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Toxins Jul 2016Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic... (Comparative Study)
Comparative Study
Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic.
Topics: Anabasine; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Tolerance; Humans; Membrane Potentials; Muscle Cells; Neurons; Nicotine; Nicotinic Agonists; Pyridines; Receptors, Nicotinic; Risk Assessment; Teratogens; Time Factors
PubMed: 27384586
DOI: 10.3390/toxins8070204 -
Plant Physiology Aug 2016Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer's disease,...
Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer's disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase.
Topics: Alkaloids; Arabidopsis; Biosynthetic Pathways; Carboxy-Lyases; Decarboxylation; Evolution, Molecular; Huperzia; Lycopodium; Lysine; Mutagenesis, Site-Directed; Onions; Ornithine Decarboxylase; Phylogeny; Plant Leaves; Plant Proteins; Plant Roots; Plants, Genetically Modified; Recombinant Proteins; Nicotiana
PubMed: 27303024
DOI: 10.1104/pp.16.00639 -
PloS One 2016The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most...
The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most ecosystems. Here we describe a role of non-neuronal acetylcholine (ACh) for breeding of Apis mellifera carnica and a so far unknown effect of neonicotinoids on non-target insects. Royal jelly or larval food are produced by the hypopharyngeal gland of nursing bees and contain unusually high ACh concentrations (4-8 mM). ACh is extremely well conserved in royal jelly or brood food because of the acidic pH of 4.0. This condition protects ACh from degradation thus ensuring delivery of intact ACh to larvae. Raising the pH to ≥5.5 and applying cholinesterase reduced the content of ACh substantially (by 75-90%) in larval food. When this manipulated brood was tested in artificial larval breeding experiments, the survival rate was higher with food supplemented by 100% with ACh (6 mM) than with food not supplemented with ACh. ACh release from the hypopharyngeal gland and its content in brood food declined by 80%, when honeybee colonies were exposed for 4 weeks to high concentrations of the neonicotinoids clothianidin (100 parts per billion [ppb]) or thiacloprid (8,800 ppb). Under these conditions the secretory cells of the gland were markedly damaged and brood development was severely compromised. Even field-relevant low concentrations of thiacloprid (200 ppb) or clothianidin (1 and 10 ppb) reduced ACh level in the brood food and showed initial adverse effects on brood development. Our findings indicate a hitherto unknown target of neonicotinoids to induce adverse effects on non-neuronal ACh which should be considered when re-assessing the environmental risks of these compounds. To our knowledge this is a new biological mechanism, and we suggest that, in addition to their well documented neurotoxic effects, neonicotinoids may contribute to honeybee colony losses consecutive to a reduction of the ACh content in the brood food.
Topics: Acetylcholine; Anabasine; Animals; Bees; Choline O-Acetyltransferase; Female; Guinea Pigs; Hypopharynx; Insecticides; Larva; Male; Muscle Contraction; Muscle, Smooth; Neurons; Nitro Compounds; Pollination; Reproduction
PubMed: 27285384
DOI: 10.1371/journal.pone.0156886 -
Chemical Research in Toxicology Mar 2016Nicotine is the most abundant alkaloid in tobacco accounting for 95% of the alkaloid content. There are also several minor tobacco alkaloids; among these are...
Nicotine is the most abundant alkaloid in tobacco accounting for 95% of the alkaloid content. There are also several minor tobacco alkaloids; among these are nornicotine, anatabine, and anabasine. We developed and applied a 96 well plate-based capillary LC-tandem mass spectrometry method for the analysis of nornicotine, anatabine, and anabasine in urine. The method was validated with regard to accuracy and precision. Anabasine was quantifiable to low levels with a limit of quantitation (LOQ) of 0.2 ng/mL even when nicotine, which is isobaric, is present at concentrations >2500-fold higher than anabasine. This attribute of the method is important since anatabine and anabasine in urine have been proposed as biomarkers of tobacco use for individuals using nicotine replacement therapies. In the present study, we analyzed the three minor tobacco alkaloids in urine from 827 smokers with a wide range of tobacco exposures. Nornicotine (LOQ 0.6 ng/mL) was detected in all samples, and anatabine (LOQ, 0.15 ng/mL) and anabasine were detected in 97.7% of the samples. The median urinary concentrations of nornicotine, anatabine, and anabasine were 98.9, 4.02, and 5.53 ng/mL. Total nicotine equivalents (TNE) were well correlated with anatabine (r(2) = 0.714) and anabasine (r(2) = 0.760). TNE was most highly correlated with nornicotine, which is also a metabolite of nicotine. Urine samples from a subset of subjects (n = 110) were analyzed for the presence of glucuronide conjugates by quantifying any increase in anatabine and anabasine concentrations after β-glucuronidase treatment. The median ratio of the glucuronidated to free anatabine was 0.74 (range, 0.1 to 10.9), and the median ratio of glucuronidated to free anabasine was 0.3 (range, 0.1 to 2.9). To our knowledge, this is the largest population of smokers for whom the urinary concentrations of these three tobacco alkaloids has been reported.
Topics: Adolescent; Alkaloids; Anabasine; Chromatography, High Pressure Liquid; Humans; Molecular Structure; Nicotine; Pyridines; Smoking; Tandem Mass Spectrometry; Nicotiana
PubMed: 26825008
DOI: 10.1021/acs.chemrestox.5b00521 -
Insect Molecular Biology Apr 2016Honey bees, Apis mellifera, are markedly less sensitive to neonicotinoid insecticides containing a cyanoimino pharmacophore than to those with a nitroimino group....
Honey bees, Apis mellifera, are markedly less sensitive to neonicotinoid insecticides containing a cyanoimino pharmacophore than to those with a nitroimino group. Although previous work has suggested that this results from enhanced metabolism of the former by detoxification enzymes, the specific enzyme(s) involved remain to be characterized. In this work, a pretreatment of honey bees with a sublethal dose of thiacloprid resulted in induced insensitivity to the same compound immediately following thiacloprid feeding. A longer pretreatment time resulted in no, or increased, sensitivity. Transcriptome profiling, using microarrays, identified a number of genes encoding detoxification enzymes that were over-expressed significantly in insecticide-treated bees compared with untreated controls. These included five P450s, CYP6BE1, CYP305D1, CYP6AS5, CYP315A1, CYP301A1, and a carboxyl/cholinesterase (CCE) CCE8. Four of these P450s were functionally expressed in Escherichia coli and their ability to metabolize thiacloprid examined by liquid chromatography-mass spectrometry (LC-MS) analysis.
Topics: Anabasine; Animals; Bees; Cytochrome P-450 Enzyme System; Gene Expression Regulation; Inactivation, Metabolic; Insecticides; Neonicotinoids; Pyridines; Thiazines; Transcriptional Activation
PubMed: 26790026
DOI: 10.1111/imb.12211 -
Journal of Chromatography. A Jan 2016The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of...
Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts.
The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs.
Topics: Alkaloids; Anabasine; Binding Sites; Chemistry Techniques, Analytical; Chromatography, Affinity; Fabaceae; Lycopodiaceae; Nicotine; Plant Extracts; Receptors, Nicotinic; Smoke
PubMed: 26774122
DOI: 10.1016/j.chroma.2015.12.065 -
European Journal of Medicinal Chemistry Jan 2016We report the design, synthesis and pharmacological screening of a group of analogues of anabaseine 2, a naturally occurring unselective nicotinic agonist. The novel...
We report the design, synthesis and pharmacological screening of a group of analogues of anabaseine 2, a naturally occurring unselective nicotinic agonist. The novel nAChR ligands 5-15 were planned following a molecular modeling analysis which suggested the replacement of the pyridine ring of 2 with a 3-substituted benzene ring as a means to gain selectivity for the α3β4 nAChR subtype. Overall, from binding experiments, the synthesized compounds showed high values of α3β4 affinity and α3β4 vs α4β2 selectivity, although they poorly discriminated the homomeric α7 subtype. The three analogues 6, 12 and 13 were also evaluated in electrophysiological assays, and 12 [6-(3-iodophenyl)-2,3,4,5-tetrahydropyridine] emerged as a rather interesting nicotinic ligand. Indeed, in addition to a noteworthy affinity (Ki = 4.7 nM) for the α3β4 subtype and to an excellent α3β4 vs α4β2 subtype selectivity (806-fold), compound 12 selectively activated the α3β4 nAChR (EC50 = 7.4 μM) while eliciting a negligible response at the α7 subtype and no effect at the α4β2 subtype.
Topics: Anabasine; Binding Sites; Cell Line; Dose-Response Relationship, Drug; Humans; Models, Molecular; Molecular Structure; Pyridines; Receptors, Nicotinic; Structure-Activity Relationship
PubMed: 26706350
DOI: 10.1016/j.ejmech.2015.11.045 -
Journal of Occupational Health 2016Neonicotinoid insecticides have been widely used around the world since the 1990s. Reports have been made since the 1990s of rice paddy farmers in the North Central...
OBJECTIVES
Neonicotinoid insecticides have been widely used around the world since the 1990s. Reports have been made since the 1990s of rice paddy farmers in the North Central Region (NCR) of Sri Lanka suffering from chronic kidney disease with unknown etiology (CKDu). A preliminary evaluation of the exposure of local farmers in the NCR of Sri Lanka to neonicotinoids was performed.
METHODS
We analyzed neonicotinoid and neonicotinoid metabolite concentrations in spot urine samples. We selected 40 samples, 10 from farmers with CKDu and 10 from controls from each of two areas, Medawachchiya and Girandurukotte.
RESULTS
Imidacloprid and desmethyl-acetamiprid were found at significantly higher concentrations in the control samples (with medians of 51 ng/l and 340 ng/l, respectively) than in the CKDu samples (medians of 15 ng/l and 150 ng/l, respectively) when the results were not adjusted for the creatinine contents. None of the six compounds that were measured in the urine samples were found at significantly higher concentrations in the CKDu samples than in the control samples. None of the neonicotinoid concentrations in the samples analyzed in this study exceeded the concentrations that have been found in samples from the general population of Japan.
CONCLUSIONS
Farmers (both with and without CKDu) living in CKDu-endemic areas in the NCR of Sri Lanka are exposed to lower neonicotinoid concentrations than non-occupationally exposed residents of Japan.
Topics: Adult; Agriculture; Anabasine; Case-Control Studies; Female; Humans; Imidazoles; Insecticides; Japan; Male; Middle Aged; Neonicotinoids; Nitro Compounds; Occupational Exposure; Pyridines; Renal Insufficiency, Chronic; Sri Lanka
PubMed: 26549834
DOI: 10.1539/joh.15-0140-BR -
PloS One 2015The impact of consuming biologically active compounds is often dose-dependent, where small quantities can be medicinal while larger doses are toxic. The consumption of...
The impact of consuming biologically active compounds is often dose-dependent, where small quantities can be medicinal while larger doses are toxic. The consumption of plant secondary compounds can be toxic to herbivores in large doses, but can also improve survival in parasitized herbivores. In addition, recent studies have found that consuming nectar secondary compounds may decrease parasite loads in pollinators. However, the effect of compound dose on bee survival and parasite loads has not been assessed. To determine how secondary compound consumption affects survival and pathogen load in Bombus impatiens, we manipulated the presence of a common gut parasite, Crithidia bombi, and dietary concentration of anabasine, a nectar alkaloid produced by Nicotiana spp. using four concentrations naturally observed in floral nectar. We hypothesized that increased consumption of secondary compounds at concentrations found in nature would decrease survival of uninfected bees, but improve survival and ameliorate parasite loads in infected bees. We found medicinal effects of anabasine in infected bees; the high-anabasine diet decreased parasite loads and increased the probability of clearing the infection entirely. However, survival time was not affected by any level of anabasine concentration, or by interactive effects of anabasine concentration and infection. Crithidia infection reduced survival time by more than two days, but this effect was not significant. Our results support a medicinal role for anabasine at the highest concentration; moreover, we found no evidence for a survival-related cost of anabasine consumption across the concentration range found in nectar. Our results suggest that consuming anabasine at the higher levels of the natural range could reduce or clear pathogen loads without incurring costs for healthy bees.
Topics: Anabasine; Animals; Bees; Crithidia; Dose-Response Relationship, Drug; Herbivory; Host-Parasite Interactions; Parasite Load; Plant Nectar; Plants, Medicinal; Nicotiana
PubMed: 26545106
DOI: 10.1371/journal.pone.0142496 -
Scientific Reports Oct 2015Neonicotinoids are often applied as systemic seed treatments to crops and have reported negative impact on pollinators when they appear in floral nectar and pollen....
Neonicotinoids are often applied as systemic seed treatments to crops and have reported negative impact on pollinators when they appear in floral nectar and pollen. Recently, we found that bees in a two-choice assay prefer to consume solutions containing field-relevant doses of the neonicotinoid pesticides, imidacloprid (IMD) and thiamethoxam (TMX), to sucrose alone. This suggests that neonicotinoids enhance the rewarding properties of sucrose and that low, acute doses could improve learning and memory in bees. To test this, we trained foraging-age honeybees to learn to associate floral scent with a reward containing nectar-relevant concentrations of IMD and TMX and tested their short (STM) and long-term (LTM) olfactory memories. Contrary to our predictions, we found that none of the solutions enhanced the rate of olfactory learning and some of them impaired it. In particular, the effect of 10 nM IMD was observed by the second conditioning trial and persisted 24 h later. In most other groups, exposure to IMD and TMX affected STM but not LTM. Our data show that negative impacts of low doses of IMD and TMX do not require long-term exposure and suggest that impacts of neonicotinoids on olfaction are greater than their effects on rewarding memories.
Topics: Anabasine; Animals; Bees; Feeding Behavior; Learning; Memory, Short-Term; Olfactory Perception; Pesticides; Reward
PubMed: 26477973
DOI: 10.1038/srep15322