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Genes Nov 2019Nicotine, the most abundant pyridine alkaloid in cultivated tobacco ( L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain...
Nicotine, the most abundant pyridine alkaloid in cultivated tobacco ( L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain function. Nicotine biosynthesis is controlled developmentally and can be induced by abiotic and biotic stressors via a jasmonic acid (JA)-mediated signal transduction mechanism involving members of the APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH) transcription factor (TF) families. AP2/ERF and bHLH TFs work combinatorically to control nicotine biosynthesis and its subsequent accumulation in tobacco leaves. Here, we demonstrate that overexpression of the tobacco NtERF32, NtERF221/ORC1, and NtMYC2a TFs leads to significant increases in nicotine accumulation in T2 transgenic K326 tobacco plants before topping. Up to 9-fold higher nicotine production was achieved in transgenics overexpressing NtERF221/ORC1 under the control of a constitutive GmUBI3 gene promoter compared to wild-type plants. The constitutive 2XCaMV35S promoter and a novel JA-inducible 4XGAG promoter were less effective in driving high-level nicotine formation. Methyljasmonic acid (MeJA) treatment further elevated nicotine production in all transgenic lines. Our results show that targeted manipulation of NtERF221/ORC1 is an effective strategy for elevating leaf nicotine levels in commercial tobacco for use in the preparation of reduced risk tobacco products for smoking replacement therapeutics.
Topics: Acetates; Alkaloids; Anabasine; Cyclopentanes; Ethylenes; Gene Expression Regulation, Plant; Helix-Loop-Helix Motifs; Nicotine; Oxylipins; Plant Growth Regulators; Plant Leaves; Plants, Genetically Modified; Promoter Regions, Genetic; Pyridines; Nicotiana; Tobacco Products; Transcription Factors
PubMed: 31739571
DOI: 10.3390/genes10110930 -
Marine Drugs Oct 2019Three major forms of the nicotinic agonist toxin anabaseine (cyclic iminium, cyclic imine and the monocationic open-chain ammonium-ketone) co-exist in almost equal...
Three major forms of the nicotinic agonist toxin anabaseine (cyclic iminium, cyclic imine and the monocationic open-chain ammonium-ketone) co-exist in almost equal concentrations at physiological pH. We asked the question: Which of these forms is pharmacologically active? First, we investigated the pH dependence of anabaseine inhibition of [H]-methylcarbamylcholine binding at rat brain α4β2 nicotinic acetylcholine receptors (nAChRs). These experiments indicated that one or both monocationic forms interact with the orthosteric binding site for ACh. However, since they occur at equal concentrations near physiological pH, we employed another approach, preparing a stable analog of each form and examining its agonist activities and binding affinities at several vertebrate brain and neuromuscular nAChRs. Only 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine monohydrogen chloride (PTHP), the cyclic iminium analog, displayed nAChR potencies and binding affinities similar to anabaseine. The cyclic imine analog 2,3'-bipyridyl and the open-chain ammonium-ketone analog 5-methylamino-1-(3-pyridyl)-1-pentanone (MAPP), displayed ≤1% of the activity predicted if the one form was solely active. The lower potency of weakly basic 2,3'-bipyridyl can be explained by the presence of a small concentration of its monocationic form. Since the open chain ammonium-ketone monocationic form of anabaseine has some structural similarity to the neurotransmitter GABA, we also tested the ability of anabaseine and its 1,2-dehydropyrrolidinyl analog myosmine to activate a mammalian GABA receptor, but no activity was detected. We conclude that the monocationic cyclic iminium is the form which avidly binds and activates vertebrate nAChRs.
Topics: Anabasine; Animals; Binding Sites; Brain; Cell Line; Humans; Nicotinic Agonists; Rats; Receptors, GABA; Receptors, Nicotinic; Structure-Activity Relationship
PubMed: 31671780
DOI: 10.3390/md17110614 -
Addiction Science & Clinical Practice Sep 2019Tobacco use is a chronic relapsing disease, and remains the leading cause of preventable death in much of the world. Increasingly, tobacco use, chiefly cigarette...
Tobacco use is a chronic relapsing disease, and remains the leading cause of preventable death in much of the world. Increasingly, tobacco use, chiefly cigarette smoking, is being framed as a chronic disease, with periods of use and periods of abstinence. An implicit component of this conceptualization is that treatment-both counseling and pharmacotherapy-may be needed at various intervals for extended periods of time, perhaps over an individual's lifetime. This would mirror the treatment of other chronic conditions, such as diabetes, hypertension, or hyperlipidemia. Yet, clinical trials of tobacco dependence treatment still generally model outcome measures in terms of cessation, abstinence, or quitting, measured at discrete time points. This reinforces the notion that smoking, or tobacco dependence, is a dichotomous condition, and that one is either "cured," or not. Although the goal of treating tobacco dependence is to ensure long-term abstinence (i.e. "quitting"), this model is discordant with clinical reality, in which of periods of tobacco use are interspersed with periods of abstinence. Hence, the goal of treatment is to lengthen the duration of the latter, while shortening the duration of the former. In the clinical arena, this dichotomous model of tobacco use is reflected in electronic health records, where smoking is generally categorized as current, former, or never. We propose that clinicians move away from the dichotomous categorization of tobacco use, and adopt methods used to categorize the status of other chronic conditions. Specifically, biomarkers such as carbon monoxide, cotinine, and anabasine, measured at regular intervals, can provide clinicians with much clearer, clinically relevant and actionable assessments of current tobacco use by their patients. This can be done without making reference to dichotomous states such as current or former use of tobacco. In psychological terms, one can frame tobacco use in terms of states, attributes in specific situations at discrete moments in time, rather than the more durable traits.
Topics: Biomarkers; Chronic Disease; Humans; Patient Care Planning; Smoking Cessation; Tobacco Smoking; Tobacco Use Disorder
PubMed: 31474229
DOI: 10.1186/s13722-019-0159-z -
International Journal of Environmental... Aug 2019E-liquid manufacturers are under scrutiny concerning the purity and concentration accuracy of nicotine and the minor nicotine-related alkaloids (NRAs) packaged in their... (Comparative Study)
Comparative Study
UNLABELLED
E-liquid manufacturers are under scrutiny concerning the purity and concentration accuracy of nicotine and the minor nicotine-related alkaloids (NRAs) packaged in their products. In this communication we report concentrations of nicotine and five NRAs (nornicotine, cotinine, anabasine, anatabine, myosmine) from locally purchased E-liquids.
METHODS
Five brands of E-liquids (three bottles each) were purchased locally. Additionally, three bottles of reference E-liquid were prepared. Concentrations of nicotine and NRAs from each bottle were measured by HPLC. Concentrations of these alkaloids were also determined from electronic cigarette-generated aerosol and traditional cigarette smoke.
RESULTS
Nicotine concentrations in E-liquid brands 1, 2, 3, 4, 5 and in the reference E-liquid were 17.8 ± 4.1, 23.2 ± 0.7, 24.0 ± 0.9, 24.9 ± 0.2, 19.7 ± 0.3 and 20.4 ± 0.1 mg/mL, respectively. Concentrations normalized to 100% of product label were 74%, 97%, 100%, 104%, 109% and 102%, respectively. E-liquid brand 1 showed significance ( < 0.001) between bottles, while the reference showed the least variability. Similar results were obtained for the NRAs. Results also indicated the NRAs in aerosol of the reference E-liquid are lower than in cigarette smoke.
CONCLUSIONS
The amounts of NRAs present in E-liquids and E-liquid aerosol are less compared to cigarettes, however, inconsistencies and variation in nicotine concentrations supports the need for regulatory oversight.
Topics: Aerosols; Alkaloids; Chromatography, High Pressure Liquid; Electronic Nicotine Delivery Systems; Smoke; Nicotiana
PubMed: 31438499
DOI: 10.3390/ijerph16173015 -
Food and Chemical Toxicology : An... Oct 2019Within the traditional pharmacopeia, tobacco (Nicotiana spp.) is often cited as an efficient pesticide. This activity is generally attributed to nicotine, but tobacco...
Within the traditional pharmacopeia, tobacco (Nicotiana spp.) is often cited as an efficient pesticide. This activity is generally attributed to nicotine, but tobacco plants contain other alkaloids that could potentially contribute to this effect. In this study, we tested methanolic extracts of N. glutinosa, N. glauca, N. debneyi, and N. tabacum (putrescine N-methyltransferase line, burley TN90 and Stella, Virginia ITB 683 and K326), selected according to alkaloid content. Their antiparasitic activity was evaluated in bioassays against adult fleas (Ctenocephalides felis), blowfly (Lucilia cuprina) larvae, nematodes (Caenorhabditis elegans), and ticks (Rhipicephalus sanguineus larvae and adults, Ixodes ricinus nymphs). None of the extracts killed fleas and blowfly larvae effectively at the concentrations tested. Only N. tabacum K326 and N. glutinosa exhibited moderate anthelmintic activity. All extracts significantly repelled R. sanguineus ticks, but not I. ricinus, and the nicotine-rich extracts rapidly knocked down all tick species and stages at high concentrations. The link between nicotine and tick knockdown was confirmed by successfully testing the pure alkaloid at concentrations found in the tobacco extracts. In contrast, repellent activity could not be correlated to the individually tested alkaloids (nicotine, nornicotine, anabasine, anatabine), although anatabine and nornicotine were active in the tick bioassay at high concentrations.
Topics: Animals; Antiparasitic Agents; Biological Assay; Female; Insecta; Nematoda; Plant Extracts; Plant Leaves; Ticks; Nicotiana
PubMed: 31276744
DOI: 10.1016/j.fct.2019.110660 -
Ecology Oct 2019Bee populations have experienced declines in recent years, due in part to increased disease incidence. Multiple factors influence bee-pathogen interactions, including...
Bee populations have experienced declines in recent years, due in part to increased disease incidence. Multiple factors influence bee-pathogen interactions, including nectar and pollen quality and secondary metabolites. However, we lack an understanding of how plant interactions with their environment shape bee diet quality. We examined how plant interactions with the belowground environment alter floral rewards and, in turn, bee-pathogen interactions. Soil-dwelling mycorrhizal fungi are considered plant mutualists, although the outcome of the relationship depends on environmental conditions such as nutrients. In a 2 × 2 factorial design, we asked whether mycorrhizal fungi and nutrients affect concentrations of nectar and pollen alkaloids (anabasine and nicotine) previously shown to reduce infection by the gut pathogen Crithidia in the native bumble bee Bombus impatiens. To ask how plant interactions affect this common bee pathogen, we fed pollen and nectar from our treatment plants, and from a wildflower pollen control with artificial nectar, to bees infected with Crithidia. Mycorrhizal fungi and fertilizer both influenced flowering phenology and floral chemistry. While we found no anabasine or nicotine in nectar, high fertilizer increased anabasine and nicotine in pollen. Arbuscular mycorrhizal fungi (AMF) decreased nicotine concentrations, but the reduction due to AMF was stronger in high than low-nutrient conditions. AMF and nutrients also had interactive effects on bee pathogens via changes in nectar and pollen. High fertilizer reduced Crithidia cell counts relative to low fertilizer in AMF plants, but increased Crithidia in non-AMF plants. These results did not correspond with effects of fertilizer and AMF on pollen alkaloid concentrations, suggesting that other components of pollen or nectar were affected by treatments and shaped pathogen counts. Our results indicate that soil biotic and abiotic environment can alter bee-pathogen interactions via changes in floral rewards, and underscore the importance of integrative studies to predict disease dynamics and ecological outcomes.
Topics: Animals; Bees; Crithidia; Mycorrhizae; Nutrients; Parasites; Soil
PubMed: 31234229
DOI: 10.1002/ecy.2801 -
Heliyon May 2019N'-Nitrosonornicotine (NNN), a carcinogenic tobacco-specific N'-nitrosamine (TSNA), is on the FDA list of harmful and potentially harmful constituents (HPHCs)....
N'-Nitrosonornicotine (NNN), a carcinogenic tobacco-specific N'-nitrosamine (TSNA), is on the FDA list of harmful and potentially harmful constituents (HPHCs). Nornicotine, a product of the demethylation of nicotine, is the immediate alkaloid precursor for NNN formation. Nicotine, nornicotine and NNN are optically active. The accumulation of the isomers of nicotine, nornicotine, and NNN impacts their biological activity. In this paper, we report the determination of tobacco alkaloid enantiomers (including nicotine, nornicotine, anabasine, and anatabine) in samples of different tobacco lines using a reversed phase ultra-performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS) method. Current method demonstates excellent detection capability for all alkaloid enantiomers, with correlation coefficients (r) > 0.996 within their linear dynamic ranges. The limit of detection (LOD) and limit of quantitation (LOQ) of all analytes are less than 10 ng/mL and 30 ng/mL, respectively. In addition, their recovery and coefficient of variation (CV%) are within 100-115% and 0.2-3.7%, respectively. The method validated in this paper is simple, fast, and sensitive for the quantification of alkaloid enantiomers in tobacco leaf and has been applied to investigations of tobacco alkaloid enantiomer ratios in different tobacco lines and tobacco products.
PubMed: 31193304
DOI: 10.1016/j.heliyon.2019.e01719 -
BMJ Open May 2019Electronic cigarettes (EC) mainly with nicotine content are widely used worldwide. Although the number of publications about its use is increasing exponentially,... (Comparative Study)
Comparative Study Randomized Controlled Trial
Randomised, placebo-controlled, double-blind, double-dummy, multicentre trial comparing electronic cigarettes with nicotine to varenicline and to electronic cigarettes without nicotine: the ECSMOKE trial protocol.
INTRODUCTION
Electronic cigarettes (EC) mainly with nicotine content are widely used worldwide. Although the number of publications about its use is increasing exponentially, evidence-based, unbiased, conclusive, head-to-head comparisons about its efficacy and safety as an aid for smoking cessation are lacking. METHODS AND ANALYSIS: randomised, placebo and reference treatment-controlled, multicentre, double-blind, double-dummy, parallel-group trial. smokers smoking at least 10 cigarettes/day in the past year and motivated to quit, aged 18-70 years. : (A) EC without nicotine (ECwoN) plus placebo tablets of varenicline administered by oral route: , (B) EC with nicotine (ECwN) plus placebo tablets of varenicline: Voltage regulated EC will be used with liquid containing 12 mg/mL of nicotine for ad libitum use. : blond tobacco. (C) : ECwoN plus 0.5 mg varenicline tablets: Varenicline administered according to the marketing authorisationauthorisation. : 1 week+3 months. continuous smoking abstinence rate (CAR) (abstinence from conventional/combustible cigarettes) during the last 4 weeks (weeks 9-12) of the treatment period defined as self-report of no smoking during the previous 2 weeks and expired air carbon monoxide ≤8 at visit 4 at week 10 after target quit date (TQD), that is, 11 weeks after treatment initiation AND at visit 5, week 12 after TQD, that is, 13 weeks after treatment initiation. : safety profile; point prevalence abstinence rate; CAR confirmed by urinary anabasine concentration; changes in cigarettes/day consumption; craving for tobacco and withdrawal symptoms with respect of baseline.
ETHICS AND DISSEMINATION
The ethics committee approval was obtained on 17 April 2018. All data collected about the study participants will be anonymised. Investigators will communicate trial results to participants, health authorities, healthcare professionals, the public and other relevant groups without any publication restrictions.
TRIAL REGISTRATION NUMBER
NCT03630614; Pre-results.
Topics: Adolescent; Adult; Aged; Double-Blind Method; Electronic Nicotine Delivery Systems; Female; Humans; Male; Middle Aged; Nicotine; Nicotinic Agonists; Research Design; Smoking Cessation; Treatment Outcome; Varenicline; Young Adult
PubMed: 31129603
DOI: 10.1136/bmjopen-2018-028832 -
Analytical Sciences : the International... Aug 2019One method based on QuEChERS sample preparation is presented in this study, which leads to simultaneously detect nine alkaloids in tobacco and tobacco products....
One method based on QuEChERS sample preparation is presented in this study, which leads to simultaneously detect nine alkaloids in tobacco and tobacco products. Nicotine, nornicotine, myosmine, N-methyl anabasine, β-nicotyrine, anabasine, anatabine, isonicotenine and cotinine can all be found in fresh tobacco leaves, cigars, Virginia-type and blended-type cigarettes. The samples were purified via a certain proportion of adsorbents consisting of anhydrous magnesium sulfate, PSA and carbon after extracting, then centrifuged and filtered before analyzing by GC-MS. The matrix effects were all among 88 - 105%. The limit of detection of all were within the range of 0.0065 - 0.1509 μg/g and limit of quantification were among 0.0217 - 0.5031 μg/g. The recovery rates were higher than 89%. This is the first time that the QuEChERS sample preparation method has been applied for tobacco alkaloids, where more varieties of alkaloids could be quantified regarding sensitivity and reproducibility.
Topics: Alkaloids; Gas Chromatography-Mass Spectrometry; Solid Phase Extraction; Nicotiana; Tobacco Products
PubMed: 30930354
DOI: 10.2116/analsci.19P063 -
International Journal For Parasitology.... Dec 2018The control of parasitic nematodes impacting animal health relies on the use of broad spectrum anthelmintics. However, intensive use of these drugs has led to the...
The control of parasitic nematodes impacting animal health relies on the use of broad spectrum anthelmintics. However, intensive use of these drugs has led to the selection of resistant parasites in livestock industry. In that respect, there is currently an urgent need for novel compounds able to control resistant parasites. Nicotine has also historically been used as a de-wormer but was removed from the market when modern anthelmintics became available. The pharmacological target of nicotine has been identified in nematodes as acetylcholine-gated ion channels. Nicotinic-sensitive acetylcholine receptors (N-AChRs) therefore represent validated pharmacological targets that remain largely under-exploited. In the present study, using an automated larval migration assay (ALMA), we report that nicotinic derivatives efficiently paralyzed a multiple (benzimidazoles/levamisole/pyrantel/ivermectin) resistant field isolate of H. contortus. Using C. elegans as a model we confirmed that N-AChRs are preferential targets for nornicotine and anabasine. Functional expression of the homomeric N-AChR from C. elegans and the distantly related horse parasite Parascaris equorum in Xenopus oocytes highlighted some striking differences in their respective pharmacological properties towards nicotine derivative sensitivity. This work validates the exploitation of the nicotine receptors of parasitic nematodes as targets for the development of resistance-breaking compounds.
Topics: Animals; Anthelmintics; Antinematodal Agents; Ascaridoidea; Caenorhabditis elegans; Drug Delivery Systems; Drug Resistance, Multiple; Haemonchus; Helminth Proteins; Horses; Larva; Levamisole; Livestock; Nematoda; Nematode Infections; Nicotine; Protein Subunits; Receptors, Nicotinic; Sheep; Xenopus laevis
PubMed: 30502120
DOI: 10.1016/j.ijpddr.2018.11.003