Alpha‐phellandrene‐induced apoptosis in mice leukemia WEHI‐3 cells in vitro

Although reports have shown that α‐phellandrene (α‐PA) is one of the monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α‐PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α‐PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α‐PA on the cell morphology, viability, cell cycle distribution, and apoptosis in mice leukemia WEHI‐3 cells in vitro. Results indicated that α‐PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub‐G1 phase (apoptosis) in WEHI‐3 cells. α‐PA increased the productions of reactive oxygen species (ROS) and Ca²⁺ and decreased the levels of mitochondrial membrane potential (ΔΨ_m) in dose‐ and time‐dependent manners in WEHI‐3 cells that were analyzed by flow cytometer. Results from confocal laser microscopic system examinations show that α‐PA promoted the release of cytochrome c, AIF, and Endo G from mitochondria in WEHI‐3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α‐PA induces cell cycle arrest and apoptosis in WEHI‐3 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1640–1651, 2016.     

α‐Lipoic acid protects against microcystin‐LR induced hepatotoxicity through regeneration of glutathione via activation of Nrf2

Microcystins (MCs), as the most dominant bloom‐forming strains in eutrophic surface water, can induce hepatotoxicity by oxidative stress. Alpha‐lipoic acid (α‐LA) is a super antioxidant that can induce the synthesis of antioxidants, such as glutathione (GSH), by nuclear factor erythroid 2‐related factor 2 (Nrf2). However, the potential molecular mechanism of α‐LA regeneration of GSH remains unclear. The present study aimed to investigate whether α‐LA could reduce the toxicity of MCs induced in human hepatoma (HepG2), Bel7420 cells, and BALB/c mice by activating Nrf2 to regenerate GSH. Results showed that exposure to 10 μM microcystin‐leucine arginine (MC‐LR) reduced viability of HepG2 and Bel7402 cells and promoted the formation of reactive oxygen species (ROS) compared with untreated cells. Moreover, the protection of α‐LA included reducing the level of ROS, increasing superoxide dismutase activity, and decreasing malondialdehyde. Levels of reduced glutathione (rGSH) and rGSH/oxidized glutathione were significantly increased in cells cotreated with α‐LA and MC‐LR compared to those treated with MC‐LR alone, indicating an ability of α‐LA to attenuate oxidative stress and MC‐LR‐induced cytotoxicity by increasing the amount of rGSH. α‐LA can mediate GSH regeneration through the Nrf2 pathway under the action of glutathione reductase in MC‐LR cell lines. Furthermore, the data also showed that α‐LA‐induced cytoprotection against MC‐LR is associated with Nrf2 mediate pathway in vivo. These findings demonstrated the potential of α‐LA to resist MC‐LR‐induced oxidative damage of liver.     

Physiological Effects of a Novel Immune Stimulator Drug, (1,4)‐α‐d‐Glucan,in Rats

Abstract: The (1,4)‐α‐d ‐glucan (α‐d ‐glucan), derived from medicinal plant, Tinospora cordifolia, activates human lymphocytes with downstream synthesis of the pro‐ and anti‐inflammatory cytokines, in vitro. We investigated physiological and immunological effects of a low and a high dose of α‐d ‐glucan (0.5 and 10 mg/kg), in vivo, testing the hypothesis that intravenous administration of α‐d ‐glucan does not affect haemodynamic, respiratory, haematological, and immune responses in normal rats. Male rats (300–400 g) were anaesthetized, tracheostomized, and catheterized in one femoral artery and vein. The mean arterial blood pressure and heart rate were continuously recorded. The baselines for gas exchange, differential blood cell count, and plasma concentration of TNF‐α, IL‐1β, IL‐4, IL‐6, and IFN‐γ were determined. Rats were then randomly assigned to controls (n = 7), a low dose (0.5 mg/kg; n = 10), and a high dose (10 mg/kg; n = 7) of α‐d ‐glucan for a six 6 hr study period. Gas exchange, differential cell count, plasma concentration of TNF‐α, IL‐1β, IL‐4, IL‐6, and IFN‐γ, and mean arterial blood pressure values remained within physiological range. Intravenous administration of 10 mg/kg α‐d ‐glucan created tachycardia, associated with hyperventilation, and significant reductions in the blood haemoglobin and haematocrit concentrations. We suggest that these in vivo effects of α‐d ‐glucan should be considered for future clinical and/or experimental trials.     

The synthetic cathinone psychostimulant α‐PPP antagonizes serotonin 5‐HT2A receptors: In vitro and in vivo evidence

Synthetic cathinones (SCs) are β‐keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5‐HT_2A receptors (5‐HT_2AR) and muscarinic M₁ receptors (M₁R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M₁R (minimal displacement of [~K_d] [³H]scopolamine up to 10 μM). However, two SCs, α‐pyrrolidinopropiophenone (α‐PPP) and 4‐methyl‐α‐PPP, had low μM K_i values at 5‐HT_2AR. In 5‐HT_2AR–phosphoinositide hydrolysis assays, α‐PPP and 4‐methyl‐α‐PPP displayed inverse agonist activity. We further assessed the 5‐HT_2AR functional activity of α‐PPP, and observed it competitively antagonized 5‐HT_2AR signaling stimulated by the 5‐HT₂R agonist (±)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI; K_b = 851 nM). To assess in vivo 5‐HT_2AR activity, we examined the effects of α‐PPP on the DOI‐elicited head‐twitch response (HTR) in mice. α‐PPP dose‐dependently blocked the HTR with maximal suppression at 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α‐PPP. To corroborate a 5‐HT_2AR mechanism, we also tested 3,4‐methylenedioxy‐α‐PPP (MDPPP) and 3‐bromomethcathinone (3‐BMC), SCs that we observed had 5‐HT_2AR K_is > 10 μM. Neither MDPPP nor 3‐BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α‐PPP has antagonist interactions at 5‐HT_2AR in vitro that may translate at physiologically‐relevant doses in vivo. Considering 5‐HT_2AR antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α‐PPPs unpopularity compared to other monoamine transporter inhibitors.     

Radiochemical and radiopharmacological characterization of a 64Cu‐labeled α‐MSH analog conjugated with different chelators

Radiolabeled α‐melanocyte‐stimulating hormone (α‐MSH) derivatives have a high potential for diagnosis and treatment of melanoma, because of high specificity and binding affinity to the melanocortin‐1 receptor (MC1R). Hence, the α‐MSH‐derived peptide NAP‐NS1 with a β‐Ala linker (ε‐Ahx‐β‐Ala‐Nle‐Asp‐His‐D‐Phe‐Arg‐Trp‐Gly‐NH₂) was conjugated to different chelators: either to NOTA (p‐SCN‐Bn‐1,4,7‐triazacyclononane‐1,4,7‐triacetic acid), to a hexadentate bispidine carbonate derivative (dimethyl‐9‐(((4‐nitrophenoxy)carbonyl)oxy)‐2,4‐di(pyridin‐2‐yl)‐3,7‐bis(pyridin‐2‐ylmethyl)‐3,7‐diazabicyclo[3.3.1]nonane‐1,5‐dicarboxylate), or to DMPTACN (p‐SCN‐Ph‐bis(2‐pyridyl‐methyl)‐1,4,7‐triaza‐cyclononane), labeled with ⁶⁴Cu, and investigated in terms of radiochemical and radiopharmacological properties. For the three ⁶⁴Cu‐labeled conjugates negligible transchelation, suitable buffer and serum stability, as well as appropriate water solubility, was determined. The three conjugates exhibited high binding affinity (low nanomolar range) in murine B16F10, human MeWo, and human TXM13 cells. The B_max values of [⁶⁴Cu]Cu‐bispidine‐NAP‐NS1 ([⁶⁴Cu]Cu‐ 2 ) and [⁶⁴Cu]Cu‐DMPTACN‐NAP‐NS1 ([⁶⁴Cu]Cu‐ 3 ) were higher than those of [⁶⁴Cu]Cu‐NOTA‐NAP‐NS1 ([⁶⁴Cu]Cu‐ 1 ), implying that different charged chelate units might have an impact on binding capacity. Preliminary in vivo biodistribution studies suggested the main excretion pathway of [⁶⁴Cu]Cu‐ 1 and [⁶⁴Cu]Cu‐ 3 to be renal, while that of [⁶⁴Cu]Cu‐ 2 seemed to be both renal and hepatobiliary. An initial moderate uptake in the kidney decreased clearly after 60 minutes. All three ⁶⁴Cu‐labeled conjugates should be considered for further in vivo investigations using a suitable xenograft mouse model.     

A trend in the therapy of Amanita phalloides poisoning

Recent experimental evidences have been produced on the protection afforded by penicillin G in rats poisoned by Amanita phalloides extracts.A therapeutic trend which combines penicillin G infusions to the classical supportive measures was applied to 33 cases of severe A. phalloides poisoning, with 100% survival rates.The possible mechanism of the protective effect of penicillin G in A. phalloides poisoning is discussed.

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Zusammenfassung Tierexperimentell wurde eine Verminderung des toxischen Effekts von Amanita phalloides-Extrakten durch Penicillin G bei Ratten festgestellt.Auf Grund dieser Untersuchungen wurden zusätzlich zu den üblichen intensivmedizinischen Maßnahmen 250 mg/kg Penicillin G als tägliche Dauerinfusion gegeben. 33 schwere Vergiftungsfalle, die früh genug in klinische Behandlung kamen, überlebten.Der mögliche Schutzmechanismus wird diskutiert.

     

Metabolic studies of 1‐testosterone in horses

1‐Testosterone (17β‐hydroxy‐5α‐androst‐1‐en‐3‐one), a synthetic anabolic steroid, has been described as one of the most effective muscle‐building supplements currently on the market. It has an anabolic potency of 200 as compared to 26 for testosterone. Apart from its abuse in human sports, it can also be a doping agent in racehorses. Metabolic studies on 1‐testosterone have only been reported for human in the early seventies, whereas little is known about its metabolic fate in horses. This paper describes the studies of in vitro and in vivo metabolism of 1‐testosterone in horses, with the aim of identifying the most appropriate target metabolites to be monitored for controlling the misuse or abuse of 1‐testosterone in racehorses. Six in vitro metabolites, namely 5α‐androst‐1‐ene‐3α,17β‐diol (T1a), 5α‐androstane‐3β,17β‐diol (T2), epiandrosterone (T3), 16,17‐dihydroxy‐5α‐androst‐1‐ene‐3‐one (T4 & T5), and 5α‐androst‐1‐ene‐3,17‐dione (T6), were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 800 mg of 1‐testosterone by stomach tubing. The results revealed that the parent drug and eight metabolites were detected in urine. Besides the four in vitro metabolites (T1a, T2, T3, and T5), four other urinary metabolites, namely 5α‐androst‐1‐ene‐3β,17α‐diol (T1b), 5α‐androst‐1‐ene‐3β,17β‐diol (T1c), 5α‐androstane‐3α,17α‐diol (T7) and 5α‐androstane‐3β,17α‐diol (T8) were identified. This study shows that the detection of 1‐testosterone administration is best achieved by monitoring the parent drug, which could be detected for up to 30 h post‐administration. Copyright © 2012 John Wiley & Sons, Ltd.     

GRK2/β‐arrestin mediates arginine vasopressin‐induced cardiac fibroblast proliferation

Cardiac fibrosis is a pathological feature commonly found in hearts exposed to haemodynamic orneurohormonal stress. Elevated levels of arginine vasopressin (AVP) are closely associated with the progression of heart failure and could be an underlying cause of cardiac fibrosis. The aim of this study is to characterize the effect of AVP on neonatal rat cardiac fibroblasts (NRCFs) and to illustrate its signalling mechanism. The proliferative effect of AVP was assessed by methylthiazolyldiphenyl‐tetrazolium assay and 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay, and the amounts of cellular signalling proteins α‐smooth muscle actin (α‐SMA), matrix metalloproteinase (MMP) 2, MMP9, and phosphorylated ERK_1/2 were determined by western blotting. AVP, in a time‐ and concentration‐dependent manner, promoted NRCF proliferation and the expression of MMP2 and MMP9. Inhibition of G protein‐coupled receptor kinase2 (GRK2) by the inhibitory peptide GRK2‐Ct or knock‐down of GRK2 suppressed AVP‐induced BrdU incorporation and the expression of MMP2 and α‐SMA in NRCFs. Moreover, shRNA‐mediated silencing of β‐arrestin1 or β‐arrestin 2 abolished AVP‐induced BrdU incorporation and MMP2 expression. AVP‐induced NRCF proliferation depended on the phosphorylation of ERK_1/2, and inhibition of GRK2 or silencing of β‐arrestins blocked AVP‐induced ERK_1/2 phosphorylation. The effects of AVP on NRCF proliferation and α‐SMA expression were blocked by SR45059, a vasopressin receptor type1A (V_1AR) selective antagonist. In conclusion, AVP promotes NRCF proliferation through V_1AR‐mediated GRK2/β‐arrestin/ERK_1/2 signalling.     

Genotoxic effects of a particular mixture of acetamiprid and α‐cypermethrin on chromosome aberration,sister chromatid exchange,and micronucleus formation in human peripheral blood lymphocytes

The genotoxic effects of a particular mixture of acetamiprid (Acm, neonicotinoid insecticide) and α‐cypermethrin (α‐cyp, pyrethroid insecticide) on human peripheral lymphocytes were examined in vitro by chromosomal aberrations (CAs), sister chromatid exchange (SCE), and micronucleus (MN) tests. The human peripheral lymphocytes were treated with 12.5 + 2.5, 15 + 5, 17.5 + 7.5, and 20 + 10 μg/mL of Acm+α‐cyp, respectively, for 24 and 48 h. The mixture of Acm+α‐cyp induced the CAs and SCEs at all concentrations and treatment times when compared with both the control and solvent control and these increases were concentration‐dependent in both treatment times. MN formation was significantly induced at 12.5 + 2.5, 15 + 5, 17.5 + 7.5, μg/mL of Acm+α‐cyp when compared with both controls although these increases were not concentration‐dependent. Binuclear cells could not be detected sufficiently in the highest concentration of the mixture (20 + 10 μg/mL) for both the 24‐ and 48‐h treatment times. Mitotic index (MI), proliferation index (PI) and nuclear division index (NDI) significantly decreased because of the cytotoxic and cytostatic effects of the mixture, at all concentrations for two treatment periods. Significant decreases in MI and PI were concentration dependent at both treatment times. The decrease in NDI was also concentration‐dependent at 48‐h treatment period. In general, Acm+α‐cyp inhibited nuclear division more than positive control, mitomycin C (MMC) and showed a higher cytostatic effect than MMC. Furthermore, in this article, the results of combined effects of Acm+α‐cyp were compared with the results of single effects of Acm or α‐cyp (Kocaman and Topaktas, 2007 , 2009 , respectively). In conclusion, the particular mixture of Acm+α‐cyp synergistically induced the genotoxicity/cytotoxicity in human peripheral blood lymphocytes. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

The Anti‐tumor Effects of Androstene Steroids Exhibit a Strict Structure–Activity Relationship Dependent upon the Orientation of the Hydroxyl Group on Carbon‐17

Androstene steroids are metabolites of dehydroepiandrosterone and exist as androstene‐diols or ‐triols in α‐ and β‐epimeric forms based upon the placement of the hydroxyl groups relative to the plane of the Δ⁵cycloperhydrophenanthrene ring. 5‐Androstene‐3β,17β‐diol (3β,17β‐AED) functions to upregulate immunity and the addition of a third hydroxyl group at C‐7 in the α‐ or β‐orientation (3β,7α,17β‐AET and 3β,7β,17β‐AET, respectively) enhances the immunological activity of the molecule. In contrast, 5‐androstene‐3β,17α‐diol (3β,17α‐AED) possesses potent anti‐tumor activity. We synthesized a new androstene by adding a third hydroxyl group at C‐7 to make 5‐androstene‐3β,7α,17α‐triol (3β,7α,17α‐AET) and compared the anti‐tumor activity of this steroid to the four existing androstenes. The results showed that this modification reduced the activity of 3β,17α‐AED. The ranking of the anti‐tumor activities of these steroids and their IC50 on human glioblastoma and lymphoma cells was: 3β,17α‐AED (～10 μm ) > 3β,7α,17α‐AET (～30 μm ) >> 3β,7α,17β‐AET (～150 μm )> 3β,7β,17β‐AET (not achievable) ≥ 3β,17β‐AED (not achievable). 3β,17α‐AED and 3β,7α,17α‐AET induced autophagy in T98G glioblastoma cells and apoptosis in U937 lymphoma cells. These results indicate that the position of the hydroxyl group on C‐17 dictates the anti‐tumor activity of the androstenes and must be in the α‐configuration, demonstrating a strict structure–activity relationship.     

Immunological Response to (1,4)‐α‐d‐Glucan in the Lung and Spleen of Endotoxin‐Stimulated Juvenile Rats

Abstract: We investigated the effects of (1,4)‐α‐D‐glucan (α‐DG), a novel immune stimulatory drug from Tinospora cordifolia, on the concentration of pro‐ and anti‐inflammatory cytokines (interleukin [IL]‐1β, IL‐6, tumour necrosis factor‐α [TNF‐α], γ‐interferon [IFN‐γ] and IL‐10) in the lung and spleen of endotoxin‐stimulated juvenile rats. Experimental groups (n = 16/group) included controls with an intraperitoneal injection of saline, endotoxaemic rats with a non‐lethal dose of 10 mg/kg Escherichia coli endotoxin, and endotoxaemic rats treated with two doses of 10 mg/kg α‐DG, intraperitoneally, 2 and 4 hr after endotoxin injection. At 24 hr of treatment, rats were euthanized and lungs and spleen were removed for cytokines determination and lung injury. Endotoxaemia increased IL‐1β concentration by fivefold in both organs, while creating a moderate pulmonary hypercellularity (demonstrated by about 11% increase in the alveolar‐septal thickening and 11% decrease in the alveolar‐interstitial space ratio). In the lung, α‐DG treatment reduced concentrations of IL‐1β by 30% (p > 0.05), IL‐6 by 43% (p < 0.01), IFN‐γ by 46% (p < 0.01) and the anti‐inflammatory cytokine, IL‐10, by 31% (p > 0.05) compared to endotoxaemia. In the spleen, α‐DG treatment decreased the ratio of IL‐1β to IL‐10 by 55% (p < 0.05), demonstrating an anti‐inflammatory trend. These data suggest that α‐DG differentially modulates cytokine response in the lung and spleen and modifies the pro‐ and anti‐inflammatory balance during an early period of endotoxaemia in juvenile rats.     

Synthesis and biological evaluation of novel N‐aryl‐ω‐(benzoazol‐2‐yl)‐sulfanylalkanamides as dual inhibitors of α‐glucosidase and protein tyrosine phosphatase 1B

α‐Glucosidase is known to catalyze the digestion of carbohydrates and release free glucose into the digestive tract. Protein tyrosine phosphatase 1B (PTP1B) is engaged in the dephosphorylation of the insulin receptor and regulation of insulin sensitivity. Therefore, dual antagonists by targeting both α‐glucosidase and PTP1B may be potential candidates for type 2 diabetes therapy. In this work, three series of novel N‐aryl‐ω‐(benzoazol‐2‐yl)‐sulfanylalkanamides were synthesized and assayed for their α‐glucosidase and PTP1B inhibitory activities, respectively. Compound 3l , exhibiting the most effective α‐glucosidase inhibitory activity (IC₅₀ = 10.96 μm ( 3l ), IC₅₀ = 51.32 μm (Acarbose), IC₅₀ = 18.22 μm (Ursolic acid)) and potent PTP1B inhibitory activity (IC₅₀ = 13.46 μm ( 3l ), IC₅₀ = 14.50 μm (Ursolic acid)), was identified as a novel dual inhibitor of α‐glucosidase and PTP1B. Furthermore, 3l is a highly selective PTP1B inhibitor because no inhibition was showed by 3l at 100 μm against PTP‐MEG2, TCPTP, SHP2, or SHP1. Subsequent kinetic analysis revealed 3l inhibited α‐glucosidase in a reversible and mixed manner. Molecular docking study indicated that hydrogen bonds, van der Waals, charge interactions and Pi‐cation interactions all contributed to affinity between 3l and α‐glucosidase/PTP1B.     

Alpha‐phellandrene‐induced DNA damage and affect DNA repair protein expression in WEHI‐3 murine leukemia cells in vitro

Although there are few reports regarding α‐phellandrene (α‐PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α‐PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α‐PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α‐PA on total cell viability and the results indicated that α‐PA induced cell death. Comet assay and 4,6‐diamidino‐2‐phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α‐PA induced DNA damage and condensation in a concentration‐dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α‐PA induced DNA damage in WEHI‐3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α‐PA increased p‐p53, p‐H2A.X, 14‐3‐3‐σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA‐PK, and BRCA‐1. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1322–1330, 2015.     

Ameliorative effect of N‐acetyl cysteine on alpha‐cypermethrin‐induced pulmonary toxicity in male rats

Alpha‐cypermethrin (α‐CYP) is one of the most widely used insecticides. It may become an air pollutant and adversely affect the health. The present study was designed to determine whether treatment with N‐acetyl cysteine (NAC), a well‐known antioxidant, can be useful for the management of the deleterious effects of α‐CYP on lung tissues. For this purpose, thirty two male rats were divided into four different groups (eight rats for each). Group (I) gavaged with corn oil (control group), group (II) gavaged daily with NAC (150 mg kg^?1 body weight), group (III) gavaged with α‐CYP (14.5 mg kg^?1 body weight/day, dissolved in corn oil), group (IV) gavaged with NAC then with α‐CYP 2 h later for 12 weeks. α‐CYP significantly increased serum lactate dehydrogenase (LDH) and pulmonary malondialdehyde (MDA) levels, while decreased the activities of catalase (CAT) and superoxide dismutase (SOD) as well as reduced glutathione (GSH) content in lung. It also provoked higher levels of serum nitric oxide (NO), lung interleukin‐1 beta (IL‐1β), tumor necrosis factor‐alpha (TNF‐α), hydroxyproline (Hyp) as well as heme oxygenase‐1 (HO‐1), inducible nitric oxide synthase (iNOS) and nuclear factor‐kappa B (NF‐_КB) gene expression in lung tissues. Histopathological alterations in lung with congestion, cellular infiltration, necrotic changes and thickening of inter‐alveolar septa were observed following α‐CYP administration. NAC reduced the adverse effects of α‐CYP on lung tissues and improved the histological architecture of lung since it showed antioxidant, anti‐inflammatory and antifibrotic effects on lung tissues. Our results indicate that NAC exerts a potent protective effect against α‐CYP‐induced oxidative damage and inflammation in lung tissues. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 26–43, 2015.     

Detection and metabolic investigations of a novel designer steroid: 3‐chloro‐17α‐methyl‐5α‐androstan‐17β‐ol

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid‐related compounds. Subsequent gas chromatography–mass spectrometry (GC‐MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α‐ and 3β‐ isomers of the novel compound; 3‐chloro‐17α‐methyl‐5α‐androstan‐17β‐ol. Synthesis of authentic reference materials followed by comparison of NMR, GC‐MS and gas chromatography‐tandem mass spectrometry (GC‐MS/MS) data confirmed the finding of a new ‘designer’ steroid. Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid. Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α‐chloro‐17α‐methyl‐5α‐androstan‐17β‐ol using equine and human S9 liver fractions were performed. For equine, GC‐MS/MS analysis identified the diagnostic 3α‐chloro‐17α‐methyl‐5α‐androstane‐16α,17β‐diol metabolite. For human, the 17α‐methyl‐5α‐androstane‐3α,17β‐diol metabolite was found. Results from these studies were used to verify the ability of GC‐MS/MS precursor‐ion scanning techniques to support untargeted detection strategies for designer steroids in anti‐doping analyses. Copyright © 2015 John Wiley & Sons, Ltd.     

Phytoestrogen α‐Zearalanol exerts Antiapoptotic Effects in Differentiated PC12 Cells via Oestrogen Receptor α

Our previous studies have demonstrated that phytoestrogen α‐zearalanol (α‐ZAL) possesses potential benefits in alleviating cell apoptotic death just like oestrogen. However, the underlying mechanism is not fully understood. This study was designed to test the hypothesis that the neuroprotective effect of α‐ZAL is mediated by oestrogen receptor (ER) as α‐ZAL owns the benzene ring structure may interact with ER. The present results showed a significant increase in apoptosis in differentiated PC12 cells after a 24‐hr exposure to amyloid β‐peptide fragment 25‐35 (Aβ_25‐35), accompanied by decreasing of bcl‐2 expression and increasing bax expression, whereas a pre‐treatment with α‐ZAL ameliorated these changes induced by Aβ_25‐35. In addition, the α‐ZAL‐mediated cytoprotection was abrogated by ERα antagonist but not by ERβ antagonist. In summary, these data suggest that α‐ZAL intervenes against Aβ‐induced apoptosis via intersecting bcl‐2‐bax apoptotic pathway in an ERα‐sensitive manner.     

Early morphological and functional alterations in canine hepatocytes due to α-amanitin,a major toxin of <Emphasis Type="Italic">Amanita phalloides</Emphasis>

The toadstool death cap (Amanita phalloides) and its subspecies, destroying angel (A. virosa) and death angel (A. verna) are responsible for nearly 95% of all fatal mushroom poisonings. High mortality rate in A. phalloides intoxications is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amanitins, the major toxins of this mushroom. This study evaluated early morphological and functional alterations in hepatocytes exposed to different concentrations of α-amanitin (α-AMA). All experiments were performed on cultured canine hepatocytes since intoxicated with A. phalloides dogs have clinical course and pathological findings similar to those seen in humans. The overall functional integrity and viability of cultured hepatocytes were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and by measurements of lactate dehydrogenase (LDH), total protein, and urea levels. Our results showed that the course of α-AMA toxicity in cultured dog hepatocytes is divided into two phases. The first phase comprises functional cell impairments expressed by significant increase of LDH activity and inhibition of protein and urea synthesis when compared with the control group. This is followed by discrete changes in hepatocyte ultrastructure, including marginalization and condensation of nuclear chromatin, as well as formation of the foamlike cytoplasm. The second stage is lethal and is characterized by ongoing necrosis, and/or apoptosis. This may be related to dose of toxin and time of exposure.     

The Biological Consequences of Replacing d‐Ala in Biphalin with Amphiphilic α‐Alkylserines

Biphalin, a synthetic opioid peptide with a broad affinity for all opioid receptors (δ, μ, and κ) and high antinociceptive activity, has been under extensive study as a potential analgesic drug. This study presents the synthesis and biological properties of four new analogues of biphalin containing amphiphilic α‐alkylserines in position 2 and 2′. The incorporation of bulky α,α‐disubstituted amino acids in the peptide chain using standard peptide chemistry is often unsuccessful. We synthesized depsipeptides, and then, the desired peptides were obtained by internal O,N‐migration of the acyl residue from the hydroxyl to the amino group under mild basic conditions. The potency and selectivity of the new analogues were evaluated by a competitive receptor‐binding assay in the rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). Their binding affinity is strongly dependent on the chirality of α‐alkylserine, as analogues containing (R)‐α‐alkylserines displayed higher μ receptor affinity and selectivity than those incorporating the (S)‐isomers.     

Synthesis and mode of action studies of novel {2‐(3‐R‐1H‐1,2,4‐triazol‐5‐yl)phenyl}amines to combat pathogenic fungi

Due to their high specificity and efficacy, triazoles have become versatile antifungals to treat fungal infections in human healthcare and to control phytopathogenic fungi in agriculture. However, azole resistance is an emerging problem affecting human health as well as food security. Here we describe the synthesis of 10 novel {2‐(3‐R‐1H‐1,2,4‐triazol‐5‐yl)phenyl}amines. Their structure was ascertained by liquid chromatography–mass spectrometry, ¹H and ¹³C NMR, and elemental analysis data. Applying an in vitro growth assay, these triazoles show moderate to significant antifungal activity against the opportunistic pathogen Aspergillus niger, 12 fungi (Fusarium oxysporum, Fusarium fujikuroi, Colletotrichum higginsianum, Gaeumannomyces graminis, Colletotrichum coccodes, Claviceps purpurea, Alternaria alternata, Mucor indicus, Fusarium graminearum, Verticillium lecanii, Botrytis cinerea, Penicillium digitatum) and three oomycetes (Phytophtora infestans GL‐1, P. infestans 4/91; R+ and 4/91; R?) in the concentration range from 1 to 50 µg/ml (0.003–2.1 μM). Frontier molecular orbital energies were determined to predict their genotoxic potential. Molecular docking calculations taking into account six common fungal enzymes point to 14α‐demethylase (CYP51) and N‐myristoyltransferase as the most probable fungal targets. With respect to effectiveness, structure–activity calculations revealed the strong enhancing impact of adamantyl residues. The shown nonmutagenicity in the Salmonella reverse‐mutagenicity assay and no violations of drug‐likeness parameters suggest the good bioavailability and attractive ecotoxicological profile of the studied triazoles.