Potential for use of nicotinic acid as a selective agent for isolation of high nicotine-producing lines of Nicotiana rustica hairy root cultures

The addition of exogenous nicotinic acid, nicotinamide or nicotine was studied with reference to their effects on growth and alkaloid production by hairy root cultures of Nicotiana rustica. Nicotinic acid and nicotinamide were toxic (50% phytostatic dose being 2.4 and 9 mM respectively) while nicotine was not toxic below 10 mM. Nicotinic acid (up to 5 mM) was found to be phytostatic rather than phytotoxic. Roots exposed to increasing nicotinic acid or nicotinamide levels had altered alkaloid accumulation patterns relative to the controls. The principal effects were to increase the intracellular and extracellular levels of anatabine and nicotine, with a markedly greater proportion of anatabine being produced. The use of nicotinic acid as a selection agent for the recovery of higher alkaloid-producing lines is identified and discussed.     

Biotransformation of nicotine alkaloids by tobacco shooty teratomas induced by a Ti plasmid mutant

Tobacco shooty or rooty teratomas and hairy roots were induced by Agrobacterium tumefaciens (pGV 3845), A. tumefaciens (pGV 3304) and A. rhizogenes (pRi 8196), respectively. The tobacco alkaloids, nicotine, nornicotine and anatabine, were produced in hairy roots and in rooty teratomas but not in shooty teratomas. However, the shooty teratomas have the ability to accumulate the alkaloids and to biotransform nicotine to nornicotine. These were established by co-culture experiments incubating hairy roots and shooty teratomas in a same dish and by biotransformation experiments with shooty teratomas.     

Chronic Anatabine Treatment Reduces Alzheimer’s Disease (AD)-Like Pathology and Improves Socio-Behavioral Deficits in a Transgenic Mouse Model of AD

Anatabine is a minor tobacco alkaloid, which is also found in plants of the Solanaceae family and displays a chemical structure similarity with nicotine. We have shown previously that anatabine displays some anti-inflammatory properties and reduces microgliosis and tau phosphorylation in a pure mouse model of tauopathy. We therefore investigated the effects of a chronic oral treatment with anatabine in a transgenic mouse model (Tg PS1/APPswe) of Alzheimer’s disease (AD) which displays pathological Aβ deposits, neuroinflammation and behavioral deficits. In the elevated plus maze, Tg PS1/APPswe mice exhibited hyperactivity and disinhibition compared to wild-type mice. Six and a half months of chronic oral anatabine treatment, suppressed hyperactivity and disinhibition in Tg PS1/APPswe mice compared to Tg PS1/APPswe receiving regular drinking water. Tg PS1/APPswe mice also elicited profound social interaction and social memory deficits, which were both alleviated by the anatabine treatment. We found that anatabine reduces the activation of STAT3 and NFκB in the vicinity of Aβ deposits in Tg PS1/APPswe mice resulting in a reduction of the expression of some of their target genes including Bace1, iNOS and Cox-2. In addition, a significant reduction in microgliosis and pathological deposition of Aβ was observed in the brain of Tg PS1/APPswe mice treated with anatabine. This is the first study to investigate the impact of chronic anatabine treatment on AD-like pathology and behavior in a transgenic mouse model of AD. Overall, our data show that anatabine reduces β-amyloidosis, neuroinflammation and alleviates some behavioral deficits in Tg PS1/APPswe, supporting further exploration of anatabine as a possible disease modifying agent for the treatment of AD.     

Studies on the Chemical Regulation of Alkaloid Biosynthesis in Tobacco Plants

Effect of auxin treatment upon the alkaloid content of Nicotiana was examined using hydroponically grown plants. Nicotine content of Nicotiana tabacum was reduced by the addition of 3-indoleacetic acid or 2, 4-dichlorophenoxyacetic acid to nutrient solution, an the incorporation of nitrate-¹⁵N into nicotine was decreased by the addition of 2, 4-dichloro-phenoxyacetic acid. Contents of nicotine, nornicotine, anabasine and anatabine in Nicotiana glutinosa were decreased by 2, 4-dichlorophenoxyacetic acid added to the nutrient solution. Foliar applications of eleven kinds of synthetic auxins were effective in reducing nicotine content of Nicotiana tabacum and the effectiveness of the compounds seemed to be concerned with their auxin activities. Foliar application of auxin tended to increase the dry weight of stems whereas decreased that of roots, and, moreover, higher content of soluble sugars and lower content of starch in leaves were observed by the treatment. A possibility of a practical application of auxin to reduce the nicotine content of tobacco plants were drawn from this work.     

Role of Acetylcholine Receptors and Dopamine Transporter in Regulation of Extracellular Dopamine in Rat Carotid Body Cultures Grown in Chronic Hypoxia or Nicotine

Abstract: Using dissociated rat carotid body (CB) cultures, we compared levels of extracellular dopamine (DA) around oxygen-sensitive glomus cells grown for ～12 days in normoxia (Nox; 20% O₂), chronic hypoxia (CHox; 6% O₂), or chronic nicotine (CNic; 10 µM nicotine, 20% O₂), with or without acetylcholine (ACh) receptor (AChR) agonists/antagonists and blockers of DA uptake. In Nox cultures, extracellular DA, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was augmented by acute (～15-min) exposure to hypoxia (5% O₂; ～6× basal), high extracellular K⁺ (30 mM; ～10× basal), nomifensine (1 µM; a selective DA uptake inhibitor; ～3× basal), and nicotine (100 µM; ～5× basal), but not methylcholine (300 µM; a specific muscarinic agonist). In contrast, in CHox cultures where basal DA release is markedly elevated (～9× control), the stimulatory effect of high K⁺ (3–4× basal) and acute hypoxia (～2× basal) on DA release persisted, but nicotine and nomifensine were no longer effective and methylcholine had a partial inhibitory effect. In CNic cultures, basal DA levels were also elevated (～9× control), similar to that in CHox cultures; however, although acute hypoxia had a stimulatory effect on DA release (～2× basal), nicotine, nomifensine, and high K⁺ were ineffective. The elevated basal DA in both CHox and CNic cultures was attenuated by acute or chronic treatment with mecamylamine (100 µM), a nicotinic AChR (nAChR) antagonist. In addition, long-term (16-h), but not acute (15-min), treatment with the muscarinic antagonist atropine (1 µM) produced an additional enhancement of basal DA levels in CHox cultures. Thus, after chronic hypoxia or nicotine in vitro, extracellular DA levels around CB chemoreceptor cell clusters appear to be set by a variety of factors including released ACh, positive and negative feedback regulation via nAChRs and muscarinic AChRs, respectively, and modulation of DA transporters. These results provide insight into roles of endogenous transmitters in the adaptation of CB chemoreceptors to chronic hypoxia and suggest pathways by which neuroactive drugs, e.g., nicotine, can interfere with the protective chemoreflex response against hypoxia.     

Determination of tobacco-specific N-nitrosamines in urine of smokers and non-smokers

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN), N′-nitrosoanabasine (NAB) and N′-nitrosoanatabine (NAT) and are found in tobacco and tobacco smoke. TSNA are of interest for biomonitoring of tobacco-smoke exposure as they are associated with carcinogenesis. Both NNK and NNN are classified by IARC as Group 1 carcinogens. Samples of 24?h urine collections (n?=?108) were analysed from smokers and non-smokers, using a newly developed and validated LC-MS/MS method for determining total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major metabolite of NNK), and total NNN, NAB and NAT. TSNA levels in smokers’ urine were significantly higher than in non-smokers. In smokers, urinary excretion of total TSNA correlated significantly (r?>?0.5) with markers of smoking dose, such as daily cigarette consumption, salivary cotinine and urinary nicotine equivalents and increased with the ISO tar yield of cigarettes smoked. The correlation between urinary total NNN and the smoking dose was weaker (r?=?0.4–0.5). In conclusion, this new method is suitable for assessing tobacco use-related exposure to NNK, NNN, NAB and NAT.     

Nicotine degradation enhancement by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> ZCJ during aging process of tobacco leaves

Nicotine is a key harmful component of tobacco and cigarettes, and the development of low-nicotine cigarettes is of increasing importance in the market. The objectives of this study are to isolate native nicotine-degrading strains and evaluate their feasibility for nicotine reduction during the aging (or fermentation) of tobacco leaves. A novel nicotine-degrading strain was isolated and identified as Pseudomonas stutzeri ZCJ based on its 16S rDNA sequence and morphological-biochemical characteristics. In submerged cultures, P. stutzeri ZCJ could tolerate 4.5 g/L nicotine and completely degrade 1.5 g/L nicotine within 24 h at 37°C and pH 7.4. The addition of glucose (1 g/L) could improve nicotine degradation by P. stutzeri ZCJ in submerged cultures. After submerged culturing, the cell suspension of P. stutzeri ZCJ could be utilized to improve nicotine reduction in tobacco leaves during solid-state fermentation. The nicotine content of tobacco leaves decreased by as much as 32.24% after 7 days of solid-state fermentation by P. stutzeri ZCJ, suggesting the industrial application potential of the native strain to enhance nicotine degradation during the aging of tobacco leaves.     

Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco

Tobacco (Nicotiana tabacum) plants synthesize nicotine and related pyridine-type alkaloids, such as anatabine, in their roots and accumulate them in their aerial parts as chemical defenses against herbivores. Herbivory-induced jasmonate signaling activates structural genes for nicotine biosynthesis and transport by way of the NICOTINE (NIC) regulatory loci. The biosynthesis of tobacco alkaloids involves the condensation of an unidentified nicotinic acid-derived metabolite with the N-methylpyrrolinium cation or with itself, but the exact enzymatic reactions and enzymes involved remain unclear. Here, we report that jasmonate-inducible tobacco genes encoding flavin-containing oxidases of the berberine bridge enzyme family (BBLs) are expressed in the roots and regulated by the NIC loci. When expression of the BBL genes was suppressed in tobacco hairy roots or in tobacco plants, nicotine production was highly reduced, with a gradual accumulation of a novel nicotine metabolite, dihydromethanicotine. In the jasmonate-elicited cultured tobacco cells, suppression of BBL expression efficiently inhibited the formation of anatabine and other pyridine alkaloids. Subcellular fractionation and localization of green fluorescent protein-tagged BBLs showed that BBLs are localized in the vacuoles. These results indicate that BBLs are involved in a late oxidation step subsequent to the pyridine ring condensation reaction in the biosynthesis of tobacco alkaloids.     

Validation of a LC-MS/MS Method for Quantifying Urinary Nicotine,Six Nicotine Metabolites and the Minor Tobacco Alkaloids—Anatabine and Anabasine—in Smokers' Urine

Tobacco use is a major contributor to premature morbidity and mortality. The measurement of nicotine and its metabolites in urine is a valuable tool for evaluating nicotine exposure and for nicotine metabolic profiling—i.e., metabolite ratios. In addition, the minor tobacco alkaloids—anabasine and anatabine—can be useful for monitoring compliance in smoking cessation programs that use nicotine replacement therapy. Because of an increasing demand for the measurement of urinary nicotine metabolites, we developed a rapid, low-cost method that uses isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneously quantifying nicotine, six nicotine metabolites, and two minor tobacco alkaloids in smokers'' urine. This method enzymatically hydrolyzes conjugated nicotine (primarily glucuronides) and its metabolites. We then use acetone pretreatment to precipitate matrix components (endogenous proteins, salts, phospholipids, and exogenous enzyme) that may interfere with LC-MS/MS analysis. Subsequently, analytes (nicotine, cotinine, hydroxycotinine, norcotinine, nornicotine, cotinine N-oxide, nicotine 1′-N-oxide, anatabine, and anabasine) are chromatographically resolved within a cycle time of 13.5 minutes. The optimized assay produces linear responses across the analyte concentrations typically found in urine collected from daily smokers. Because matrix ion suppression may influence accuracy, we include a discussion of conventions employed in this procedure to minimize matrix interferences. Simplicity, low cost, low maintenance combined with high mean metabolite recovery (76–99%), specificity, accuracy (0–10% bias) and reproducibility (2–9% C.V.) make this method ideal for large high through-put studies.     

Enantiomeric analysis of anatabine, nornicotine and anabasine in commercial tobacco by multi-dimensional gas chromatography and mass spectrometry

A fully automated multi-dimensional gas chromatography (MDGC) system with a megabore precolumn and cyclodextrin-based analytical column was developed to analyze the enantiomeric compositions of anatabine, nornicotine and anabasine in commercial tobacco. The enantiomer abundances of anatabine and nornicotine varied among different tobacco. S-(-)-anatabine, as a proportion of total anatabine, was 86.6% for flue-cured, 86.0% for burley and 77.5% for oriental tobacco. S-(-)-nornicotine, as a proportion of total nornicotine, was 90.8% in oriental tobacco and higher than in burley (69.4%) and flue-cured (58.7%) tobacco. S-(-)-anabasine, as a proportion of total anabasine, was relatively constant for flue-cured (60.1%), burley (65.1%) and oriental (61.7%) tobacco. A simple solvent extraction with dichloromethane followed by derivatisation with trifluoroacetic anhydride gave relative standard deviations of less than 1.5% for the determination of the S-(-)-isomers of all three alkaloids. The study also indicated that, a higher proportion of S-(-)-nornicotine is related to the more active nicotine demethylation in the leaf.     

The alkaloid contents of sixty Nicotiana species

The alkaloid content (nicotine, nornicotine, anabasine, and anatabine) in leaves and roots of 60 Nicotiana species were analyzed by GC. All species contained alkaloids, the amounts varying with the species. There was no clearcut correlation between alkaloid amounts and subgeneric or sectional classification. The alkaloid content in the floral parts and immature and mature fruits of Nicotiana tabacum were also analyzed.     

The incorporation of [5,6-13C2]Nicotinic acid into the tobacco alkaloids examined by the use of 13C nuclear magnetic resonance

[5,6-¹⁴C,¹³C₂]Nicotinic acid was prepared from [¹⁴C,¹³C]methyl iodide via nitromethane, 2-nitroacetaldehyde oxime, 3-nitroquinoline, 3-aminoquinoline, and quinoline in 20% overall yield. Administration of this material to Nicotiana tabacum and N. glauca afforded labeled anabasine, anatabine, nicotine, and nornicotine. Qualitative and quantitative incorporation (0.07–4.5% specific incorporation) was determined by radioactive assay and by examination of the ¹³C NMR spectra of these alkaloids. Satellites due to spin-spin coupling of the incorporated contiguous ¹³C atoms were observed at the resonances due to C-5 and C-6 in anabasine, nicotine, and nornicotine. In anatabine, satellites were found at C-5, C-6, C-5′, and C-6′.     

Correlation between the nicotine content of tobacco plants and callus cultures

Callus cultures of two low-alkaloid lines of Nicotiana tabacum L. had considerably lower nicotine contents than cultures from the respective highalkaloid cultivars which were isogenic except for the two loci for alkaloid accumulation. Thus, there was a strong correlation between the nicotine content of callus cultures and the plants from which they were derived.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine