Myosmine

LC/MS analysis of three-dimensional model cells exposed to cigarette smoke or aerosol from a novel tobacco vapor product.[Pubmed:33268677]

J Toxicol Sci. 2020;45(12):769-782.

A novel tobacco vapor product (NTV) contains tobacco leaves and generates nicotine-containing aerosols using heating elements. Subchronic biological effects have been evaluated previously using three-dimensional bronchial epithelial model cells by repeated exposure to cigarette smoke (CS) and the NTV aerosols; however, the intracellular exposure characteristics have not been studied in detail. In this study, cells were initially exposed to an aqueous extract (AqE) of cigarette smoke (CS) at two concentration levels, and the cell lysate underwent untargeted analysis by LC-high resolution mass spectrometry to determine the exogenous compounds present in the cells. Among the thousands of peaks detected, four peaks showed a CS-dependency, which were reproducibly detected. Two of the peaks were nicotine and nicotine N-oxide, and the other two putative compounds were Myosmine and norharman. The cells were then exposed to an AqE of CS in various combinations of exposure and post-exposure culture durations. Post-exposure culturing of cells with fresh medium markedly decreased the peak areas of the four compounds. The in-vitro switching study of CS to NTV aerosols was investigated by intermittently exposing cells to an AqE of CS four times, followed by exposure to either an AqE of CS, NTV aerosol or medium another four times. Switching to NTV reduced Myosmine and norharman levels, which are known CS constituents. The results indicate that extracellular compounds inside cells reflect the exposure state outside cells. Thus, monitoring functional changes to cells in these exposure experiments is feasible.

Analysis of Nicotine and Non-nicotine Tobacco Constituents in Aqueous Smoke/Aerosol Extracts by UHPLC and Ultraperformance Convergence Chromatography-Tandem Mass Spectrometry.[Pubmed:33226218]

Chem Res Toxicol. 2020 Nov 23.

The non-nicotine constituents of tobacco may alter the reinforcing effects of nicotine, but the quantitative and qualitative profiles of these chemicals in tobacco products such as electronic cigarettes (e-cigarettes), cigars, and waterpipe tobacco are not well characterized. The objective of this work was to develop and validate analytical methods to utilize saline both as an extraction solvent for smoke condensates from cigarettes, little cigars, and waterpipe tobacco and aerosols from e-cigarettes and as a delivery vehicle of nicotine and non-nicotine constitents for nonclinical pharmacological studies. Ultrahigh-performance liquid chromatography was used to analyze nicotine and acetaldehyde, and a novel ultraperformance convergence chromatography-tandem mass spectrometry method was developed to analyze anabasine, anatabine, cotinine, Myosmine, nornicotine, harmane, and norharmane. Linearity was confirmed for each standard curve with correlation coefficients (r) >/= 0.99, and relative errors (RE) for the standards were /= 80%) indicated that the saline formulations of all four products could be considered stable for up to approximately 45 days at 4-8 degrees C. Therefore, the use of saline both as an extraction solvent and as a delivery vehicle adds versatility and improved performance in the study of the pharmacological effects of constituents from mainstream smoke and aerosols generated from cigarettes, little cigars, waterpipes, and e-cigarettes.

Alkaloid chemophenetics and transcriptomics of the Nicotiana genus.[Pubmed:32526514]

Phytochemistry. 2020 Sep;177:112424.

In this study, we determined the pyridine alkaloid content (nicotine, nornicotine, anabasine, anatabine, cotinine, and Myosmine) of 58 species and 2 subspecies of the Nicotiana genus by ultra-high-performance liquid chromatography coupled with mass spectrometry. We observed clear correlation between Noctiflorae and Suaveolentes sections and their above average accumulation of anabasine in the genus. In addition, the results demonstrated the presence of not only trace amounts but quantifiable levels of Myosmine, an alkaloid previously detected in only minute quantities, in the leaves and roots of 16 species. In this study, analysis of gene expression of 58 species and 2 subspecies from the Nicotiana genus by mRNA sequencing was performed for the first time. Sequencing reads were mapped against annotated genes of a Nicotiana tabacum reference genome and expression values were subsequently calculated. Hierarchical clustering of alkaloid biosynthesis pathway genes and alkaloid content composition revealed patterns clearly segregating Nicotiana sections. Correlation of gene expression with alkaloid accumulation phenotypes was evident, including low putrescine methyltransferase expression for all species in the Suaveolentes section or clear correlation of nicotine demethylase with conversion rates of nicotine to nornicotine in the majority of species. Multiple additional correlations between alkaloid accumulation and gene expression values were identified, which makes this study an important fundament toward future scientific exploration of the Nicotiana genus.

Simultaneous determination of six alkaloids in tobacco and tobacco products by direct analysis of real-time triple quadrupole mass spectrometry with a modified pretreatment method.[Pubmed:32034866]

J Sep Sci. 2020 Apr;43(8):1603-1613.

In order to determine six alkaloids (mass fraction) of nicotine, nornicotine, Myosmine, anatabine, anabasine, and nicotyrine in tobacco and tobacco products quickly, accurately, and simultaneously, a novel method based on direct analysis of real-time model in situ ionization technique combined tandem mass spectrometry with a modified sample pretreatment was established, in which experimental parameters such as the type and amount of extraction solvent and injection rate were optimized, respectively. The samples of five commercial cigarettes and five kinds of tobacco leaves were analyzed by the established method, and the determined values were compared with those obtained using a gas chromatography with mass spectrometry method: (1) Under optimized conditions (30 mL ultrapure water as extraction solvent and with extraction rate of 0.6 mm/s), analysis could be completed within 10 min. (2) The linear range of the method was 0.002-2000 mug/g with R 2 = 0.9957 , the recovery ranged from 86.8 to 105.6%, and the limit of detection and the limit of quantification were 0.004-0.835 mug/g and 0.013-2.787 mug/g, respectively. (3) The relative standard deviation between direct analysis of real-time method and the gas chromatography with mass spectrometry method was 0.34-8.83%. The established method is rapid, reliable, and suitable for the ultrafast determination of six alkaloids in tobacco and tobacco products.

Investigation of the Possible Pharmacologically Active Forms of the Nicotinic Acetylcholine Receptor Agonist Anabaseine.[Pubmed:31671780]

Mar Drugs. 2019 Oct 29;17(11). pii: md17110614.

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 [(3)H]-methylcarbamylcholine binding at rat brain alpha4beta2 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 Myosmine to activate a mammalian GABAA receptor, but no activity was detected. We conclude that the monocationic cyclic iminium is the form which avidly binds and activates vertebrate nAChRs.

The Use of HPLC-PDA in Determining Nicotine and Nicotine-Related Alkaloids from E-Liquids: A Comparison of Five E-Liquid Brands Purchased Locally.[Pubmed:31438499]

Int J Environ Res Public Health. 2019 Aug 21;16(17). pii: ijerph16173015.

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 (p < 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.

Exploring Matrix Effects on Binding Properties and Characterization of Cotinine Molecularly Imprinted Polymer on Paper-Based Scaffold.[Pubmed:30960554]

Polymers (Basel). 2019 Mar 26;11(3). pii: polym11030570.

Commercially available sorbent materials for solid-phase extraction are widely used in analytical laboratories. However, non-selective binding is a major obstacle for sample analysis. To overcome this problem, molecularly imprinted polymers (MIPs) were used as selective adsorbent materials prior to determining target analysts. In this study, the use of non-covalent molecularly imprinted polymers (MIPs) for cotinine adsorption on a paper-based scaffold was studied. Fiberglass paper was used as a paper scaffold for cotinine-selective MIP adsorption with the use of 0.5% agarose gel. The effects of salt, pH, sample matrix, and solvent on the cotinine adsorption and extraction process were investigated. Under optimal conditions, the adsorption isotherm of synthesized MIPs increased to 125.41 microg/g, whereas the maximum adsorption isotherm of non-imprinted polymers (NIPs) was stable at 42.86 microg/g. The ability of the MIP paper scaffold to absorb cotinine in water medium was approximately 1.8(-)2.8-fold higher than that of the NIP scaffold. From Scatchard analysis, two dissociation constants of MIPs were calculated to be 2.56 and 27.03 microM. Nicotine, Myosmine, and N-nitrosonornicotine were used for selectivity testing, and the calculated selectivity factor of cotinine to nicotine, Myosmine, and N-nitrosonornicotine was 1.56, 2.69, and 2.05, respectively. Overall, the MIP paper scaffold is promising for simple onsite sampling of cotinine and can be used to assess tobacco smoke exposure.

Gas Chromatography-Mass Spectrometry Method for Simultaneous Detection of Nine Alkaloids in Tobacco and Tobacco Products by QuEChERS Sample Preparation.[Pubmed:30930354]

Anal Sci. 2019 Aug 10;35(8):849-854.

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, beta-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 mug/g and limit of quantification were among 0.0217 - 0.5031 mug/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.

Computational Insight into the Activation Mechanism of Carcinogenic N'-Nitrosonornicotine (NNN) Catalyzed by Cytochrome P450.[Pubmed:30209943]

Environ Sci Technol. 2018 Oct 16;52(20):11838-11847.

Tobacco-specific N'-nitrosonornicotine (NNN), a genotoxic nitrosamine classified as Group 1 carcinogen, is also present in atmospheric particulate matter and has even been detected as a new disinfection byproduct in wastewaters. NNN generally requires metabolic activation by cytochrome P450 enzymes to exert its genotoxicity, but the respective biotransformation pathways have not been described in detail. In this work, we performed density functional theory (DFT) calculations to unravel possible NNN activation pathways including alpha-hydroxylation, beta-hydroxylation, pyridine N-oxidation, and norcotinine formation. The results reveal an initial rate-determining Halpha-atom abstraction step for alpha-hydroxylation, followed by an unexpected kinetic competition between denitrosation and OH rebound, leading to ( iso-)Myosmine as a detoxified product and alpha-hydroxyNNNs as the precursor of carcinogenic diazohydroxides, respectively. Further detoxification routes are given by beta-hydroxylation with relative high reaction barrier and N-oxidation with comparable barrier to the toxifying alpha-hydroxylation. Moreover, we show for the first time how norcotinine can be generated as a minor NNN metabolite that is formed from iso-Myosmine through a unique porphyrin-assisted H atom 1,2-transfer mechanism. These results demonstrate that the carcinogenic potential of NNN is subject to a kinetic competition between activating and deactivating metabolic routes, and identify respective biomarkers to inform about the individual risk associated with NNN exposure.

Simultaneous determination of tobacco minor alkaloids and tobacco-specific nitrosamines in mainstream smoke by dispersive solid-phase extraction coupled with ultra-performance liquid chromatography/tandem orbitrap mass spectrometry.[Pubmed:29964303]

Rapid Commun Mass Spectrom. 2018 Oct 30;32(20):1791-1798.

RATIONALE: The minor alkaloids in tobacco play an important role in the chemical composition of cigarette smoke, and they are precursors of tobacco-specific nitrosamines (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT)). NNN and NNK are classified as group 1 carcinogens. A method quantitating both tobacco minor alkaloids and tobacco-specific nitrosamines in mainstream smoke has not been reported. METHODS: Tobacco minor alkaloids and tobacco-specific nitrosamines in cigarette mainstream smoke were extracted by sonication. The extract was cleaned up by dispersive solid-phase extraction, and separation was achieved via ultra-performance liquid chromatography/tandem orbitrap mass spectrometry. RESULTS: The method was validated by analysis of six replicate samples spiked with three levels of the analyses. The mean recoveries for the six replicates were from 84.7% to 118% with less than 15% relative standard deviation except Myosmine at the low spiked level and the calculated detection limits were 0.066 to 13.2 ng/cig, respectively. The mean concentrations of nicotyrine, anabasine, nornicotine, anatabine, Myosmine, 2,3-bipyridine, cotinine, nicotelline, N-formylnornicotine, NNK, NNN, NAB and NAT in 30 different brands of commercial cigarette smoke under the ISO smoking regimen were 2.50 mug/cig, 2.34 mug/cig, 3.21 mug/cig, 5.78 mug/cig, 2.83 mug/cig, 1.05 mug/cig, 1.55 mug/cig, 0.55 mug/cig, 2.48 mug/cig, 6.06 ng/cig, 3.62 ng/cig, 0.40 ng/cig and 6.15 ng/cig, respectively. CONCLUSIONS: The proposed method was suitable for analysis of tobacco minor alkaloids and tobacco-specific nitrosamines in cigarette mainstream smoke.

Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.[Pubmed:29812904]

Biochemistry. 2018 Jul 3;57(26):3741-3751.

Nicotine oxidoreductase (NicA2) is a bacterial flavoenzyme, which catalyzes the first step of nicotine catabolism by oxidizing S-nicotine into N-methyl-Myosmine. It has been proposed as a biotherapeutic for nicotine addiction because of its nanomolar substrate binding affinity. The first crystal structure of NicA2 has been reported, establishing NicA2 as a member of the monoamine oxidase (MAO) family. However, substrate specificity and structural determinants of substrate binding and/or catalysis have not been explored. Herein, analysis of the pH-rate profile, single-turnover kinetics, and binding data establish that pH does not significantly affect the catalytic rate and product release is not rate-limiting. The X-ray crystal structure of NicA2 with S-nicotine refined to 2.65 A resolution reveals a hydrophobic binding site with a solvent exclusive cavity. Hydrophobic interactions predominantly orient the substrate, promoting the binding of a deprotonated species and supporting a hydride-transfer mechanism. Notably, NicA2 showed no activity against neurotransmitters oxidized by the two isoforms of human MAO. To further probe the substrate range of NicA2, enzyme activity was evaluated using a series of substrate analogues, indicating that S-nicotine is the optimal substrate and substitutions within the pyridyl ring abolish NicA2 activity. Moreover, mutagenesis and kinetic analysis of active-site residues reveal that removal of a hydrogen bond between the pyridyl ring of S-nicotine and the hydroxyl group of T381 has a 10-fold effect on KM, supporting the role of this bond in positioning the catalytically competent form of the substrate. Together, crystallography combined with kinetic analysis provides a deeper understanding of this enzyme's remarkable specificity.

Nicotine alkaloid levels, and nicotine to nornicotine conversion, in Australian Nicotiana species used as chewing tobacco.[Pubmed:29264422]

Heliyon. 2017 Dec 1;3(11):e00469.

A range of endemic Nicotiana species are chewed as a smokeless tobacco by several Aboriginal populations of Australia. In tobacco research, nicotine to nornicotine conversion is important because nornicotine lowers tobacco quality and is detrimental to health. A diverse group of cytochrome P450 genes with different transcriptional regulations are involved in this conversion. The primary aims of this study were to quantify the pyridine alkaloids and investigate nicotine to nornicotine conversion in laboratory-grown Australian Nicotiana spp. Nicotine, nornicotine, anatabine, anabasine, Myosmine and cotinine were quantified in fresh leaves of 24 out of the 26 recognised Australian Nicotiana taxa. Conserved regions of CYP82E related genes were PCR amplified in all studied taxa. The conversion process in fresh leaves was compared with that in leaves that underwent a simulated curing process for species that we identified as being high converters (N. cavicola, N. goodspeedii, N. velutina) and low converters (N. benthamiana, N. excelsior, N. gossei). Agarose gel electrophoretic analysis of CYP82E related genes obtained from the PCR amplification of the cDNA in fresh versus leaves with simulated curing showed about a 3-fold increase in transcript accumulation levels in cured leaves of the high converter species, while the transcript accumulation in N. gossei and N. excelsior maintained a steady basal level and increased by a small amount in N. benthamiana. This suggests the presence of functional loci that are triggered by curing in only high converter species and indicates a potential risk for chewers of high converter species.

Photochemical Transformation of Nicotine in Wastewater Effluent.[Pubmed:28942634]

Environ Sci Technol. 2017 Oct 17;51(20):11718-11730.

Nicotine is a highly toxic tobacco alkaloid that is ubiquitous in wastewater effluent. For the first time, we report the identification of the products and the pathways for the photodegradation of nicotine in an effluent matrix under simulated solar irradiation. Nicotine was found to be degraded by triplet-state organic matter ((3)OM*), thus indicating that electron transfer is a preferred reaction mechanism. Using the multivariate statistical strategies orthogonal projection to latent structures discriminant analysis (OPLS-DA) and hierarchical clustering, 49 potential transformation products (TPs) of nicotine were successfully extracted from the water matrix via high-resolution ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). Overall, 30 TPs, including 4 groups of nonseparated isomeric photo TPs, were identified with various levels of confidence based on the tandem mass spectrometry information on standard compounds and the isotope-labeling method (using rac-nicotine-2',3',3'-D3, rac-nicotine-(13)CD3, and rac-nicotine-D4) under air-saturated conditions. The pyrrolidine ring of nicotine was found to be the reactive site under sunlight irradiation. Pseudooxynicotine was the main primary TP from nicotine, with a maximum transformation ratio of 64%. Nicotinic acid, cotinine, 3'-hydroxycotinine, and Myosmine were the final stable TPs after 72 h of solar irradiation, with yields of 13%, 3%, 5%, and 5%, respectively.

Tobacco's minor alkaloids: Effects on place conditioning and nucleus accumbens dopamine release in adult and adolescent rats.[Pubmed:28844873]

Eur J Pharmacol. 2017 Nov 5;814:196-206.

Tobacco products are some of the most commonly used psychoactive drugs worldwide. Besides nicotine, alkaloids in tobacco include cotinine, Myosmine, and anatabine. Scientific investigation of these constituents and their contribution to tobacco dependence is less well developed than for nicotine. The present study evaluated the nucleus accumbens dopamine-releasing properties and rewarding and/or aversive properties of nicotine (0.2-0.8mg/kg), cotinine (0.5-5.0mg/kg), anatabine (0.5-5.0mg/kg), and Myosmine (5.0-20.0mg/kg) through in vivo microdialysis and place conditioning, respectively, in adult and adolescent male rats. Nicotine increased dopamine release at both ages, and anatabine and Myosmine increased dopamine release in adults, but not adolescents. The dopamine release results were not related to place conditioning, as nicotine and cotinine had no effect on place conditioning, whereas anatabine and Myosmine produced aversion in both ages. While the nucleus accumbens shell is hypothesized to play a role in strengthening drug-context associations following initiation of drug use, it may have little involvement in the motivational effects of tobacco constituents once these associations have been acquired. Effects of Myosmine and anatabine on dopamine release may require a fully developed dopamine system, since no effects of these tobacco alkaloids were observed during adolescence. In summary, while anatabine and Myosmine-induced dopamine release in nucleus accumbens may play a role in tobacco dependence in adults, the nature of that role remains to be elucidated.

Liquid chromatography with tandem mass spectrometry method for the determination of nicotine and minor tobacco alkaloids in electronic cigarette refill liquids and second-hand generated aerosol.[Pubmed:28012240]

J Sep Sci. 2017 Mar;40(5):1049-1056.

A liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of nicotine and seven minor tobacco alkaloids in both refill liquids for electronic cigarettes and their generated aerosol was developed and validated. The limit of detection and limit of quantification values were 0.3-20.0 and 1.0-31.8 ng/mL, respectively. Within-laboratory reproducibility was 8.2-14.2% at limit of quantification values and 4.8-12.7% at other concentration levels. Interday recovery was 75.8-116.4%. The method was applied to evaluate the compliance of commercial liquids (n = 95) with their labels and to assess levels of minor alkaloids. Levels of nicotine and its corresponding compounds were also evaluated in generated aerosol. About 47% of samples showed differences above +/-10 % of the stated nicotine concentration. About 78% of the "zero nicotine" liquids showed traces in the range of 1.3 +/- 0.1-254.0 +/- 14.6 mug/mL. Nicotine-N'-oxides, Myosmine, and anatabine were the most common minor alkaloids in liquids containing nicotine. Nicotine and N'-oxides were detected in all air samples when aerosol was generated from liquids containing nicotine. Nicotine average emissions from electronic cigarette (2.7 +/- 0.9 mug/m(3) ) were significantly lower (p < 0.01, t-test) with respect to conventional cigarette (30.2 +/- 1.5 mug/m(3) ).