Petra Mikšátková

Emerging toxicity of 5,6-methylenedioxy-2-aminoindane (MDAI): Pharmacokinetics, behaviour, thermoregulation and LD50 in rats.

MDAI (5,6-Methylenedioxy-2-aminoindane) has a reputation as a non-neurotoxic ecstasy replacement amongst recreational users, however the drug has been implicated in some severe and lethal intoxications. Due to this, and the fact that the drug is almost unexplored scientifically we investigated a broad range of effects of acute MDAI administration: pharmacokinetics (in sera, brain, liver and lung); behaviour (open field; prepulse inhibition, PPI); acute effects on thermoregulation (in group-/individually-housed rats); and systemic toxicity (median lethal dose, LD50) in Wistar rats. Pharmacokinetics of MDAI was rapid, maximum median concentration in serum and brain was attained 30min and almost returned to zero 6h after subcutaneous (sc.) administration of 10mg/kg MDAI; brain/serum ratio was ~4. MDAI particularly accumulated in lung tissue. In the open field, MDAI (5, 10, 20 and 40mg/kg sc.) increased exploratory activity, induced signs of behavioural serotonin syndrome and reduced locomotor habituation, although by 60min some effects had diminished. All doses of MDAI significantly disrupted PPI and the effect was present during the onset of its action as well as 60min after treatment. Unexpectedly, 40mg/kg MDAI killed 90% of animals in the first behavioural test, hence LD50 tests were conducted which yielded 28.33mg/kg sc. and 35mg/kg intravenous but was not established up to 40mg/kg after gastric administration. Disseminated intravascular coagulopathy (DIC) with brain oedema was concluded as a direct cause of death in sc. treated animals. Finally, MDAI (10, 20mg/kg sc.) caused hyperthermia and perspiration in group-housed rats. In conclusion, the drug had fast pharmacokinetics and accumulated in lipohilic tissues. Behavioural findings were consistent with mild, transient stimulation with anxiolysis and disruption of sensorimotor processing. Together with hyperthermia, the drug had a similar profile to related entactogens, especially 3,4-metyhlenedioxymethamphetamine (MDMA, ecstasy) and paramethoxymethamphetamine (PMMA). Surprisingly subcutaneous MDAI appears to be more lethal than previously thought and its serotonergic toxicity is likely exacerbated by group housing conditions. MDAI therefore poses greater risks to physical and mental health than recognised hitherto.

The presence of monoiodinated derivates of daidzein and genistein in human urine and its effect on thyroid gland function.

Dietary supplements used by women during menopause are usually based on plant extracts containing isoflavonoids, daidzein and genistein. Genistein is a known inhibitor of many enzymes, including thyroid peroxidase (TPO). In the thyroid follicle, genistein acts as its alternate substrate for the formation of genistein iodinated derivatives. The aim of this study was to search for daidzein- and genistein-iodinated derivatives in urine of isoflavonoid-supplemented women. Additionally, selected phytoestrogens, steroid and thyroid hormones before and after three months of phytoestrogen supplementation were estimated. Urinary levels of free phytoestrogen increased significantly after therapy. They ranged between 0.3-1600, 0.6-670 and 0-206 nmol/L for daidzein, genistein and S-equol, respectively. Monoiodinated derivatives of genistein were observed (0-504 pmol/L) in 60% of the investigated samples. Steroid and thyroid hormone levels were within the normal range and were not significantly altered. The presence of monoiodinated derivates in human urine confirmed that genistein and daidzein may enter human thyroid follicles and influence TPO. Since the levels of the free thyroid hormones were not affected, we propose that the use of phytoestrogen dietary supplements is not associated with the development of thyroid-gland disorders in subjects with adequate iodine intake.

Isoflavonoids in the Amaryllidaceae family

Plants of the Amaryllidaceae family are known as producers of biologically active alkaloids. Besides these a variety of flavonoids, including flavones, chalcones and chromones, have been detected in the Amaryllidaceous plants. In this study, we have analysed 16 representatives of the family for the presence of isoflavonoids. The water/ethanolic extracts were analysed with HPLC-ESI-MS both without any pre-treatment and after immunoaffinity chromatography as a clean-up step. Four individual immunosorbents specific for biochanin A, daidzein and genistein were used. In addition, five enzyme-linked immunosorbent assays specific for the above-mentioned isoflavonoids and their derivatives have been used for the analysis of the extracts after fractionation by semi-preparative HPLC. Fifteen selected isoflavonoids were detected in the studied samples, and the amount of individual compounds ranged between ca. 0.8 and 400 ng/g of dry weight. This study extends the number of known isoflavonoid-producing families within the monocotyledonous plants.

Individual prolactin reactivity modulates response of nucleus accumbens to erotic stimuli during acute cannabis intoxication: an fMRI pilot study

RationaleSelf-report studies indicate that cannabis could increase sexual desire in some users. We hypothesized that intoxication increases activation of brain areas responsive to visual erotica, which could be useful in the treatment of hypoactive sexual desire disorder, a condition marked by a lack of sexual desire.ObjectivesThe aim of this study is to assess the aphrodisiacal properties of cannabis.MethodsWe conducted an open-randomized study with 21 heterosexual casual cannabis users. A 3T MRI was used to measure brain activation in response to erotic pictures. Blood samples were collected to determine the serum levels of cannabinoids, cortisol and prolactin. Participants were grouped according to whether they had ever experienced any aphrodisiacal effects during intoxication (Group A) or not (Group non-A).ResultsIntoxication was found to significantly increase activation in the right nucleus accumbens in the Group A while significantly decreasing activation in the Group non-A. There was also a significant interaction between the group and intoxication, with elevated prolactin in the Group non-A during intoxication. No intoxication-related differences in subjective picture evaluations were found.ConclusionCannabis intoxication increases activation of the right nucleus accumbens to erotic stimuli. This effect is limited to users whose prolactin is not elevated in response to intoxication. This effect may be useful in the treatment of low sexual desire.

Highly sensitive avidin-biotin ELISA for detection of nandrolone and testosterone in dietary supplements

Avidin-biotin technology was used for the implementation of an enzyme-linked immunosorbent assay (AB-ELISA) as a sensitive method for the detection of anabolic androgenic steroids (AAS) present in dietary supplements. Using click chemistry, novel haptens (linker-optimized biotinylated nandrolone (NT) and testosterone (T) at positions C-3 and C-17, respectively) were designed and synthesized to be then applied as four different immobilized competitors in a proposed set of four indirect competitive AB-ELISAs. Four rabbit polyclonal antibodies of various specificities were prepared using four different immunogens synthesized from C-3 and C-17 carboxymethyloxime and hemisuccinate derivatives of NT and T, respectively. Assembled AB-ELISAs were characterized to establish method parameters such as a half-maximum inhibition concentration (0.18-12.99 ng/mL), limit of detection (0.004-0.032 ng/mL) and linear working range (the best with 0.02-1.38 ng/mL). The stability of the set simulating storage in different conditions was demonstrated. Cross reactivity (CR) was tested for 59 steroids including both endogenous and synthetic analogues in four assembled AB-systems. The focus was placed on the practical use of the method in detection of various AAS in 49 samples of counterfeit dietary supplements. The concordance between ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the CR corrected data from AB-ELISA indicated the potential of this method even to quantification of T propionate, NT phenyl propionate, and NT decanoate in such a complex matter. Copyright

Determination of Flavonoids in Stellaria by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

The genus Stellaria (Caryophyllaceae) presents widely distributed plants often used in traditional medicine. Flavonoids are highly active plant secondary metabolites that may be involved in some of the effects of Stellaria plants. In this study, two new high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS-MS) methods were developed for the determination of flavonoids and isoflavonoids in plant material. The separations were performed on a reverse-phase C18 column with gradient elution using methanol and water with 0.5% acetic acid as the mobile phase. Multiple reaction monitoring was used for the tandem mass spectrometry detection and the two most intensive transitions were chosen for the identification of each analyte. The limits of detection and the limits of quantification were between 0.2 and 15.0 ng/mL and 0.6 and 50.0 ng/mL. The developed methods were successfully used for the analyses of four representatives of the genus Stellaria. All the studied herbs contained luteolin and its 7-O-glycosides, naringenin, kaempferol, quercetin, its glycoside rutin, apigenin, and its 7-O-glycoside. One coumarin, scopoletin, was also found. Isoflavones were primarily represented by genistein, genistin, and ononin. Some of the analytes were detected for the first time in Stellaria sp. The findings support that these methods are suitable for analyses of plant material.

Lateral flow immunoassay and enzyme linked immunosorbent assay as effective immunomethods for the detection of synthetic cannabinoid JWH-200 based on the newly synthesized hapten

Graphical abstract

Plectin controls biliary tree architecture and stability in cholestasis

BACKGROUND & AIMS Plectin, a highly versatile cytolinker protein, controls intermediate filament cytoarchitecture and cellular stress response. In the present study, we investigate the role of plectin in the liver under basal conditions and in experimental cholestasis. METHODS We generated liver-specific plectin knockout (PleΔalb) mice and analyzed them using two cholestatic liver injury models: bile duct ligation (BDL) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Primary hepatocytes and a cholangiocyte cell line were used to address the impact of plectin on keratin filament organization and stability in vitro. RESULTS Plectin deficiency in hepatocytes and biliary epithelial cells led to aberrant keratin filament network organization, biliary tree malformations, and collapse of bile ducts and ductules. Further, plectin ablation significantly aggravated biliary damage upon cholestatic challenge. Coincidently, we observed a significant expansion of A6-positive progenitor cells in PleΔalb livers. After BDL, plectin-deficient bile ducts were prominently dilated with more frequent ruptures corresponding to an increased number of bile infarcts. In addition, more abundant keratin aggregates indicated less stable keratin filaments in PleΔalb hepatocytes. A transmission electron microscopy analysis revealed a compromised tight junction formation in plectin-deficient biliary epithelial cells. In addition, protein profiling showed increased expression of the adherens junction protein E-Cadherin, and inefficient upregulation of the desmosomal protein desmoplakin in response to BDL. In vitro analyses revealed a higher susceptibility of plectin-deficient keratin networks to stress-induced collapse, paralleled by elevated activation of p38 MAP kinase. CONCLUSION Our study shows that by maintaining proper keratin network cytoarchitecture and biliary epithelial stability, plectin plays a critical role in protecting the liver from stress elicited by cholestasis. LAY SUMMARY Plectin is a cytolinker protein capable of interconnecting all three cytoskeletal filament systems and linking them to plasma membrane-bound junctional complexes. In liver, the plectin-controlled cytoskeleton mechanically stabilizes epithelial cells and provides them with the capacity to adapt to increased bile pressure under cholestasis.

Sensitive enzyme immunoassay for screening methandienone in dietary supplements

ABSTRACT Methandienone is a synthetic exogenous steroid which, like other anabolic steroids, is strictly regulated in many countries. In recent years, increasing numbers have been detected of illegal additions into dietary supplements of methandienone and other anabolic androgenic steroids (AAS). In this work, a competitive indirect enzyme-linked immunosorbent assay (ELISA) has been constructed for the detection of methandienone using an antiserum against methandienone. Under optimal experimental conditions, the ELISA achieved a limit of detection of 0.04 ± 0.01 µg.g−1. The obtained intra- and inter-day coefficients of variation were less than 8%. The developed ELISA was applied in the analysis of real dietary supplement samples. To minimise the effect of the sample matrix, the sample extracts were simply diluted before addition into the immunoassay. The achieved recovery values were around 100%. Results obtained from the ELISA correlated well, both in terms of accuracy and precision, with those obtained by UHPLC-MS/MS (reference method). The presented ELISA could be successfully applied for the simple screening of dietary supplements. Graphical Abstract

Immunoassay for determination of trilobolide.

Graphical Abstract Figure. No Caption available. HighlightsNovel conjugates of trilobolide with bovine serum albumin and biotin were prepared.Trilobolide specific polyclonal antibodies were obtained by immunization of rabbits.Indirect competitive ELISA for determination of trilobolide was developed.Immunoassay was used to quantify trilobolide in different parts of Laser trilobum. Abstract Trilobolide (Tb) is a pharmacologically interesting sesquiterpene lactone isolated from Laser trilobum (L.) Borkh. Structural relation to a sarco/endoplasmic reticulum Ca2+‐ATPase inhibitor thapsigargin bring promising prospects for Tb to be used in the development of new anti‐cancer drugs. As long as there are still unanswered questions regarding its investigation, a need for novel analytical tools emerge. Since immunoassays serve as one of powerful tools within the investigation of natural products, the development of indirect competitive enzyme‐linked immunosorbent assay (ELISA) utilizing coating based on avidin‐biotin technology is described. In our set‐up of ELISA, newly synthesized biotinylated Tb served as immobilized competitor. Tb‐carboxymethyloxime‐bovine serum albumin (BSA) and Tb‐succinoyl‐BSA conjugates were used separately for immunization of rabbits. Two sets of polyclonal antibodies (RAbs) were obtained. Antibodies against Tb‐succinoyl‐BSA conjugate (RAb No. 206) were chosen as the best. Under optimized conditions, limit of detection and 50% intercept of our ELISA were 849 pg/mL and 8.89 ng/mL, respectively. The cross‐reactivity (CR) was tested on 10 structurally related compounds and CR did not exceed 6.1%. The reproducibility of the system is expressed as intra‐ and inter‐assay coefficients of variation (9.7% and 11.4%, respectively). Based on conducted experiments, we proposed the use of ELISA for quantification of Tb in complex biological matrices such as plant extracts. A method was applied to analyze three extracts obtained from different parts of L. trilobum. Data obtained were compared to those acquired by UHPLC‐MS/MS. The concordance between the methods (103–87%) showed the ability of ELISA to quantify Tb.