Glycolytic interference blocks influenza A virus propagation by impairing viral polymerase-driven synthesis of genomic vRNA.

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell's metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-d-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme's function from transcription to replication.

Integrated data from intravital imaging and HPLC-MS/MS analysis reveal large interspecies differences in AFB1 metabolism in mice and rats.

Large interspecies differences between rats and mice concerning the hepatotoxicity and carcinogenicity of aflatoxin B1 (AFB1) are known, with mice being more resistant. However, a comprehensive interspecies comparison including subcellular liver tissue compartments has not yet been performed. In this study, we performed spatio-temporal intravital analysis of AFB1 kinetics in the livers of anesthetized mice and rats. This was supported by time-dependent analysis of the parent compound as well as metabolites and adducts in blood, urine, and bile of both species by HPLC-MS/MS. The integrated data from intravital imaging and HPLC-MS/MS analysis revealed major interspecies differences between rats and mice: (1) AFB1-associated fluorescence persisted much longer in the nuclei of rat than mouse hepatocytes; (2) in the sinusoidal blood, AFB1-associated fluorescence was rapidly cleared in mice, while a time-dependent increase was observed in rats in the first three hours after injection followed by a plateau that lasted until the end of the observation period of six hours; (3) this coincided with a far stronger increase of AFB1-lysine adducts in the blood of rats compared to mice; (4) the AFB1-guanine adduct was detected at much higher concentrations in bile and urine of rats than mice. In both species, the AFB1-glutathione conjugate was efficiently excreted via bile, where it reached concentrations at least three orders of magnitude higher compared to blood. In conclusion, major differences between mice and rats were observed, concerning the nuclear persistence, formation of AFB1-lysine adducts, and the AFB1-guanine adducts.

Comparative metabolism of aflatoxin B1 in mouse, rat and human primary hepatocytes using HPLC-MS/MS.

Aflatoxin B1 (AFB1) is a highly hepatotoxic and carcinogenic mycotoxin produced by Aspergillus species. The compound is mainly metabolized in the liver and its metabolism varies between species. The present study quantified relevant AFB1- metabolites formed by mouse, rat, and human primary hepatocytes after treatment with 1 µM and 10 µM AFB1. The use of liquid chromatographic separation coupled with tandem mass spectrometric detection enabled the selective and sensitive determination of phase I and phase II metabolites of AFB1 over incubation times of up to 24 h. The binding of AFB1 to macromolecules was also considered. The fastest metabolism of AFB1 was observed in mouse hepatocytes which formed aflatoxin P1 as a major metabolite and also its glucuronidated form, while AFP1 occurred only in traces in the other species. Aflatoxin M1 was formed in all species and was, together with aflatoxin Q1 and aflatoxicol, the main metabolite in human cells. Effective epoxidation led to high amounts of DNA adducts already 30 min post-treatment, especially in rat hepatocytes. Lower levels of DNA adducts and fast DNA repair were found in mouse hepatocytes. Also, protein adducts arising from reactive intermediates were formed rapidly in all three species. Detoxification via glutathione conjugation and subsequent formation of the N-acetylcysteine derivative appeared to be similar in mice and in rats and strongly differed from human hepatocytes which did not form these metabolites at all. The use of qualitative reference material of a multitude of metabolites and the comparison of hepatocyte metabolism in three species using advanced methods enabled considerations on toxification and detoxification mechanisms of AFB1. In addition to glutathione conjugation, phase I metabolism is strongly involved in the detoxification of AFB1.

Monoanionic C

A family of Pt(II) complexes bearing monoanionic C^N^N ligands as luminophoric units as well as a set of monodentate ligands derived from allenylidene and carbene species were synthesized and characterized in terms of structure and photophysical properties. In addition, we present the extraordinary molecular structure of a phosphorescent complex carrying an allenylidene ligand. Depending on the co-ligand, an effect can be observed in the photoluminescence lifetimes and quantum yields as well as in the radiative and radiation less deactivation rate constants. Their correlation with the substitution pattern was analyzed by comparing the photoluminescence in fluid solution at room temperature and in frozen glassy matrices at 77 K. Moreover, in order to gain a deeper understanding of the electronic states responsible for the optical properties, density functional theory calculations were performed. Finally, the cytotoxicity of the complexes was evaluated in vitro, showing that the cationic complexes exhibit strong effects at low micromolar concentrations. The calculated half-maximum effective concentrations (EC50 values) were 4 times lower in comparison to the established antitumor agent oxaliplatin. In contrast, the neutral species are less toxic, rendering them as potential bioimaging agents.

Metabolic profiling as a powerful tool for the analysis of cellular alterations caused by 20 mycotoxins in HepG2 cells.

Mycotoxins are secondary fungal metabolites which exhibit toxic effects in low concentrations. Several mycotoxins are described as carcinogenic or immunosuppressive, but their underlying modes of action especially on molecular level have not yet been entirely elucidated. Metabolic profiling as part of the omics methods is a powerful tool to study the toxicity and the mode of action of xenobiotics. The use of hydrophilic interaction chromatography in combination with targeted mass spectrometric detection enables the selective and sensitive analysis of more than 100 polar and ionic metabolites and allows the evaluation of metabolic alterations caused by xenobiotics such as mycotoxins. For metabolic profiling, the hepato-cellular carcinoma cell line HepG2 was treated with sub-cytotoxic concentrations of 20 mycotoxins. Moniliformin and citrinin significantly affected target elements of the citric acid cycle, but also influenced glycolytic pathways and energy metabolism. Penitrem A, zearalenone, and T2 toxin mainly interfered with the urea cycle and the amino acid homeostasis. The formation of reactive oxygen species seemed to be influenced by T2 toxin and gliotoxin. Glycolysis was altered by ochratoxin A and DNA synthesis was affected by several mycotoxins. The observed effects were not limited to these metabolic reactions as the metabolic pathways are closely interrelated. In general, metabolic profiling proved to be a highly sensitive tool for hazard identification in comparison to single-target cytotoxicity assays as metabolic alterations were already observed at sub-toxic concentrations. Metabolic profiling could therefore be a powerful tool for the overall evaluation of the toxic properties of xenobiotics.

Insights into Ergochromes of the Plant Pathogen Claviceps purpurea.

Claviceps purpurea is an ergot fungus known for its neurotropic alkaloids, which have been identified as the main cause of ergotism, a livestock and human disease triggered by ergot consumption. Tetrahydroxanthone dimers, the so-called ergopigments, presumably also contribute to this toxic effect. Overexpression of the cluster-specific transcription factor responsible for the formation of these pigments in C. purpurea led to the isolation of three new metabolites (8-10). The new pigments were characterized utilizing HRMS, NMR techniques, and CD spectroscopy and shown to be xanthone dimers. Secalonic acid A and its 2,4'- and 4,4'-linked isomers were also isolated, and their absolute configuration was investigated. The contribution of secalonic acid A, its isomers, and new metabolites to the toxicity of C. purpurea was investigated in HepG2 and CCF-STTG1 cells. Along with cytotoxic properties, secalonic acid A was found to inhibit topoisomerase I and II activity.

Topoisomerase poisoning by the flavonoid nevadensin triggers DNA damage and apoptosis in human colon carcinoma HT29 cells.

Nevadensin, an abundant polyphenol of basil, is reported to reduce alkenylbenzene DNA adduct formation. Furthermore, it has a wide spectrum of further pharmacological properties. The presented study focuses the impact of nevadensin on topoisomerases (TOPO) in vitro. Considering the DNA-intercalating properties of flavonoids, first, minor groove binding properties (IC50 = 31.63 µM), as well as DNA intercalation (IC50 = 296.91 µM) of nevadensin, was found. To determine potential in vitro effects on TOPO I and TOPO IIα, the relaxation and decatenation assay was performed in a concentration range of 1-500 µM nevadensin. A partial inhibition was detected for TOPO I at concentrations  ≥ 100 µM, whereas TOPO IIα activity is only inhibited at concentrations  ≥ 250 µM. To clarify the mode of action, the isolating in vivo complex of enzyme assay was carried out using human colon carcinoma HT29 cells. After 1 h of incubation, the amount of TOPO I linked to DNA was significantly increased by nevadensin (500 µM), why nevadensin was characterized as TOPO I poison. However, no effects on TOPO IIα were detected in the cellular test system. As a subsequent cellular response to TOPO I poisoning, a highly significant increase of DNA damage after 2 h and a decrease of cell viability after 48 h at the same concentration range were found. Furthermore, after 24 h of incubation a G2/M arrest was observed at concentrations ≥ 100 µM by flow cytometry. The analysis of cell death revealed that nevadensin induces the intrinsic apoptotic pathway via activation of caspase-9 and caspase-3. The results suggest that cell cycle disruption and apoptotic events play key roles in the cellular response to TOPO I poisoning caused by nevadensin in HT29 cells.

α-Asarone, ß-asarone, and γ-asarone: Current status of toxicological evaluation.

Asarone isomers are naturally occurring in Acorus calamus Linné, Guatteria gaumeri Greenman, and Aniba hostmanniana Nees. These secondary plant metabolites belong to the class of phenylpropenes (phenylpropanoids or alkenylbenzenes). They are further chemically classified into the propenylic trans- and cis-isomers α-asarone and ß-asarone and the allylic γ-asarone. Flavoring, as well as potentially pharmacologically useful properties, enables the application of asarone isomers in fragrances, food, and traditional phytomedicine not only since their isolation in the 1950s. However, efficacy and safety in humans are still not known. Preclinical evidence has not been systematically studied, and several pharmacological effects have been reported for extracts of Acorus calamus and propenylic asarone isomers. Toxicological data are rare and not critically evaluated altogether in the 21st century yet. Therefore, within this review, available toxicological data of asarone isomers were assessed in detail. This assessment revealed that cardiotoxicity, hepatotoxicity, reproductive toxicity, and mutagenicity as well as carcinogenicity were described for propenylic asarone isomers with varying levels of reliability. The toxicodynamic profile of γ-asarone is unknown except for mutagenicity. Based on the estimated daily exposure and reported adverse effects, officials restricted or published recommendations for the use of ß-asarone and preparations of Acorus calamus. In contrast, α-asarone and γ-asarone were not directly addressed due to a limited data situation.

Methyleugenol and oxidative metabolites induce DNA damage and interact with human topoisomerases.

Methyleugenol is a substituted alkenylbenzene found in several herbs and spices. It is classified by the European Union's Scientific Committee on Food as a genotoxic carcinogen. We addressed the biological mechanism of the genotoxic properties of methyleugenol and its oxidative metabolites. Methyleugenol and the oxidative metabolites significantly enhanced the DNA damage in human colon carcinoma cells (HT29). Methyleugenol did not affect the protein status of γH2AX, a biomarker of DNA double-strand breaks, whereas its metabolites methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol significantly increased the cellular phosphorylated H2AX level. Both of these metabolites also showed a significant induction of micronuclei in HT29 cells. Furthermore, we investigated whether topoisomerase interaction contribute to the observed effect on DNA integrity. Methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol inhibited the activity of recombinant topoisomerase I. In HT29 cells, neither methyleugenol nor the metabolites affected the level of topoisomerase protein bound to DNA, excluding a topoisomerase poisoning mode of action. In addition, 3'-oxomethylisoeugenol potently diminished the level of camptothecin-stabilized topoisomerase I/DNA intermediates and camptothecin-induced DNA strand breaks. In conclusion, it could be suggested that 3'-oxomethylisoeugenol may also interact with classical or food-borne topoisomerase I poisons, diminishing their poisoning effectiveness.

A sensitive method for the determination of per- and polyfluoroalkyl substances in food and food contact material using high-performance liquid chromatography coupled with tandem mass spectrometry.

Dietary intake is the major pathway of human exposure to per- and polyfluoroalkyl substances (PFAS). Due to their generally very low concentrations in food, especially for foods of plant origin, and their toxicological relevance, demand is growing for improved selective and sensitive analytical methods for the determination of PFAS in the lower ng/kg range. The relevance is pointed out due to the fact that the European Commission has published limits of quantification (LOQs) in the lower ng/kg range for different food matrices in Recommendation (EU) 2022/1431 on the monitoring of perfluoroalkyl substances in food. For example, LOQs of 2 ng/kg for perfluorooctanesulfonic acid (PFOS), 1 ng/kg for perfluorooctanoic acid (PFOA), 1 ng/kg for perfluorononanoic acid (PFNA) and 4 ng/kg for perfluorohexanesulfonic acid (PFHxS) in fruit, vegetables and baby foods are required. A new, very sensitive method is presented here for the determination of 22 PFAS in food and food contact materials. The method is based on liquid-solid extraction and automated clean-up using two solid phase extraction techniques. The analytes are separated and detected by HPLC-MS/MS. A limit of detection (LOD) of 0.33 ng/kg and an LOQ of 1.0 ng/kg are attained for plant foods such as fruits and vegetables as well as for milk and baby food. For foods of animal origin such as egg, meat, fish and paper-based food contact materials an LOD of 1.6 ng/kg as well as an LOQ of 5.0 ng/kg are attained. PFOS and PFOA were the most abundant compounds in the food samples with concentration as high as 1,051 ng/kg of PFOA in sea weed samples and 772 ng/kg of PFOS in eggs samples. In food contact material samples, higher levels were found with a maximum of 310,000 ng/kg PFHxA. In sum the presented method firstly allows determination of PFAS in a wide variety of foodstuffs and paper-based food contact materials at EU-required concentration ranges.

Anthocyanins suppress the cleavable complex formation by irinotecan and diminish its DNA-strand-breaking activity in the colon of Wistar rats.

In the present study, the question was addressed whether anthocyanins interfere with the topoisomerase I poison irinotecan in vivo. In vivo complexes of enzyme to DNA bioassay was used to detect irinotecan-induced stabilization of topoisomerase I/DNA complexes and single cell gel electrophoresis to determine DNA-strand-break induction in the colon of male Wistar rats. Furthermore, analysis of anthocyanin concentrations in rat plasma and rat colon was included in the testing, demonstrating that anthocyanins reach the colon and the concentrations do not differ between rats that only received anthocyanins and the anthocyanin/irinotecan group. Blackberry extract was found to significantly reduce irinotecan-mediated topoisomerase I/DNA cleavable complex formation. Overall, anthocyanins did not notably increase cleavable complex formation. However, a significant increase of DNA damage was shown after a single dose of irinotecan as well as the single compounds cyanidin (cy) and cyanidin-3-glucoside (cy-3-g). Furthermore, a significant reduction of irinotecan-induced DNA-strand breaks after a pretreatment with cy, cy-3-g and blackberry extract was observed. Thus, the question arises whether anthocyanin-rich preparations might interfere with chemotherapy or whether, due to low systemic bioavailability, the preparations might provide protective potential in the gastrointestinal tract.

Genotoxic properties of cyclopentenone prostaglandins and the onset of glutathione depletion.

Prostaglandins are endogenous mediators formed from arachidonic acid by cyclooxygenases and prostaglandin synthases during inflammatory processes. The five-membered ring can be dehydrated, and α,ß-unsaturated cyclopentenone PGs (cyPGs) are generated. Recent studies have been focused on their potential pharmacological use against inflammation and cancer. However, little is known so far about possible adverse health effects of cyPGs. We addressed the question whether selected cyPGs at a concentration range of 0.1-10 µM exhibit mutagenic and genotoxic properties in the hamster lung fibroblast V79 cell line and whether these effects are accompanied by a depletion of intracellular glutathione (GSH). The cyPGs 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) and prostaglandin A2 (PGA2) significantly induced DNA damage in V79 cells after 1 h of incubation. Furthermore, a more pronounced increase in formamidopyrimidine-DNA glycosylase (FPG) sensitive sites, indicative of oxidative DNA-damage, was observed. The findings on DNA-damaging properties were supported by our results that 15dPGJ(2) acts as an aneugenic agent which induces the amount of kinetochore positive micronuclei associated with an increase of apoptosis. The strong potency of cyPGs to rapidly bind GSH measured in a chemical assay and to significantly reduce the GSH level after only 1 h of incubation may contribute to the observed oxidative DNA strand breaks, whereas directly induced oxidative stress via reactive oxygen species could be excluded. However, after an extended incubation time of 24 h no genotoxicity could be measured, this may contribute to the lack of mutagenicity in the hypoxanthine phosphorybosyltransferase (HPRT) assay. In conclusion, potential in vitro genotoxicity of cyPG and a strong impact on GSH homeostasis have been demonstrated, which may be involved in carcinogenesis mediated by chronic inflammation.

Polyphenols Cause Structure Dependent Effects on the Metabolic Profile of Human Hepatocarcinogenic Cells.

SCOPE: Although many beneficial health effects are attributed to polyphenols their influence on the human metabolome has not been elucidated yet. The ubiquitous occurrence of polyphenols in the human diet demands comprehensive knowledge about physiological and toxicological effects of these compounds on human cells. METHODS AND RESULTS: The human hepatocarcinogenic cell line HepG2 is used to elucidate the effects of 13 polyphenols and three respective phenolic degradation products on the human metabolome using HPLC-MS/MS. To investigate structure-activity-relationships, structurally related examples of polyphenols from different compound classes are selected. The analysis of catechins points toward a relation between the degree of hydroxylation and the extent of metabolic effects particularly on the urea cycle and the pentose phosphate pathway (PPP). A correlation between the modulation of the PPP and the stability of the compounds is demonstrated, which may be caused by reactive oxygen species (ROS). The incubation of flavones and alkenylbenzenes demonstrates reduced activity of methoxylated compounds and no impact of the B-ring position. CONCLUSION: In general, polyphenols induce a multitude of metabolic effects, for example, on energy metabolism, PPP, and urea cycle. These metabolic alterations may be related to the widely reported bioactivity of these compounds such as the anticarcinogenic effects.

Lithium Difluorophosphate: A Boon for High Voltage Li Ion Batteries and a Bane for High Thermal Stability/Low Toxicity: Towards Synergistic Dual Additives to Circumvent this Dilemma.

The specific energy/energy density of state-of-the-art (SOTA) Li-ion batteries can be increased by raising the upper charge voltage. However, instability of SOTA cathodes (i. e., LiNiy Cox Mny O2 ; x+y+z=1; NCM) triggers electrode crosstalk through enhanced transition metal (TM) dissolution and contributes to severe capacity fade; in the worst case, to a sudden death ("roll-over failure"). Lithium difluorophosphate (LiDFP) as electrolyte additive is able to boost high voltage performance by scavenging dissolved TMs. However, LiDFP is chemically unstable and rapidly decomposes to toxic (oligo)organofluorophosphates (OFPs) at elevated temperatures; a process that can be precisely analyzed by means of high-performance liquid chromatography-high resolution mass spectroscopy. The toxicity of LiDFP can be proven by the well-known acetylcholinesterase inhibition test. Interestingly, although fluoroethylene carbonate (FEC) is inappropriate for high voltage applications as a single electrolyte additive due to rollover failure, it is able to suppress formation of toxic OFPs. Based on this, a synergistic LiDFP/FEC dual-additive approach is suggested in this work, showing characteristic benefits of both individual additives (good capacity retention at high voltage in the presence of LiDFP and decreased OFP formation/toxicity induced by FEC).

Cellular Uptake of Epigallocatechin Gallate in Comparison to Its Major Oxidation Products and Their Antioxidant Capacity In Vitro.

Depletion of reactive oxygen species and reduction of oxidative stress have been identified as key parameters in the prevention of cellular aging. In previous in vitro studies, the tea catechin epigallocatechin gallate (EGCG) was found to have both pro- and antioxidant properties, disregarding the low stability under cell culture conditions. Besides hydrogen peroxide, theasinensin dimers amongst other oxidation products are formed. Exact quantities, cellular uptake and antioxidant capacities of these dimeric oxidation products remain unknown. Via high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS), formation kinetics and cellular uptake of EGCG and its major oxidation products are quantified. The antioxidant capacity is determined on a cellular level using a modified dichlorofluorescein (DCF) approach. As a first result, oxidation product quantities of up to 21 µM each are measured after incubation of 50 µM EGCG. While EGCG is taken up equimolarly, its major oxidation products are accumulated in hepatocarcinoma HepG2 cells at millimolar concentrations, especially theasinensin A (TSA). Lastly, the oxidation products show higher antioxidant properties than the monomer EGCG. In correlation with cellular uptake, TSA displays the highest capacity of all tested analytes. The findings reveal the strong influence of EGCG oxidation products on its bioactivity in vitro.

Permeability of dopamine D2 receptor agonist hordenine across the intestinal and blood-brain barrier in vitro.

Hordenine, a bioactive food compound, has several pharmacological properties and has recently been identified as a dopamine D2 receptor (D2R) agonist. Since the pharmacokinetic profile of hordenine has been described to a limited extent, the present study focused on the transfer and transport of hordenine across the intestinal epithelium and the blood-brain barrier (BBB) in vitro. Hordenine was quickly transferred through the Caco-2 monolayer in only a few hours, indicating a rapid oral uptake. However, the high bioavailability may be reduced by the observed efflux transport of hordenine from the bloodstream back into the intestinal lumen and by first pass metabolism in intestinal epithelial cells. To determine the biotransformation rate of hordenine, the metabolite hordenine sulfate was synthesized as reference standard for analytical purposes. In addition, transfer studies using primary porcine brain capillary endothelial cells (PBCEC) showed that hordenine is able to rapidly penetrate the BBB and potentially accumulate in the brain. Thus, a D2R interaction of hordenine and activation of dopaminergic signaling is conceivable, assuming that the intestinal barrier can be circumvented by a route of administration alternative to oral uptake.

Can the presence of additives result in false positive errors for microplastics in infant feeding bottles?

In recent years, it has been shown that food contact materials can be a potential source of microplastics (MP). Recently, it was reported that more than 16 million polypropylene (PP) particles L-1 may be released from infant feeding bottles (IFBs) made of PP. In the present study seven different IFBs were investigated by the same method used in the aforementioned publication. In our tests, however, only one IFB showed a level of MP above the limit of detection. More importantly, the MP detected were not of the same material as the bottle and are more likely the result of contamination. In addition, there was a notable difference in released MP particles when the water simulant was filtered for µ-Raman spectroscopy at hot temperature (70°C) instead of filtering it after cooling down to room temperature. Thermal desorption gas chromatography mass spectrometry showed that these differences may be the result of migration and precipitation of additives such as fatty acid esters, often used as release agents in bottle production. These observations, that migrating additives could result in false positive errors for MP, indicate the need for critical consideration when polymers have been subjected to heat.

Metabolic conjugation reduces in vitro toxicity of the flavonoid nevadensin.

The present study focuses on the association between metabolic capacity and toxicity of the natural occurring flavonoid nevadensin in vitro. Human colon (HT29), liver (HepG2) and bone marrow (KG1) carcinoma cells were used and strong cell line dependent differences in toxic effect strength were found. HepG2 and KG1 cells were more sensitive against nevadensin treatment in comparison to HT29 cells. High resolution mass spectrometry experiments showed that nevadensin is rapidly glucuronidated in HT29 cells, whereas KG1 cells do not metabolize nevadensin, thus glucuronidation was supposed to be a crucial metabolic pathway in vitro. To proof this suggestion, nevadensin glucuronides were isolated from pig liver microsomes und structurally elucidated via NMR spectroscopy. In HepG2 cells a cellular enrichment of nevadensin itself as well as nevadensin-7-O-glucuronide was determined by tandem mass spectrometry. A proteomic screening of uridine 5'-diphospho (UDP)-glucuronosyltransferase (UGT) in HT29 and HepG2 cells provided first hints that the isoforms UGT1A6 and UGT1A1 are responsible for nevadensin glucuronidation. Additionally, nevadensin was found to be a potent SULT inhibitor in HepG2 cells. In sum, the present study clearly illustrates the importance of obtaining detailed information about metabolic competence of cell lines which should be considered in the evaluation of toxic endpoints.

Phase II Metabolism of Asarone Isomers In Vitro and in Humans Using HPLC-MS/MS and HPLC-qToF/MS.

(1) Background: Metabolism data of asarone isomers, in particular phase II, in vitro and in humans is limited so far. For the first time, phase II metabolites of asarone isomers were characterized and human kinetic as well as excretion data after oral intake of asarone-containing tea infusion was determined. (2) Methods: A high pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-qTOF-MS) approach was used to identify phase II metabolites using liver microsomes of different species and in human urine samples. For quantitation of the respective glucuronides, a beta-glucuronidase treatment was performed prior to analysis via high pressure liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). (3) Results: Ingested beta-asarone and erythro and threo-asarone diols were excreted as diols and respective diol glucuronide conjugates within 24 h. An excretion rate about 42% was estimated. O-Demethylation of beta-asarone was also indicated as a human metabolic pathway because a corresponding glucuronic acid conjugate was suggested. (4) Conclusions: Already reported O-demethylation and epoxide-derived diols formation in phase I metabolism of beta-asarone in vitro was verified in humans and glucuronidation was characterized as main conjugation reaction. The excretion rate of 42% as erythro and threo-asarone diols and respective asarone diol glucuronides suggests that epoxide formation is a key step in beta-asarone metabolism, but further, as yet unknown metabolites should also be taken into consideration.

Quantitative Analysis of ß-Asarone Derivatives in Acorus calamus and Herbal Food Products by HPLC-MS/MS.

α-Asarone and ß-asarone are reported as bioactive constituents of Acorus calamus. Phase I metabolism of asarone isomers results in a multiple spectrum of genotoxic metabolites. Thus, the question arises whether structural analogues of the known phase I metabolites also naturally occur in A. calamus-based food products. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for three product classes, herbal infusions, alcoholic beverages, and food supplements. High asarone contents were detected in herbal infusions (total mean 9.13 mg/kg, n = 8) and food supplements (total mean 14.52 mg/kg, n = 6); hence, these food products can highly contribute to human exposure to genotoxic asarone derivatives. Also, the occurrence of asarone oxidation products found in food and food supplements has to be taken under consideration because data on toxicity is limited so far.