Identification of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole, in cell culture medium, as a factor that controls the background aryl hydrocarbon receptor activity.

The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.     

The tryptophan derivative 6-formylindolo[3,2-b]carbazole,FICZ, a dynamic mediator of endogenous aryl hydrocarbon receptor signaling,balances cell growth and differentiation

Abstract

The aryl hydrocarbon receptor (AHR) is not essential to survival, but does act as a key regulator of many normal physiological events. The role of this receptor in toxicological processes has been studied extensively, primarily employing the high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, regulation of physiological responses by endogenous AHR ligands remains to be elucidated. Here, we review developments in this field, with a focus on 6-formylindolo[3,2-b]carbazole (FICZ), the endogenous ligand with the highest affinity to the receptor reported to date. The binding of FICZ to different isoforms of the AHR seems to be evolutionarily well conserved and there is a feedback loop that controls AHR activity through metabolic degradation of FICZ via the highly inducible cytochrome P450 1A1. Several investigations provide strong evidence that FICZ plays a critical role in normal physiological processes and can ameliorate immune diseases with remarkable efficiency. Low levels of FICZ are pro-inflammatory, providing resistance to pathogenic bacteria, stimulating the anti-tumor functions, and promoting the differentiation of cancer cells by repressing genes in cancer stem cells. In contrast, at high concentrations FICZ behaves in a manner similar to TCDD, exhibiting toxicity toward fish and bird embryos, immune suppression, and activation of cancer progression. The findings are indicative of a dual role for endogenously activated AHR in barrier tissues, aiding clearance of infections and suppressing immunity to terminate a vicious cycle that might otherwise lead to disease. There is not much support for the AHR ligand-specific immune responses proposed, the differences between FICZ and TCDD in this context appear to be explained by the rapid metabolism of FICZ.     

Structural elucidation of hydroxylated metabolites of the isoflavan equol by gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry.

Equol has, as have other isoflavonoids, recently gained considerable interest due to its possible health effects. However, detailed studies on the metabolism of equol are scarce. Therefore, we investigated the phase I metabolism of equol using liver microsomes from Aroclor-treated male Wistar rats as well as from a male human. The identification of the metabolites formed was elucidated using high performance liquid chromatography (HPLC) with diode array detection, HPLC/atmospheric pressure ionization electrospray mass spectrometry, and gas chromatography-mass spectrometry, as well as reference compounds. (+/-)-Equol was converted to 11 metabolites by the liver microsomes from Aroclor-pretreated rats comprising three aromatic monohydroxylated and four aliphatic monohydroxylated as well as four dihydroxylated products. The main metabolite was identified as 3'-hydroxy-equol. Using human liver microsomes, equol was converted to six metabolites with 3'-hydroxy- and 6-hydroxy-equol as main products. Furthermore, the aliphatic hydroxylated metabolite 4-hydroxyequol, which was recently detected in human urine after soy consumption, was formed. On the basis of these findings, it is suggested that phase I metabolism of equol is part of a complex biotransformation of the soy isoflavone daidzein in humans in vivo.     

Synthesis and substance P receptor binding activity of androstano[3,2-b]pyrimido[1,2-a]benzimidazoles.

Several heterosteroids containing a dihydroethisterone skeleton were prepared and shown to displace substance P in a receptor binding assay. Further biochemical (kinetic and Scatchard analyses) and pharmacological evaluation (substance P-induced plasma extravasation and salivation in the rat) of a representative example in this series (5a) established that these compounds are competitive antagonists at the substance P receptor.     

Characterization of the in vitro metabolic profile of amlodipine in rat using liquid chromatography-mass spectrometry.

In the present study, the metabolic profile of amlodipine, a well-known calcium channel blocker, was investigated employing liquid chromatography-mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers - a triple-quadrupole (QqQ) and a quadrupole time-of-flight (Q-TOF) mass spectrometer - were utilized to acquire structural information on amlodipine metabolites. The metabolites were produced by incubation of amlodipine with primary cultures of rat hepatocytes. Incubations from rat hepatocytes were analyzed with LC-MS/MS, and 21 phase I and phase II metabolites were detected. Their product ion spectra were acquired and interpreted, and structures were proposed. Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites and thus to confirm the proposed structures of these metabolites. Mainly phase I metabolic changes were observed including dehydrogenation of the dihydropyridine core, as well as reactions of side chains, such as hydrolysis of ester bonds, hydroxylation, N-acetylation, oxidative deamination, and their combinations. The only phase II metabolite detected was the glucuronide of a dehydrogenated, deaminated metabolite of amlodipine. We propose several in vitro metabolic pathways of amlodipine in rat, based on our analysis of the metabolites detected and characterized.     

Comparative study of aryl hydrocarbon receptor ligand activities of six chemicals in vitro and in vivo

The aryl hydrocarbon receptor (AhR) ligand activities of six known AhR ligands were compared in vivo and in vitro. The in vivo ligand activity was estimated in terms of induction of cytochrome P450 1A1/2 activities, i.e., ethoxyresorufin-O-dealkylase (EROD) and methoxyresorufin-O-dealkylase (MROD) activities, and in vitro ligand activity was evaluated with a recombinant yeast reporter gene assay. The test chemicals were 3-methylcholanthrene (MC), β-naphthoflavone (β-NF), indirubin, indigo, 3,3′-diindolylmethane (DIM) and diphenyl-p-phenylenediamine (DPPD). The first four showed potent AhR ligand activity in vitro, comparable with that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, while DIM and DPPD showed weaker activity. Administration of MC and β-NF to mice caused significant induction of EROD and MROD activities, while indirubin, indigo and DIM also induced these activities, but less potently. DPPD also induced the activities, but was toxic at higher doses. These enhancing effects were lost or greatly reduced in Ahr-null mice (Ahr ^−/−). Our results suggest that EROD and MROD activity assays are useful for evaluating the AhR ligand activity of chemicals in vivo, where the biodynamics of the chemicals plays an important role.     

HPLC-MS/MS法研究人尿中甲磺酸桂哌齐特的主要代谢产物

目的研究甲磺酸桂哌齐特在人尿中的主要代谢产物。方法选择健康受试者12名,连续5 d单剂量静脉滴注甲磺酸桂哌齐特250 mg,分段收集0²4 h尿样,经蛋白沉淀纯化后,直接采用液相色谱-串联质谱法(HPLC-MS/MS)对尿样进行测定。结果在尿样中鉴定出5个主要代谢物,分别为3-O-去甲基-桂哌齐特-葡萄糖醛酸化物(M1)、3-O-去甲基-桂哌齐特-磺酸化物(M2)、桂哌齐特-吡咯烷衍生物(M3)、桂哌齐特-吡咯烷羧基化物(M4)和3-O-去甲基-桂哌齐特(M5)。结论该方法灵敏、选择性高,为深入研究甲磺酸桂哌齐特在人体内的代谢规律提供了可靠的方法。     

液相色谱-质谱联用法检测体液中三唑仑及其代谢产物

建立了鉴别体液中三唑仑及其主要代谢物的液相色谱 质谱联用法。采用液相色谱 电喷雾离子阱质谱法 ,得到三唑仑和羟基化代谢物及其葡萄糖苷酸型结合物的色谱、质谱信息以及特征碎片离子。三唑仑的最低检测限小于 0 1ng。用本法分析了一例怀疑服用过量三唑仑的麻醉抢劫案例所取证的血样及尿样 ,并与健康受试者服药后尿样及家兔灌胃三唑仑后所取的血样及尿样比较 ,得到了当事人服用三唑仑的可靠数据。该方法可靠、快速、简便 ,尤其适用于需快速响应的法医学和毒理学分析     

Determination of lamotrigine and its metabolites in human plasma by liquid chromatography-mass spectrometry

A method based on electrospray ionization liquid chromatography-mass spectrometry was developed for the quantitative determination of lamotrigine and three of its reported metabolites, lamotrigine-2-N-glucuronide, lamotrigine-2-N-methyl, and lamotrigine-2-N-oxide in human blood plasma. The method utilized sample preparation by precipitation of proteins with acetonitrile, chromatographic separation on a reversed-phase system by gradient elution, and monitoring of the protonated molecular ions. Two internal standards, 3,5-diamino-6-(2-methoxyphenyl)-1,2,4-triazine and morphine-3-glucuronide-D3, were utilized to achieve precise quantification. The method validation comprised a demonstration of an agreement in the quantification of lamotrigine with that of a routine HPLC-UV method. The limits of detection were between 0.05 and 0.16 micromol/L. The method was employed for the measurement of clinical samples collected from 55 patients in steady-state prior to the dose intake (trough level). Lamotrigine and the 2-N-glucuronide were typically detected, while the N-methyl and N-oxide metabolites were detected only rarely. The median lamotrigine plasma level was 24.0 micromol/L (range, 4.3 to 64 micromol/L), the median 2-N-glucuronide level was 2.4 micromol/L (range, <0.05 to 24 micromol/L), and the median lamotrigine 2-N-glucuronide/lamotrigine ratio was 0.11 (range, <0.01 to 0.64). In conclusion, this liquid chromatographic-mass spectrometric method is suitable for simultaneous determination of lamotrigine and its metabolites in human plasma.     

Synthesis and anticancer activity of new 1-substituted-6H-pyrido[4,3-b]carbazole derivatives

This study examines the synthesis and cytostatic activity of new 5,6-dimethyl-1-substituted-6H-pyrido[4,3-b]carbazole derivatives. Their structures were confirmed by (1)H-NMR and elemental analysis. Seven of the new compounds were tested by the SRB method in vitro against human lung cancer (A549) and human kidney cancer (A498) cell lines. Biological tests indicated remarkable cytostatic effects of four compounds tested in comparison with ellipticine and cisplatin as reference drugs. One particular compound 3c was about four times more active on A498 than ellipticine with similar activity on the A549 cell line, and outperformed cisplatin activity on both tumor cell lines.     

液相色谱-电喷雾离子阱质谱法研究人尿中阿奇霉素的主要代谢产物

目的：研究阿奇霉素在人尿中的主要代谢产物。方法：受试者口服阿奇霉素后收集0～144 h尿样,经Strata C18-E固相萃取小柱分离纯化后,采用液相色谱-电喷雾离子阱质谱法（LC/ESI-MSn）对样品进行分析,通过比较空白尿样和给药后尿样的总离子流色谱图和选择离子监测色谱图,分析可能的代谢产物。结果：在人尿中共发现了8种代谢产物,通过与得到的代谢产物对照品比较,鉴定出2种代谢产物的结构,并推测出其他6种代谢产物的化学结构。阿奇霉素在人体内的主要代谢途径包括O-去甲基、N-去甲基、N-去甲基后去红霉糖和羟基化。结论：本研究为深入了解阿奇霉素在人体内的代谢情况提供了可靠的方法和参考依据。     

Comparison of acute toxicities of indolo[3,2-b]carbazole (ICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in TCDD-sensitive rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons are environmental toxicants that act via the AH receptor (AHR). In vitro studies have demonstrated that some indole derivatives present in cruciferous vegetables also bind to the AHR. One of the highest AHR binding affinities is exhibited by indolo[3,2-b]carbazole (ICZ). Since exposure to these dietary indoles is quantitatively far larger than that to halogenated aromatic compounds, their potential toxic risks have raised concern. In the present study, we compared the effects of ICZ with those of a single dose of 20 microg/kg TCDD in the most TCDD-sensitive rat strain (Long-Evans [Turku AB]) (L-E). Whereas TCDD elicited the expected toxicity syndrome, ICZ, either as a single subcutaneous dose (63.5, 127 or 508 microg/kg) or with repeated sc dosing (508 microg/kg for 5 days) failed to reproduce any toxic impacts of TCDD. Furthermore, a simultaneous ICZ treatment (63.5 or 127 microg/kg for 10 days) did not interfere with TCDD (20 microg/kg; single exposure) action. A moderate hepatic induction of CYP1A1 could be triggered by repeated intragastric administration of ICZ (127 microg/kg for 4 days, the last treatment 2.5 h prior to termination). In control experiments in a reconstituted yeast system, ICZ potently and dose-dependently activated L-E rat AHR function demonstrating that it represents a bona fide high-affinity ligand for the rat receptor in vivo. Thus, the present study does not support the view that dietary exposure to ICZ would present a hazard of AHR-mediated adverse health effects to humans.     

Determination of methamphetamine and its metabolites incorporated in hair by column-switching liquid chromatography-mass spectrometry

An automated column-switching liquid chromatographic-electrospray ionization-mass spectrometric (LC-ESI-MS) method has been established for the determination of methamphetamine (MA) and its metabolites, amphetamine (AP), and p-hydroxymethamphetamine (p-OH-MA) in hair. The combination of an N-vinylacetamide-containing hydrophilic polymer online extraction column, an SCX semi-micro LC column, and an electrospray ionization interface provided the successful concentration, separations, and highly sensitive MS determinations of these analytes in a hair extract without tedious sample pretreatments. The limits of detection of these analytes were 0.02 ng/mg and 0.1-0.2 ng/mg in the selected-ion monitoring (SIM) and full-scan modes, respectively, when using a 100-microL hair extract sample that corresponds to a 2.5-mg sample of hair. The calibration curves using dibenzylamine as an internal standard were linear up to 30 ng/mg hair equivalents for all these analytes in the SIM mode. p-OH-MA, the detection of which in MA users' hair had not been previously reported, was detectable in all 22 hair specimens/sections from which 1 ng or more of MA was detected per milligram hair. The amount of p-OH-MA detected per milligram of hair is presented with those of MA and AP among the MA users population. The detection of AP and p-OH-MA, in addition to the parent drug MA with reasonable ratios, was found to be a useful indicator for distinguishing internal MA incorporation from external contamination.     

Enhancement of hypoxia-induced gene expression in fish liver by the aryl hydrocarbon receptor (AhR) ligand, benzo[a]pyrene (BaP)

Fish in polluted coastal habitats commonly suffer simultaneous exposure to both hypoxia and xenobiotics. Although the adaptive molecular responses to each stress have been described, little is known about the interaction between the signaling pathways mediating these responses. Previous studies in mammalian hepatoma cell lines have shown that hypoxia-inducible factor (HIF)- and/or aryl hydrocarbon receptor (AhR)-activated gene expression is suppressed following co-exposure to hypoxia and the hallmark AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, whether similar crosstalk exists in the non-tumor liver tissues of fish and whether other non-TCDD ligands also play the same inhibitory role in this crosstalk remain unknown. Here, the in vivo hepatic mRNA expression profiles of multiple hypoxia- and AhR-responsive genes (later gene expression=mRNA expression of the gene) were examined in the orange-spotted grouper (Epinephelus coioides) upon single and combined exposures to hypoxia and benzo[a]pyrene (BaP). Combined exposure enhanced hypoxia-induced gene expression but did not significantly alter BaP-induced gene expression. Protein carbonyl content was markedly elevated in fish subjected to combined exposure, indicating accumulation of reactive oxygen species (ROS). Application of diethyldithiocarbamate (DDC) to hypoxia-treated grouper liver explants similarly exaggerated hypoxia-induced gene expression as in the combined stress tissues in vivo. These observations suggest that ROS derived from the combined hypoxia and BaP stress have a role in enhancing hypoxia-induced gene expression.     

高效液相色谱-电喷雾质谱法研究人尿中α-生育酚的主要代谢产物

研究α-生育酚在人尿中的主要代谢物。选择健康受试者5名连续7 d单剂量口服维生素E 250 mg，d 7收集0～6 h尿样，经C₁₈小柱固相萃取(SPE)分离纯化后，直接采用液相色谱-质谱联用方法(LC-MSⁿ)对尿样进行测定。在尿样中鉴定出4个主要代谢物，它们分别为α-生育酸、2,5,7,8-四甲基-2-(2′-羧乙基)-6-硫酸酯苯并二氢吡喃、 γ-生育酸内酯和2,5,7,8-四甲基-2-(4′,8′,12′-三甲基-12′-羧十二烷基)-6-硫酸酯苯并二氢吡喃。该方法灵敏、选择性高，为深入研究α-生育酚在人体内的代谢规律提供了可靠的方法。     

Small molecule quantification by liquid chromatography-mass spectrometry for metabolites of drugs and drug candidates

Identification and quantification of the metabolites of drugs and drug candidates are routinely performed using liquid chromatography-mass spectrometry (LC-MS). The best practice is to generate a standard curve with the metabolite versus the internal standard. However, to avoid the difficulties in metabolite synthesis, standard curves are sometimes prepared using the substrate, assuming that the signal for substrate and the metabolite will be equivalent. We have tested the errors associated with this assumption using a series of very similar compounds that undergo common metabolic reactions using both conventional flow electrospray ionization LC-MS and low-flow captive spray ionization (CSI) LC-MS. The differences in standard curves for four different types of transformations (O-demethylation, N-demethylation, aromatic hydroxylation, and benzylic hydroxylation) are presented. The results demonstrate that the signals of the substrates compared with those of the metabolites are statistically different in 18 of the 20 substrate-metabolite combinations for both methods. The ratio of the slopes of the standard curves varied up to 4-fold but was slightly less for the CSI method.     

液相色谱-串联电喷雾离子阱质谱法研究人尿中埃坡霉素D的主要代谢产物

目的：研究埃坡霉素D在人尿中的主要代谢产物。方法：受试者6名静脉滴注埃坡霉素D,收集0～72h尿样,经SPE固相萃取小柱分离纯化后,采用液相色谱-串联电喷雾离子阱质谱法（LC—MS^n）对样品进行分析,通过比较空白尿样和给药后的尿样的总离子流色谱图和选择离子扫描色谱图,分析可能的代谢产物。代谢产物的结构鉴定主要依据代谢产物的各级质谱图与原药的各级质谱图的相关性推断所得。结果：在人尿中共检测到11种主要的代谢产物,根据所提供的代谢产物标准品鉴定出3种代谢产物的结构,代谢方式主要有氧化、还原、水解和结合反应。结论：本方法选择性好,为深入研究埃坡霉素类药物在人体内的代谢规律提供了可靠的方法和可参考的依据。