hydrolysis and blood/serum from three flurazepam and seven presumptive norflurazepam cases were analysed by LC–HRMS. No metabolites were detected for norflurazepam in hepatocytes, but six metabolites for flurazepam, two for fludiazepam and three for cinolazepam were found. In human specimens collected after flurazepam ingestion, a total of eight metabolites, in good agreement with hepatocyte metabolites, were detected. In specimens of presumptive norflurazepam intake, norflurazepam and its metabolites (four hydroxy metabolites and one glucuronide of a hydroxy metabolite) were found. Based on the results, hydroxy metabolites for norflurazepam, N-(hydroxyethyl), desethyl and didesethyl for flurazepam, hydroxy for fludiazepam and glucuronides and N-(hydroxyethyl) for cinolazepam are recommended for monitoring. While flurazepam, fludiazepam and cinolazepam were metabolised by hepatocytes at side chain, norflurazepam was not, which seems to indicate that hepatocytes have difficulty in modifying the benzene/diazepine rings of some 1,4-benzodiazepines. As for confirming the intake of norflurazepam, the urine ratio of 3-hydroxy-norflurazepam/norflurazepam might be the key; a high ratio might be correlated to norflurazepam intake, thereby enabling the differentiation.

Design, synthesis and evaluation of the osimertinib analogue (C-005) as potent EGFR inhibitor against NSCLC.

Osimertinib has been approved as a first-line treatment for non-small-cell lung cancer (NSCLC) patients whose tumor carries EGFR activation and / or resistant mutations. To mitigate Osimertinib's toxicity caused by AZ5104, the N-demethylation metabolite of Osimertinib, we designed and synthesized a series of Osimertinib analogs with different headpieces. In vitro and in vivo analysis rendered a potential clinical candidate C-005 which had pyrrolo-pyridine headpiece. Biochemically, C-005 and its main human hepatocyte metabolite showed over 30 fold selectivity of L858R/T790M mutant EGFR over WT EGFR. Such selectivity profile was retained at cellular level. In general, C-005 is 2-14 fold more selective than Osimertinib in a panel of WT EGFR cancer cell lines. Furthermore, C-005 demonstrated robust antitumor efficacy and good tolerability in NCI-H1975, PC-9 and HCC827 xenograft mouse models, making it a potential candidate for human test in clinical.

Identification of a xenobiotic as a potential environmental trigger in primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune-associated chronic liver disease triggered by environmental factors, such as exposure to xenobiotics, which leads to a loss of tolerance to the lipoic acid-conjugated regions of the mitochondrial pyruvate dehydrogenase complex, typically to the E2 component. We aimed to identify xenobiotics that might be involved in the environmental triggering of PBC. Urban landfill and control soil samples from a region with high PBC incidence were screened for xenobiotic activities using analytical, cell-based xenobiotic receptor activation assays and toxicity screens. A variety of potential xenobiotic classes were ubiquitously present, as identified by their interaction with xenobiotic receptors - aryl hydrocarbon receptor, androgen receptor and peroxisome proliferator activated receptor alpha - in cell-based screens. In contrast, xenoestrogens were present at higher levels in soil extracts from around an urban landfill. Furthermore, two landfill sampling sites contained a chemical(s) that inhibited mitochondrial oxidative phosphorylation and induced the apoptosis of a hepatic progenitor cell. The mitochondrial effect was also demonstrated in human liver cholangiocytes from three separate donors. The chemical was identified as the ionic liquid [3-methyl-1-octyl-1H-imidazol-3-ium]+ (M8OI) and the toxic effects were recapitulated using authentic pure chemical. A carboxylate-containing human hepatocyte metabolite of M8OI, bearing structural similarity to lipoic acid, was also enzymatically incorporated into the E2 component of the pyruvate dehydrogenase complex via the exogenous lipoylation pathway in vitro. These results identify, for the first time, a xenobiotic in the environment that may be related to and/or be a component of an environmental trigger for PBC. Therefore, further study in experimental animal models is warranted, to determine the risk of exposure to these ionic liquids. Primary biliary cholangitis is a liver disease in which most patients have antibodies to mitochondrial proteins containing lipoic acid binding site(s). This paper identified a man-made chemical present in soils around a waste site. It was then shown that this chemical was metabolized into a product with structural similarity to lipoic acid, which was capable of replacing lipoic acid in mitochondrial proteins.

Distinguishing Intake of New Synthetic Cannabinoids ADB-PINACA and 5F-ADB-PINACA with Human Hepatocyte Metabolites and High-Resolution Mass Spectrometry

ADB-PINACA and its 5-fluoropentyl analog 5F-ADB-PINACA are among the most potent synthetic cannabinoids tested to date, with several severe intoxication cases. ADB-PINACA and 5F-ADB-PINACA have a different legal status, depending on the country. Synthetic cannabinoid metabolites predominate in urine, making detection of specific metabolites the most reliable way for proving intake in clinical and forensic specimens. However, there are currently no data on ADB-PINACA and 5F-PINACA metabolism. The substitution of a single fluorine atom distinguishes the 2 molecules, which may share common major metabolites. For some legal applications, distinguishing between ADB-PINACA and 5F-PINACA intake is critical. For this reason, we determined the human metabolic fate of the 2 analogs. ADB-PINACA and 5F-PINACA were incubated for 3 h with pooled cryopreserved human hepatocytes, followed by liquid chromatography-high-resolution mass spectrometry analysis. Data were processed with Compound Discoverer. We identified 19 and 12 major ADB-PINACA and 5F-ADB-PINACA metabolites, respectively. Major metabolic reactions included pentyl hydroxylation, hydroxylation followed by oxidation (ketone formation), and glucuronidation of ADB-PINACA, and oxidative defluorination followed by carboxylation of 5F-ADB-PINACA. We recommend ADB-PINACA ketopentyl and hydroxypentyl, and ADB-PINACA 5-hydroxypentyl and pentanoic acid, as optimal markers for ADB-PINACA and 5F-ADB-PINACA intake, respectively. Since the 2 compounds present positional isomers as the primary metabolites, monitoring unique product ions and optimized chromatographic conditions are required for a clear distinction between ADB-PINACA and 5F-ADB-PINACA intake.

Pathway-specific inhibition of primaquine metabolism by chloroquine/quinine.

BackgroundThere has been some evidence to suggest that the addition of chloroquine (CQ) or quinine (QN) to 8-aminoquinoline (8-AQ) treatment regimens may increase the therapeutic efficacy of the 8-AQ and simultaneously mitigate against its haemolytic toxicity. However, both CQ and QN are considered effective, although perhaps moderate inhibitors of CYP2D6, an enzyme now regarded as necessary for primaquine (PQ) pharmacologic activity. An understanding of the influence of CQ and QN on the metabolism of PQ may shed light on the potential mechanisms of the beneficial interaction.MethodsDifferential metabolism of PQ enantiomers by recombinant human CYP2D6, monoamine oxidase A (MAO), and cryopreserved human hepatocytes in the presence/absence of CQ and QN.ResultsBoth CQ and QN significantly inhibited the activity of CYP2D6. PQ depletion by MAO and human hepatocytes was not affected significantly by the presence of CQ and QN. CYP2D6-mediated hydroxylation was largely suppressed by both CQ and QN. The formation of the primary deaminated metabolites, including carboxyprimaquine (CPQ) and cyclized side chain derivative from the aldehyde (m/z 241), was not sensitive to the presence of CQ and QN. However, the appearance of the glucuronides of CPQ and PQ alcohol were significantly suppressed. CQ and QN also inhibited the appearance of the m/z 257 metabolite with a similar pattern, suggesting that it may be derived from the CPQ conjugate. The apparent quinone-imine of CPQ (m/z 289) was only partially suppressed by both QN and CQ, but with a differential pattern of inhibition for the two drugs. The m/z 274 (quinone-imine of a ring-hydroxylated PQ metabolite) and m/z 422 (an apparent glucose conjugate of PQ) metabolites in hepatocytes were strongly suppressed by both QN and CQ, perhaps a reflection of the 2D6 inhibition by these drugs. The formation of the carbamoyl glucuronide of PQ (m/z 480) was not affected by CQ/QN.ConclusionThe metabolite-specific interactions in the current studies seem at variance with earlier reports of the dependence of PQ on CYP2D6 metabolism, and enhanced PQ anti-malarial activity/reduced toxicity in the presence of CQ/QN. These results suggest a complex picture in which CQ/QN may shift metabolite pathway balances towards a profile that retains efficacy, while reducing the formation or availability of toxic metabolites to erythrocytes. Alternatively, these drugs may alter transport or distribution of PQ metabolites in a fashion that reduces toxicity while maintaining efficacy against the parasite.

High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes.

Despite increasing prevalence of novel psychoactive substances, no human metabolism data are currently available, complicating laboratory documentation of intake in urine samples and assessment of the drugs' pharmacodynamic, pharmacokinetic, and toxicological properties. In 2014, THJ-018 and THJ-2201, synthetic cannabinoid indazole analogs of JWH-018 and AM-2201, were identified, with the National Forensic Laboratory Information System containing 220 THJ-2201 reports. Because of numerous adverse events, the Drug Enforcement Administration listed THJ-2201 as Schedule I in January 2015. We used high-resolution mass spectrometry (HR-MS) (TripleTOF 5600(+)) to identify optimal metabolite markers after incubating 10 μmol/L THJ-018 and THJ-2201 in human hepatocytes for 3 h. Data were acquired via full scan and information-dependent acquisition triggered product ion scans with mass defect filter. In silico metabolite predictions were performed with MetaSite and compared with metabolites identified in human hepatocytes. Thirteen THJ-018 metabolites were detected, with the major metabolic pathways being hydroxylation on the N-pentyl chain and further oxidation or glucuronidation. For THJ-2201, 27 metabolites were observed, predominantly oxidative defluorination plus subsequent carboxylation or glucuronidation, and glucuronidation of hydroxylated metabolites. Dihydrodiol formation on the naphthalene moiety was observed for both compounds. MetaSite prediction matched well with THJ-018 hepatocyte metabolites but underestimated THJ-2201 oxidative defluorination. With HR-MS for data acquisition and processing, we characterized THJ-018 and THJ-2201 metabolism in human hepatocytes and suggest appropriate markers for laboratories to identify THJ-018 and THJ-2201 intake and link observed adverse events to these new synthetic cannabinoids.

Preclinical metabolism and pharmacokinetics of NVS-CRF38, a potent and orally bioavailable corticotropin-releasing factor receptor 1 antagonist

1. The pharmacokinetic properties and metabolism of NVS-CRF38 [7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole], a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist, were determined in vitro and in animals.2. NVS-CRF38 undergoes near complete absorption in rats and dogs. In both species the compound has low hepatic extraction and is extensively distributed to tissues.3. In rat and human hepatic microsomes and cryopreserved hepatocytes from rat, dog, monkey and human, NVS-CRF38 was metabolised to form O-desmethyl NVS-CRF38 (M7) and several oxygen adducts (M1, M3, M4, M5 and M6). In hepatocytes further metabolites were observed, specifically the carboxylic acid (M2) and conjugates (sulphate and glucuronide) of M7.4. Formation of primary metabolites in hepatocytes was blocked by the cytochrome P450 enzyme (P450) suicide inhibitor 1-aminobenzotriazole, implicating P450 enzymes in the primary metabolism of this compound.5. NVS-CRF38 is weakly bound to plasma proteins from rat (fub = 0.19), dog (fub = 0.25), monkey (fub = 0.20) and humans (fub = 0.23). Blood-to-plasma partition for NVS-CRF38 approaches unity in rat and human blood.6. The hepatic clearance of NVS-CRF38 in humans is predicted to be low (extraction ratio ∼ 0.2) based on scaling from drug depletion profiles in hepatic microsomes.

Effects of hyperhomocysteinemia and betaine–homocysteine S-methyltransferase inhibition on hepatocyte metabolites and the proteome

Both cardiovascular disease and liver injury are major public health issues. Hyperhomocysteinemia has been linked to cardiovascular diseases, and defects in methyl group metabolism, often resulting in hyperhomocysteinemia, are among the key molecular events postulated to play a role in liver injury. We employed proteomics and metabolomics analyses of human hepatocytes in primary cell culture to explore the spectrum of proteins and associated metabolites affected by the disruption of methyl group metabolism. We treated the hepatocytes with homocysteine (Hcy, 0.1mM and 2mM) to follow the impact of hyperhomocysteinemia, and in parallel, we used a specific inhibitor of betaine–homocysteine S-methyltransferase (BHMT) to extend our understanding of the physiological functions of the enzyme. The major effect of BHMT inhibition was a 50% decrease in S-adenosylmethionine levels. The treatments with Hcy resulted in multiple changes in the metabolite levels depending on the treatment modality. The BHMT inhibition and 0.1mM Hcy treatment induced only moderate changes in the hepatocyte proteome and secretome, while the changes induced by the 2mM Hcy treatment were extensive. Phosphatidylethanolamine carboxykinase and ornithine aminotransferase were up-regulated about two fold indicating an intervention into metabolism. Cellular proliferation was suspended, secretome composition was changed and signs of apoptosis were discernible. We have detected fibrinogen gamma dimers, which might have a role as a potentially new biomarker of early liver injury. Finally, we have demonstrated the failed maturation of apolipoprotein A1, which might be a new mechanism of disruption of cholesterol efflux from tissues.

Behavioral and electroencephalographic manifestations of thioacetamide-induced encephalopathy: Possible mechanisms of neurotoxic effects

Although there is still no ideal experimental model of hepatic encephalopathy, thioacetamide is widely used for the induction of acute and chronic liver failure. Thioacetamide exerts hepatotoxic effects through the formation of toxic metabolites in hepatocytes, oxidative stress and calcium mobilization. An ideal experimental model of hepatic encephalopathy should have similar behavioral and electroencephalographic manifestations as human encephalopathy. Thioacetamide induces motor manifestations in a dose-dependent manner. Milder forms of thioacetamide-induced encephalopathy are associated with an increase in relative alpha power, while more severe forms are followed by a flattening of the electroencephalogram. liver failure-induced hyperammonemia has a pivotal role in the neurotoxic effects of thioacetamide. Hyperammonemia induces brain edema, alterations in neurotransmission, oxidative stress, mitochondrial dysfunction and neuronal death. The aim of this article is to review the behavioral and electroencephalographic manifestations of thioacetamide-induced encephalopathy, as well as to summarize potential mechanisms involved in thioacetamide neurotoxicity.

Polyamines are traps for reactive intermediates in furan metabolism.

Furan is toxic and carcinogenic in rodents. Because of the large potential for human exposure, furan is classified as a possible human carcinogen. The detailed mechanism by which furan causes toxicity and cancer is not yet known. Since furan toxicity requires cytochrome P450-catalyzed oxidation of furan, we have characterized the urinary and hepatocyte metabolites of furan to gain insight into the chemical nature of the reactive intermediate. Previous studies in hepatocytes indicated that furan is oxidized to the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA), which reacts with glutathione (GSH) to form 2-(S-glutathionyl)succinaldehyde (GSH-BDA). This intermediate forms pyrrole cross-links with cellular amines such as lysine and glutamine. In this article, we demonstrate that GSH-BDA also forms cross-links with ornithine, putrescine, and spermidine when furan is incubated with rat hepatocytes. The relative levels of these metabolites are not completely explained by hepatocellular levels of the amines or by their reactivity with GSH-BDA. Mercapturic acid derivatives of the spermidine cross-links were detected in the urine of furan-treated rats, which indicates that this metabolic pathway occurs in vivo. Their detection in furan-treated hepatocytes and in urine from furan-treated rats indicates that polyamines may play an important role in the toxicity of furan.

A Baeyer-Villiger Oxidation Specifically Catalyzed by Human Flavin-Containing Monooxygenase 5

10-((4-Hydroxypiperidin-1-yl)methyl)chromeno[4,3,2-de]phthalazin-3(2H)-one (E7016), an inhibitor of poly(ADP-ribose) polymerase, is being developed for anticancer therapy. One of the major metabolites identified in preclinical animal studies was the product of an apparent oxidation and ring opening of the 4-hydroxypiperidine. In vitro, this oxidized metabolite could not be generated by incubating E7016 with animal or human liver microsomes. Further studies revealed the formation of this unique metabolite in hepatocytes. In a NAD(P)(+)-dependent manner, this metabolite was also generated by liver S9 fractions and recombinant human flavin-containing monooxygenase (FMO) 5 that was fortified with liver cytosol fractions. In animal and human liver S9, this metabolic pathway could be inhibited by 4-methylpyrazole, bis-p-nitrophenylphosphate (BNPP), or a brief heat treatment at 50°C. Based on these results, the overall metabolic pathway was believed to involve a two-step oxidation process: dehydrogenation of the secondary alcohol in liver cytosol followed by an FMO5-mediated Baeyer-Villiger oxidation in liver microsomes. The two oxidation steps were coupled via regeneration of NAD(P)(+) and NAD(P)H. To further confirm this mechanism, the proposed ketone intermediate was independently synthesized. In an NAD(P)H-dependent manner, the synthetic ketone intermediate was metabolized to the same ring-opened metabolite in animal and human liver microsomes. This metabolic reaction was also inhibited by BNPP or a brief heat treatment at 50°C. Methimazole, the substrate/inhibitor of FMO1 and FMO3, did not inhibit this reaction. The specificity of FMO5 toward catalyzing this Baeyer-Villiger oxidation was further demonstrated by incubating the synthetic ketone intermediate in recombinant enzymes.