Characterization of the RAS/RAF/ERK Signal Cascade as a Novel Regulating Factor in Alpha-Amanitin-Induced Cytotoxicity in Huh-7 Cells.

The well-known hepatotoxicity mechanism resulting from alpha-amanitin (α-AMA) exposure arises from RNA polymerase II (RNAP II) inhibition. RNAP Ⅱ inhibition occurs through the dysregulation of mRNA synthesis. However, the signaling pathways in hepatocytes that arise from α-AMA have not yet been fully elucidated. Here, we identified that the RAS/RAF/ERK signaling pathway was activated through quantitative phosphoproteomic and molecular biological analyses in Huh-7 cells. Bioinformatics analysis showed that α-AMA exposure increased protein phosphorylation in a time-dependent α-AMA exposure. In addition, phosphorylation increased not only the components of the ERK signaling pathway but also U2AF65 and SPF45, known splicing factors. Therefore, we propose a novel mechanism of α-AMA as follows. The RAS/RAF/ERK signaling pathway involved in aberrant splicing events is activated by α-AMA exposure followed by aberrant splicing events leading to cell death in Huh-7 cells.

Pharmacokinetics of α-amanitin in mice using liquid chromatography-high resolution mass spectrometry and in vitro drug-drug interaction potentials.

The aim of this study was to determine pharmacokinetics of α-amanitin, a toxic bicyclic octapeptide isolated from the poisonous mushrooms, following intravenous (iv) or oral (po) administration in mice using a newly developed and validated liquid chromatography-high resolution mass spectrometry. The iv injected α-amanitin disappeared rapidly from the plasma with high a clearance rate (26.9-30.4 ml/min/kg) at 0.1, 0.2, or 0.4 mg/kg doses, which was consistent with a rapid and a major excretion of α-amanitin via the renal route (32.6%). After the po administration of α-amanitin at doses of 2, 5, or 10 mg/kg to mice, the absolute bioavailability of α-amanitin was 3.5-4.8%. Due to this low bioavailability, 72.5% of the po administered α-amanitin was recovered from the feces. When α-amanitin is administered po, the tissue to plasma area under the curve ratio was higher in stomach > large intestine > small intestine > lung ~ kidneys > liver but not detected in brain, heart, and spleen. The high distribution of α-amanitin to intestine, kidneys, and liver is in agreement with the previously reported major intoxicated organs following acute α-amanitin exposure. In addition, α-amanitin weakly or negligibly inhibited cytochrome P450 and 5'-diphospho-glucuronosyltransferase enzymes activity in human liver microsomes as well as major drug transport functions in mammalian cells overexpressing transporters. Data suggested remote drug interaction potential may be associated with α-amanitin exposure.

Comparative metabolism of fargesin in human, dog, monkey, mouse, and rat hepatocytes.

Fargesin, a bioactive lignan derived from Flos Magnoliae, possesses anti-inflammatory, anti-oxidative, anti-melanogenic, and anti-apoptotic effects. This study compared the metabolic profiles of fargesin in human, dog, monkey, mouse, and rat hepatocytes using liquid chromatography-high resolution mass spectrometry. In addition, we investigated the human cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes responsible for fargesin metabolism. The hepatic extraction ratio of fargesin among the five species ranged from 0.59 to 0.78, suggesting that it undergoes a moderate-to-extensive degree of hepatic metabolism. During metabolism, fargesin generates three phase 1 metabolites, including fargesin catechol (M1) and O-desmethylfargesin (M2 and M3), and 11 phase 2 metabolites, including O-methyl-M1 (M4 and M5) via catechol O-methyltransferase (COMT), glucuronides of M1, M2, M4, and M5, and sulfates of M1-M5. The production of M1 from fargesin via O-demethylenation is catalyzed by CYP2C9, CYP3A4, CYP2C19, and CYP2C8 enzymes, whereas the formation of M2 and M3 (O-desmethylfargesin) is catalyzed by CYP2C9, CYP2B6, CYP2C19, CYP3A4, CYP1A2, and CYP2D6 enzymes. M4 is metabolized to M4 glucuronide by UGT1A3, UGT1A8, UGT1A10, UGT2B15, and UGT2B17 enzymes, whereas M4 sulfate is generated by multiple SULT enzymes. Fargesin is extensively metabolized in human hepatocytes by CYP, COMT, UGT, and SULT enzymes. These findings help to elucidate the pharmacokinetics and drug interactions of fargesin.

Comparative metabolism of aschantin in human and animal hepatocytes.

Aschantin, a tetrahydrofurofuran lignan with a 1,3-benzodioxole group derived from Flos Magnoliae, exhibits antioxidant, anti-inflammatory, cytotoxic, and antimicrobial activities. This study compared the metabolic profiles of aschantin in human, dog, mouse, and rat hepatocytes using liquid chromatography-high-resolution mass spectrometry. The hepatic extraction ratio of aschantin among the four species was 0.46-0.77, suggesting that it undergoes a moderate-to-extensive degree of hepatic metabolism. Hepatocyte incubation of aschantin produced 4 phase 1 metabolites, including aschantin catechol (M1), O-desmethylaschantin (M2 and M3), and hydroxyaschantin (M4), and 14 phase 2 metabolites, including O-methyl-M1 (M5 and M6) via catechol O-methyltransferase (COMT), six glucuronides of M1, M2, M3, M5, and M6, and six sulfates of M1, M2, M3, M5, and M6. Enzyme kinetic studies using aschantin revealed that the production of M1, a major metabolite, via O-demethylenation is catalyzed by cytochrome 2C8 (CYP2C8), CYP2C9, CYP2C19, CYP3A4, and CYP3A5 enzymes; the formation of M2 (O-desmethylaschantin) is catalyzed by CYP2C9 and CYP2C19; and the formation of M4 is catalyzed by CYP3A4 enzyme. Two glutathione (GSH) conjugates of M1 were identified after incubation of aschantin with human and animal liver microsomes in the presence of nicotinamide adenine dinucleotide phosphate and GSH, but they were not detected in the hepatocytes of all species. In conclusion, aschantin is extensively metabolized, producing 18 metabolites in human and animal hepatocytes catalyzed by CYP, COMT, UDP-glucuronosyltransferase, and sulfotransferase. These results can help in clarifying the involvement of metabolizing enzymes in the pharmacokinetics and drug interactions of aschantin and in elucidating GSH conjugation associated with the reactive intermediate formed from M1 (aschantin catechol).

In Vitro Metabolism and Transport Characteristics of Zastaprazan.

Zastaprazan (JP-1366), a novel potassium-competitive acid blocker, is a new drug for the treatment of erosive esophagitis. JP-1366 is highly metabolized in human, mouse, and dog hepatocytes but moderately metabolized in rat and monkey hepatocytes when estimated from the metabolic stability of this compound in hepatocyte suspension and when 18 phase I metabolites and 5 phase II metabolites [i.e., N-dearylation (M6), hydroxylation (M1, M19, M21), dihydroxylation (M7, M8, M14, M22), trihydroxylation (M13, M18), hydroxylation and reduction (M20), dihydroxylation and reduction (M9, M16), hydrolysis (M23), hydroxylation and glucuronidation (M11, M15), hydroxylation and sulfation (M17), dihydroxylation and sulfation (M10, M12), N-dearylation and hydroxylation (M3, M4), N-dearylation and dihydroxylation (M5), and N-dearylation and trihydroxylation (M2)] were identified from JP-1366 incubation with the hepatocytes from humans, mice, rats, dogs, and monkeys. Based on the cytochrome P450 (CYP) screening test and immune-inhibition analysis with CYP antibodies, CYP3A4 and CYP3A5 played major roles in the metabolism of JP-1366 to M1, M3, M4, M6, M8, M9, M13, M14, M16, M18, M19, M21, and M22. CYP1A2, 2C8, 2C9, 2C19, and 2D6 played minor roles in the metabolism of JP-1366. UDP-glucuronosyltransferase (UGT) 2B7 and UGT2B17 were responsible for the glucuronidation of M1 to M15. However, JP-1366 and active metabolite M1 were not substrates for drug transporters such as organic cation transporter (OCT) 1/2, organic anion transporter (OAT) 1/3, organic anion transporting polypeptide (OATP)1B1/1B3, multidrug and toxic compound extrusion (MATE)1/2K, P-glycoprotein (P-gp), and breast cancer-resistant protein (BCRP). Only M1 showed substrate specificity for P-gp. The findings indicated that drug-metabolizing enzymes, particularly CYP3A4/3A5, may have a significant role in determining the pharmacokinetics of zastaprazan while drug transporters may only have a small impact on the absorption, distribution, and excretion of this compound.

Characterization of complement C3 as a marker of alpha-amanitin toxicity by comparative secretome profiling.

In the human body, proteins secreted into peripheral blood vessels are known as the secretome, and they represent the physiological or pathological status of cells. The unique response of cells to toxin exposure can be confirmed via secretome analysis, which can be used to discover toxic mechanisms or exposure markers. Alpha-amanitin (α-AMA) is the most widely studied amatoxin and inhibits transcription and protein synthesis by directly interacting with RNA polymerase II. However, secretory proteins released during hepatic failure caused by α-AMA have not been fully characterized. In this study, we analyzed the secretome of α-AMA-treated Huh-7 cells and mice using a comparative proteomics technique. Overall, 1440 and 208 proteins were quantified in cell media and mouse serum, respectively. Based on the bioinformatics results for the commonly downregulated proteins in cell media and mouse serum, we identified complement component 3 (C3) as a marker for α-AMA-induced hepatotoxicity. Through western blot in cell secretome and C3 ELISA assays in mouse serum, we validated α-AMA-induced downregulation of C3. In conclusion, using comparative proteomics and molecular biology techniques, we found that α-AMA-induced hepatotoxicity reduced C3 levels in the secretome. We expect that this study will aid in identifying new toxic mechanisms, therapeutic targets, and exposure markers of α-AMA-induced hepatotoxicity. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-022-00163-z.

Identification of α-amanitin effector proteins in hepatocytes by limited proteolysis-coupled mass spectrometry.

The misuse of poisonous mushrooms containing amatoxins causes acute liver failure (ALF) in patients and is a cause of significant mortality. Although the toxic mechanisms of α-amanitin (α-AMA) and its interactions with RNA polymerase II (RNAP II) have been studied, α-AMA effector proteins that can interact with α-AMA in hepatocytes have not been systematically studied. Limited proteolysis-coupled mass spectrometry (LiP-MS) is an advanced technology that can quickly identify protein-ligand interactions based on global comparative proteomics. This study identified the α-AMA effector proteins found in human hepatocytes, following the detection of conformotypic peptides using LiP-MS coupled with tandem mass tag (TMT) technology. Proteins that are classified into protein processing in the endoplasmic reticulum and the ribosome during the KEGG pathway can be identified through affinity evaluation, according to α-AMA concentration-dependent LiP-MS and LiP-MS in hepatocytes derived from humans and mice, respectively. The possibility of interaction between α-AMA and proteins containing conformotypic peptides was evaluated through molecular docking studies. The results of this study suggest a novel path for α-AMA to induce hepatotoxicity through interactions with various proteins involved in protein synthesis, as well as with RNAP II.

Toxicokinetics and tissue distribution of phalloidin in mice.

Phalloidin, a bicyclic heptapeptide found in Amanita mushroom, specifically binds to F-actin in the liver causing cholestatic hepatotoxicity. However, the toxicokinetics and tissue distribution properties of phalloidin as well as their underlying mechanisms have to be studied further. The area under the plasma concentration curve (AUC) of phalloidin increased in proportion to the doses (0.2, 0.4, and 0.8 mg/kg for intravenous injection and 2, 5, and 10 mg/kg for oral administration). Phalloidin exhibited dose-independent low volume of distribution (395.6-456.9 mL/kg) and clearance (21.4-25.5 mL/min/kg) and low oral bioavailability (2.4%-3.3%). This could be supported with its low absorptive permeability (0.23 ± 0.05 × 10-6 cm/s) in Caco-2 cells. The tissue-to-plasma AUC ratios of intravenously injected and orally administered phalloidin were the highest in the liver and intestines, respectively, and also high in the kidneys, suggesting that the liver, kidneys, and intestines could be susceptible to phalloidin exposure and that active transport via the hepatic and renal organic anion transporters (OATP1B1, OATP1B3, and OAT3) may contribute to the higher distribution of phalloidin in the liver and kidneys.

Derivatization-assisted LC-MS/MS method for simultaneous quantification of endogenous gamma-hydroxybutyric acid and its metabolic precursors and products in human urine.

The accurate, precise, and robust quantification of endogenous biomarkers is a challenging task because of the presence of significantly low levels of endogenous compounds in biological samples, the absence of analyte-free matrix-matched calibrators, and sample instability due to in-vitro production or degradation of the analytes. Gamma-hydroxybutyric acid (GHB), a compound often used in drug-facilitated crimes, is a human neurotransmitter produced during both the biosynthesis and metabolism of gamma-aminobutyric acid (GABA). Evidently, proving GHB intoxication through the quantification of GHB and its metabolites in biological samples is not straightforward. This study aimed to develop a sensitive and accurate quantitative method for the simultaneous determination of endogenous GHB and its metabolic precursors and products (glutamic acid, GABA, succinic acid, 2,4-dihydroxybutyric acid, 3,4-dihydroxybutyric acid, glycolic acid, and succinylcarnitine) in human urine using LC-MS/MS. For this purpose, chemical derivatization with benzoyl chloride was employed to improve the sensitivity to glutamic acid and GABA. Synthetic urine was used to prepare calibrators, and the validity of this approach was fully demonstrated, particularly focusing on the instability issues. The validation results proved the method to be selective, sensitive, accurate, and precise, with acceptable linearity within calibration ranges. Moreover, our results regarding the in-vitro production or degradation of metabolites highlight the effects of handling and storage conditions of urine samples. Finally, this effective analytical method is expected to be useful in studying the relationship between GHB intoxication and metabolic alterations and, thus, discovering practical biomarkers for GHB ingestion.

Urinary Profile of Endogenous Gamma-Hydroxybutyric Acid and its Biomarker Metabolites in Healthy Korean Females: Determination of Age-Dependent and Intra-Individual Variability and Identification of Metabolites Correlated With Gamma-Hydroxybutyric Acid.

Gamma-hydroxybutyric acid (GHB), used as a therapeutic and an illegal anesthetic, is a human neurotransmitter produced during gamma-aminobutyric acid (GABA) biosynthesis and metabolism. Potential biomarker metabolites of GHB intoxication have been identified previously; however, reference concentrations have not been set due to the lack of clinical study data. Urinary profiling of endogenous GHB and its biomarker metabolites in urine samples (n = 472) of 206 healthy females was performed based on differences in age and time of sample collection using liquid chromatography-tandem mass spectrometry following validation studies. The unadjusted and creatinine-adjusted urinary concentrations ranges were obtained after urinary profiling. The creatinine-adjusted concentrations of glutamic and succinic acids and succinylcarnitine significantly increased, whereas that of glycolic acid significantly decreased with advancing age. Significant inter-day variation of GABA concentration and intra-day variation of 3,4-dihydroxybutyric acid and succinylcarnitine concentrations were observed. The urinary concentrations of 2,4-dihydroxybutyric acid, succinic acid, and 3,4-dihydroxybutyric acid showed the highest correlation with that of GHB. Data from this study suggest population reference limits to facilitate clinical and forensic decisions related to GHB intoxication and could be useful for identification of biomarkers following comparison with urinary profiles of GHB-administered populations.

Toxicokinetics of ß-Amanitin in Mice and In Vitro Drug-Drug Interaction Potential.

The toxicokinetics of ß-amanitin, a toxic bicyclic octapeptide present abundantly in Amanitaceae mushrooms, was evaluated in mice after intravenous (iv) and oral administration. The area under plasma concentration curves (AUC) following iv injection increased in proportion to doses of 0.2, 0.4, and 0.8 mg/kg. ß-amanitin disappeared rapidly from plasma with a half-life of 18.3−33.6 min, and 52.3% of the iv dose was recovered as a parent form. After oral administration, the AUC again increased in proportion with doses of 2, 5, and 10 mg/kg. Absolute bioavailability was 7.3−9.4%, which resulted in 72.4% of fecal recovery from orally administered ß-amanitin. Tissue-to-plasma AUC ratios of orally administered ß-amanitin were the highest in the intestine and stomach. It also readily distributed to kidney > spleen > lung > liver ≈ heart. Distribution to intestines, kidneys, and the liver is in agreement with previously reported target organs after acute amatoxin poisoning. In addition, ß-amanitin weakly or negligibly inhibited major cytochrome P450 and 5'-diphospho-glucuronosyltransferase activities in human liver microsomes and suppressed drug transport functions in mammalian cells that overexpress transporters, suggesting the remote drug interaction potentials caused by ß-amanitin exposure.

No association between the dopamine D3 receptor gene and Korean alcohol dependence.

The genes encoding dopamine receptor (DR) subtypes have received considerable attention for the past several years as a potential candidate that may affect susceptibility to addictive disorder, including alcoholism. The many association studies that compared the frequencies of alleles of the dopamine D2 receptor (DRD2) gene between alcoholics and control groups have produced results, but some have been equivocal. Dopamine D3 receptor genes (DRD3) are in the same class as DRD2 but with different pharmacological properties. So we compared the distribution of genotypes and frequencies of BalI polymorphism of the DRD3 gene in alcoholics and controls to assess the role of the DRD3 gene in Korean alcoholism. For this study, 67 male probands from alcoholics and 67 age-matched normal male controls were engaged. No evidence for an allelic association was found between the A1 allele of DRD3 and alcoholism in a Korean population. These results suggest that any role played by this receptor may account for only part of the variation in susceptibility to alcoholism.