A representative retinoid X receptor antagonist UVI3003 induced teratogenesis in zebrafish embryos

Retinoid X receptor (RXR) interfering activity has been detected in different water resources. To study RXR disruptor‐induced toxicological effects on vertebrates, embryos of zebrafish (Danio rerio) were exposed to a representative RXR antagonist UVI3003. Results showed that the teratogenic index (LC₅₀/EC₅₀) of UVI3003 was as high as 5.4. UVI3003 induced multiple malformations of embryos, including deformed fins, reduced brains, small jaws, bent tails and edema in hearts, the degree of which became more severe with increasing exposure concentration. Although no significant difference was observed in the hatching rates between the exposure group and control, the whole body length was significantly reduced by 6.5% and 8.9% when exposed to 200 and 300 µg l^?1 of UVI3003, respectively. The heart rate also significantly decreased by 8.8–50.2% during exposure. Further experiments revealed that the pharyngula stage was the most sensitive development phase in terms of embryo response to UVI3003. The results demonstrated severe teratogenicity of RXR antagonist in zebrafish embryos and provided important data for ecotoxicological evaluation of RXR antagonists. Copyright © 2014 John Wiley & Sons, Ltd.     

Triterpenes from Poria cocos are revealed as potential retinoid X receptor selective agonists based on cell and in silico evidence

Poria cocos is an edible and medicinal fungus that is widely used in Traditional Chinese Medicines as well as in modern applications. Retinoid X receptor (RXR) occupies a central place in nuclear receptor signaling, and a pharmacological RXR‐dependent pathway is involved in myeloid cell function. Here, structural information for 82 triterpenes from P. cocos and 17 known RXR agonists was collected in a compound library and retrieved for a molecular docking study. Three triterpenes, 16α‐hydroxytrametenolic acid (HTA), pachymic acid (PA), and polyporenic acid C (PPAC), were identified as novel RXR‐specific agonists based on luciferase reporter assays and in silico evidence. Treatment with HTA, PA, and PPAC significantly induced differentiation of the human promyelocytic leukemia cell line HL‐60 with EC50 values of 21.0 ± 0.52, 6.7 ± 0.37, and 9.4 ± 0.65 μM, respectively. These effects were partly blocked by the RXR antagonist UVI3003, suggesting that an RXR‐dependent pathway may play an important role in their anti‐acute promyelocytic leukemia (APL) effects. Taken together, triterpenes from P. cocos are revealed as naturally occurring RXR selective agonists with the potential for anti‐cancer activity. These results suggest a novel approach to the treatment or prevention of APL.     

A Trial of Mass Spectrometric Characterization of Femto‐Molar Amount from Subtropical Islands

Many kinds of venomous principles modulate physiological responses of mammalian signal transduction systems, on which they act selectively as enhancers, inhibitors or some other kind of effectors. These toxins have become useful tools for physiological research. We have characterized paralyzing toxins from the venom of spiders, scorpions, insects, jellyfishes and sea anemones in the subtropical region including the Ryukyu Islands. Venom profiles are screened by MALDI‐TOF fingerprinting analysis prior to purification of the venomous components, then marked target toxins of small molecular mass (1000–5000) are characterized directly by means of mass spectrometric techniques such as Frit‐FAB MS/MS, PSD/CID‐TOF MS, Capil. ‐HPLC/Q‐TOF MS/MS etc. The proteinous toxins of jellyfish or sea anemone are characterized by RT‐PCR technique by the information of the cleaved peptides after the protein was hydrolyzed by appropriate peptidase and the sequence of the cleaved peptide was determined by conventional methods. The venom of Araneid spider is mainly composed of a mixture of closely related acylpolyamines. More than 90 polyamine toxins were identified from one venom sac of the Madagascan spider, Nephilengys borbonica, by Frit‐Fab MS/MS employing charge remote fragmentation technique. A novel polyamine toxin was also found from the rare wondering spider, Macrothele gigas from Iriomote Island. The structure of the toxin is an analog of polyamine toxin found in trapdoor spiders. Many kinds of cystine‐rich peptides showing various types of ion channel antagonism have been isolated from spiders. A series of toxins possessing the same mode of cystine knots was recently isolated from the saliva of assassin bugs, Peirates turpis, Isyndus obscurus, Agriophodrus dohrni. These toxins act as calcium channel blocker. Most of the scorpion toxins reported are from scorpions hazardous to humans, and they belong to the major super family Buthoidea. Among them are the well‐known genera, such as Buthus, Androctonus, Centruroides, Leiurus, or Tytius. We have investigated the minor group of scorpions from the super family Chactoidea (Scorpionidae, Ishnuridae). The venoms of these scorpions, involving the genera Heterometrus, Pandinus, Opisthacanthus, and Isometrus, contain different kinds of peptide toxins. Fingerprinting peptide analysis of the toxin profiles for these scorpions showed some difference from the profiles of Buthoidea scorpions. These venoms contain linear pore‐forming peptides and 2‐cystine‐bridged toxins in addition to 4‐cystine‐bridged toxins. The most hazardous jellyfish in Okinawa, Chiropsalmus quadrigatus, and the related box jellyfishes, Carybdea rastoni, C. alata, contain quite labile proteinaceous toxins, CqTX, CrTX and CaTX, respectively. The toxins were inactivated by adding an organic solvent such as methanol or acetonitrile, by changing the pH of the toxin solution, dialyzing the toxin solution, storing the toxin in a refrigerator, or by lyophilizing the toxin solution. However, the toxic activity was retained in the presence of sodium chloride. We purified the jellyfish toxins by adding sodium chloride through all steps of the purification procedure and finally obtained the whole primary amino acid sequence of the toxin by RT‐PCR method. The toxic protein CqTX is homologous to the other box jelly fish toxin, CrTX and CaTX. These toxins belong to a new class of proteins since they show no homology to known proteins. Another notorious and dangerous specimen in the Ryukyu Islands is Phyllodiscus semori. The venom is composed of three kinds of proteins (PsTX‐20A, PsTX‐60A, PsTX‐60B). PsTX‐20A shows homology to the proteinaceous toxin actinoporin, a cytolytic protein isolated from the genus Actinia, but PsTX‐60s has no homology to any ever cloned proteins. Further elucidation of the mechanism of toxic action of these Coelenterates is in progress.     

Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches

4‐methyl‐N‐ethcathinone (4‐MEC), the N‐ethyl homologue of mephedrone, is a novel psychoactive substance of the beta‐keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4‐MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N‐deethylation, hydroxylation of the 4‐methyl group followed by further oxidation to the corresponding 4‐carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N‐methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N‐deethyl‐dihydro isomers and the 4‐carboxy‐dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4‐MEC should be detectable in urine by the GC‐MS and LC‐MSⁿ standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.     

Proteomic analysis of ametryn toxicity in zebrafish embryos

Ametrym (AMT) is the most widely used herbicide and frequently detected in the aquatic environment. AMT also represent a potential health risk to aquatic organisms and animals, including humans. However, little data are available on their toxicity to zebrafish (Danio rerio). The aim of the present study was to evaluate the toxicological effects of AMT exposure on zebrafish embryos. In the acute toxicity test, 6 hpf embryos were exposed to various concentrations of AMT for 24 or 48 h. The results indicated that AMT induced malformation in larvae. To investigate the toxicological mechanism on the protein expression level. A proteomic approach was employed to investigate the proteome alterations of zebra fish embryos exposed to 20 mg/L AMT for 48 h. Among 2925 unique proteins identified, 298 differential proteins (> or <1.3‐fold, P < 0.05) were detected in the treated embryos as compared to the corresponding proteins in the untreated embryos. Gene ontology analysis showed that these up‐regulated proteins were most involved in glycolysis, lipid transport, protein polymerization, and nucleotide binding, and the down‐regulated proteins were related to microtubule‐based process, protein polymerization, oxygen transport. Moreover, KEGG pathway analysis indicated that tight junction, ribosome, and oxidative phosphorylation were inhibited in the treated embryos. These findings provide new insight into the mechanisms of toxicity induced by AMT.     

Studies on drug metabolism by fungi colonizing decomposing human cadavers. Part II: biotransformation of five model drugs by fungi isolated from post‐mortem material

The present study investigated the in vitro metabolic capacity of 28 fungal strains isolated from post‐mortem material towards five model drugs: amitriptyline, metoprolol, mirtazapine, promethazine, and zolpidem. Each fungal strain was incubated at 25 °C for up to 120 h with each of the five models drugs. Cunninghamella elegans was used as positive control. Aliquots of the incubation mixture were centrifuged and 50 μL of the supernatants were diluted and directly analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) with product ion scanning. The remaining mixture was analyzed by full scan gas chromatography‐mass spectrometry (GC‐MS) after liquid‐liquid extraction and acetylation. The metabolic activity was evaluated through the total number of detected metabolites (NDM) produced in each model and fungal strains and the percentage of parent drug remaining (%RPD) after up to five days of incubation. All the tested fungal strains were capable of forming mammalian phase I metabolites. Fungi from the normal fungal flora of the human body such as Candida sp., Geotrichum candidum, and Trichosporon asahii) formed up to seven metabolites at %RPD values greater than 52% but no new fungal metabolites (NFM). In contrast, some airborne fungal strains like Bjerkandera adusta, Chaetomium sp, Coriolopsis sp., Fusarium solani and Mucor plumbeus showed NDM values exceeding those of the positive control, complete metabolism of the parent drug in some models and formation of NFM. NFM (numbers in brackets) were detected in four of the five model drugs: amitriptyline (18), metoprolol (4), mirtazapine (8), and zolpidem (2). The latter NFM are potential candidates for marker substances indicating post‐mortem fungal metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Lefetamine‐derived designer drugs N‐ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA): Metabolism and detectability in rat urine using GC‐MS,LC‐MSn and LC‐HR‐MS/MS

N‐Ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA) are two designer drugs, which were confiscated in Germany in 2008. Lefetamine (N,N‐dimethyl‐1,2‐diphenylethylamine, also named L‐SPA), the pharmaceutical lead of these designer drugs, is a controlled substance in many countries. The aim of the present work was to study the phase I and phase II metabolism of these drugs in rats and to check for their detectability in urine using the authors’ standard urine screening approaches (SUSA). For the elucidation of the metabolism, rat urine samples were worked up with and without enzymatic cleavage, separated and analyzed by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). According to the identified metabolites, the following metabolic pathways for NEDPA and NPDPA could be proposed: N‐dealkylation, mono‐ and bis‐hydroxylation of the benzyl ring followed by methylation of one of the two hydroxy groups, combinations of these steps, hydroxylation of the phenyl ring after N‐dealkylation, glucuronidation and sulfation of all hydroxylated metabolites. Application of a 0.3 mg/kg BW dose of NEDPA or NPDPA, corresponding to a common lefetamine single dose, could be monitored in rat urine using the authors’ GC‐MS and LC‐MSⁿ SUSA. However, only the metabolites could be detected, namely N‐deethyl‐NEDPA, N‐deethyl‐hydroxy‐NEDPA, hydroxy‐NEDPA, and hydroxy‐methoxy‐NEDPA or N‐de‐iso‐propyl‐NPDPA, N‐de‐iso‐propyl‐hydroxy‐NPDPA, and hydroxy‐NPDPA. Assuming similar kinetics, an intake of these drugs should also be detectable in human urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Phase I metabolism of the highly potent synthetic cannabinoid MDMB‐CHMICA and detection in human urine samples

Among the recently emerged synthetic cannabinoids, MDMB‐CHMICA (methyl N ‐{[1‐(cyclohexylmethyl)‐1H ‐indol‐3‐yl]carbonyl}‐3‐methylvalinate) shows an extraordinarily high prevalence in intoxication cases, necessitating analytical methods capable of detecting drug uptake. In this study, the in vivo phase I metabolism of MDMB‐CHMICA was investigated using liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS/MS) and liquid chromatography‐electrospray ionization‐quadrupole time‐of‐flight‐mass spectrometry (LC‐ESI‐Q ToF‐MS) techniques. The main metabolites are formed by hydrolysis of the methyl ester and oxidation of the cyclohexyl methyl side chain. One monohydroxylated metabolite, the ester hydrolysis product and two further hydroxylated metabolites of the ester hydrolysis product are suggested as suitable targets for a selective and sensitive detection in urine. All detected in vivo metabolites could be verified in vitro using a human liver microsome assay. Two of the postulated main metabolites were successfully included in a comprehensive LC‐ESI‐MS/MS screening method for synthetic cannabinoid metabolites. The screening of 5717 authentic urine samples resulted in 818 cases of confirmed MDMB‐CHMICA consumption (14%). Since the most common route of administration is smoking, smoke condensates were analyzed to identify relevant thermal degradation products. Pyrolytic cleavage of the methyl ester and amide bond led to degradation products which were also formed metabolically. This is particularly important in hair analysis, where detection of metabolites is commonly considered a proof of consumption. In addition, intrinsic activity of MDMB‐CHMICA at the CB₁ receptor was determined applying a cAMP accumulation assay and showed that the compound is a potent full agonist. Based on the collected data, an enhanced interpretation of analytical findings in urine and hair is facilitated. Copyright © 2016 John Wiley & Sons, Ltd.     

The in vivo and in vitro phase I metabolism of FYL‐67, a novel oxazolidinone antibacterial drug,studied by LC‐MS/MS

In our previous study, FYL‐67, a novel linezolid analogue with the morpholinyl ring replaced by a 4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl group, was demonstrated to own an excellent activity against Gram‐positive organisms,such as methicillin‐resistant Staphylococcus aureus (MRSA). However, metabolic biotransformation was not investigated. This study was performed to identify the phase I metabolites of FYL‐67 using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The chemical structures were confirmed by comparison with corresponding chemical standards obtained internal. Primary elucidation of the metabolic pathway of FYL‐67 in vitro was performed using liver preparations (microsomes and hepatocytes) from rats and humans, and SD (Sprague Dawley, rat, rattus norvegicus) rats were used for the study of in vivo approach. To the end, two metabolites (M₁ and M₂) were detected after in vitro as well as in vivo experiments. Based on LC‐MS/MS analyses, the metabolites were demonstrated to be 5‐(aminomethyl)‐3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)oxazolidin‐2‐one (M₁) and 3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (M₂). Amide hydrolysis at acetyl group of FYL‐67 leading to the formation of M₁ was observed and suggested to play a major role in both in vivo and in vitro phase I metabolism of FYL‐67. M₁ was demonstrated to undergo a further oxidation to form M₂. In addition, the results indicated no species difference existing between rats and humans. The outcomes of our research can be utilized for the development and validation of the analytical method for the quantification of FYL‐67 as well as its metabolites in biological samples. Furthermore, it is helpful to conduct studies of pharmacodynamics and toxicodynamics. Copyright © 2015 John Wiley & Sons, Ltd.     

Triclocarban at environmentally relevant concentrations induces the endoplasmic reticulum stress in zebrafish

Triclocarban (TCC) is an antibacterial agent commonly found in environmental, wildlife, and human samples. However, with in‐depth study of TCC, its negative effects are increasingly presented. Toxicological studies of TCC at environmentally relevant concentrations have been conducted in zebrafish embryos and indicated that TCC leads to deformity of development causes developmental deformities. However, the molecular mechanisms underlying the toxicity of TCC in zebrafish embryos have not been entirely elucidated. We investigated whether exposure to TCC at environmentally relevant concentrations induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in zebrafish. Zebrafish embryos were grown to 32 hours post fertilization and exposed to 2.5, 5, and 10 μg/L TCC and used in whole‐mount in situ hybridization to visualize the expression of ER chaperone hspa5 and ER stress‐related apoptosis factor chop. Zebrafish livers were exposed to different concentrations of TCC to elaborate the relationships between fatty degeneration and ER stress. Then, a human hepatic cell line (HL‐7702) was used to test whether TCC induced ER stress in human livers similar to those of zebrafish. In zebrafish embryos, TCC induced high hspa5 expression, which could defend against external stimulations. Furthermore, hapa5, hsp90b1, and chop exhibited ectopic expressions in the neuromast, intestinal tract, and tail tip of zebrafish embryos. On the one hand, significant differences were observed in the mRNA and protein expressions of the ER stress molecular chaperone pPERK‐pEIF2a‐ATF4 and ATF6 pathways in HL‐7702 cells exposed to TCC. On the other hand, lipid droplet accumulation slightly increased in zebrafish livers exposed to 10 μg/L TCC in vitro. These results demonstrate that TCC not only damages the development of zebrafish embryos and structure of zebrafish liver but also influences human hepatic cells by activating ER stress and the UPR signaling pathway.     

Silver nanoparticles affect the neural development of zebrafish embryos

Silver nanoparticles (AgNPs) have been widely used in commercial products. This study aims to understand the impact of AgNPs on the early developmental stages in zebrafish (Danio rerio) embryos. Embryos were exposed to two sizes of AgNPs at three dose levels, as well to free Ag⁺ ions, for a range of 4–96 h post‐fertilization (hpf). The acute exposure study showed that exposure to AgNPs affected the neurological development, and the exposed embryos exhibited anomalies such as small head with hypoplastic hindbrain, small eye and cardiac defects. At the molecular level, AgNPs altered the expression profiles of neural development‐related genes (gfap, huC and ngn1), metal‐sensitive metallothioneins and ABCC genes in exposed embryos. The expression of AhR2 and Cyp1A, which are usually considered to mediate polycyclic aromatic hydrocarbon toxicity, were also significantly changed. A size‐dependent uptake of AgNPs was observed, whereby 4 nm AgNPs were more efficiently taken up compared with the 10 nm‐sized particles. Importantly, the head area accumulated AgNPs more efficiently than the trunk area of exposed zebrafish embryos. No free Ag⁺ ions, which can be potentially released from the AgNP solutions, were detected. This study suggests that AgNPs could affect the neural development of zebrafish embryos, and the toxicity of AgNPs may be partially attributed to the comparatively higher uptake in the head area. These results indicate the potential neurotoxicity of AgNPs and could be extended to other aquatic organisms. Copyright © 2015 John Wiley & Sons, Ltd.     

Quercetin and naringenin reduce abnormal development of mouse embryos produced by hydroxyurea

Objectives There is limited evidence about the impact of quercetin and naringenin on embryonic development. The purpose of this work was to evaluate in vitro their direct teratogenic potential as well as their protective activity against teratogenesis mediated by oxidative damage on mouse embryos. Methods Quercetin and naringenin toxicity on whole mouse cultured embryos, as well as their ability to protect embryos against hydroxyurea‐induced insult were evaluated. Key findings Quercetin 100 µm and naringenin 300 µm produced significant reduction of developmental and growth parameters, in comparison with those of the control group. Embryos exposed to the concurrent administration of quercetin or naringenin with hydroxyurea (2 µm , 2 h) were significantly protected from growth and developmental retardation, and abnormalities induced by hydroxyurea. Interestingly, embryos exposed to hydroxyurea and dimethyl sulfoxide 0.1%, the vehicle employed to dissolve flavonoids, also showed significant damage amelioration. Conclusions These results indicate that quercetin and naringenin have not only a minor toxic effect on development, but also a protective effect against hydroxyurea‐induced embryonic damage.     

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17‐epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC‐MS/MS to establish potential long‐term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M‐I to M‐XI) were detected and characterized by LC‐MS/MS. This paper provides valuable data on the ionization and fragmentation of O‐ sulfates and N‐ sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long‐term metabolite (epistanozolol‐N ‐glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis and structural identification of fluorine‐18 labeled parathyroid hormone

Parathyroid hormone (PTH) is an 84 amino acid peptide hormone that plays a key role in bone and mineral metabolism. The biological actions of PTH are mediated via the N‐terminal PTH(1–34) fragment, serving as the PTH receptor‐binding sequence, and which is therefore used clinically to treat conditions of low bone mass such as osteoporosis. In this study, PTH(1–34) was conjugated with non‐radioactive (stable F isotope) N‐succinimidyl 4‐fluorobenzoate (SFB) leading to three isomeric mono‐fluorobenzoated (FBz) PTH followed by Liquid chromatography‐Tandem mass spectrometry (LC‐MS/MS) assisted structural identification. Corresponding [¹⁸F]SFB‐labeled PTH derivatives were prepared respectively and the Lys¹³ site‐specific labeled [¹⁸F]FBz PTH was isolated by HPLC with radiochemical purity >99% and specific activity of 2.78 GBq/µmol, suitable for future application with in vivo pharmacokinetic/pharmacodynamic studies of PTH, using preclinical Positron Emission Tomography Computed Tomography (PET/CT) imaging.     

Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT,and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS,LC–MSn,and LC‐HR‐MS/MS

Many N,N‐dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5‐methoxy‐2‐methyl‐N,N‐diallyltryptamine (5‐MeO‐2‐Me‐DALT), 5‐methoxy‐2‐methyl‐N‐allyl‐N‐cyclohexyltryptamine (5‐MeO‐2‐Me‐ALCHT), and 5‐methoxy‐2‐methyl‐N,N‐diisopropyltryptamine (5‐MeO‐2‐Me‐DIPT) using gas chromatography–mass spectrometry (GC–MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSⁿ), and liquid chromatography‐high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC‐HR‐MS/MS. 5‐MeO‐2‐Me‐DALT (24 phase I and 12 phase II metabolites), 5‐MeO‐2‐Me‐ALCHT (24 phase I and 14 phase II metabolites), and 5‐MeO‐2‐Me‐DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O‐demethylation, hydroxylation, N‐dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O‐demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N‐dealkylation. For SUSAs, GC–MS, LC‐MSⁿ, and LC‐HR‐MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC–MS SUSA, both LC–MS SUSAs were able to detect an intake of 5‐MeO‐2‐Me‐ALCHT and 5‐MeO‐2‐Me‐DIPT via their metabolites following 1 mg/kg BW administrations and 5‐MeO‐2‐Me‐DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.     

All‐trans‐retinoic acid alters Smads expression in embryonic neural tissue of mice

Retinoic acid can cause malformations of the developing nervous system. Smad signaling is involved in embryonic development. The current study investigated all‐trans‐retinoic acid (ATRA)‐induced alteration of Smad expression in the developing neural tubes of mice. Pregnant mice were treated with a single dose of 50 mg/kg ATRA by oral gavage on embryonic day (E) 7. Western immunoblotting was used to examine Smads proteins, particularly phosphorylated (p‐) Smad1, total Smad1 and Smad6 in the neural tissue of the embryos on E8–E11 following treatment. Results showed that ATRA treatment significantly increased expression of both p‐Smad1 and total Smad1, while Smad6 was decreased in neural tissues of ATRA‐exposed embryos in utero from E8 to E11, a critical period for neural tube formation. Data suggest that disruption of Smad signaling may be involved in ATRA‐induced neural tube defects. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of hydroxy metabolites of cocaine in hair samples for proof of consumption

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination. This especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens, thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC‐positive samples for these compounds was performed by liquid chromatography–quadrupole time of flight–mass spectrometry (LC–QTOF–MS). Once identified, mass transitions for o‐, p‐ and m‐isomers of hydroxy COC as well as p‐ and m‐isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by liquid chromatography–tandem mass spectrometry (LC–MS/MS) which was applied to 576 hair samples. Hydroxy metabolites were present in 92.2% of COC‐positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p‐OH‐BE, m‐OH‐BE as well as p‐OH‐NCOC and m‐OH‐NCOC have been identified for the first time in COC‐positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC‐positive hair samples.     

Gambogic acid causes fin developmental defect in zebrafish embryo partially via retinoic acid signaling

Gambogic acid (GA), the major active ingredient of gamboge, has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients due to its strong anticancer activity. However, our previous research showed that GA was teratogenic against zebrafish fin development. To explore the teratogenicity and the underlying mechanisms, zebrafish (Danio rerio) embryos were used. The morphological observations revealed that GA caused fin defects in zebrafish embryos in a concentration-dependent manner. The critical exposure time of GA to reveal teratogenicity was before 8 hpf (hours post fertilization). LC/MS/MS analysis revealed that a maximum bioconcentration of GA was occurred at 4 hpf. Q-PCR data showed that GA treatment resulted in significant inactivation of RA signaling which could be partially rescued by the exogenous supply of RA. These results indicate the potential teratogenicity of GA and provide evidence for a caution in its future clinic use.