Comparison of cytotoxicity of cigarette smoke extract derived from heat-not-burn and combustion cigarettes in human vascular endothelial cells.

The present study compared the properties of mainstream smoke generated from heat-not-burn (HNB) cigarettes and a combustion cigarette (hi-lite™ brand). Three types of cigarette heating devices were used to generate cigarette smoke at different heating temperatures [Ploom S™ (200 °C), glo™ (240 °C), and IQOS™ (300-350 °C)]. Mainstream smoke was generated using the following puffing regimen: volume, 55 mL; duration, 3 s; and interval, 30 s. The rank order of particulate phase (nicotine and tar) amounts trapped on a Cambridge filter was Ploom S < glo < IQOS < hi-lite. Heated cigarette-derived smoke extract (hCSE) from the devices except for Ploom S, and burned CSE (bCSE) decreased mitochondrial metabolic activity (glo < IQOS < hi-lite) in human vascular endothelial cells. Furthermore, the cytotoxicity was reduced by removing the particulate phase from the mainstream smoke. Endothelial nitric oxide synthase activity was reduced by nicotine- and tar-free CSE of IQOS and hi-lite (IQOS < hi-lite), but not Ploom S and glo. These inhibitory effects were diminished by removing the carbonyl compounds from the mainstream smoke. These results indicated that the cytotoxicity of hCSE was lower than that of bCSE in vascular endothelial cells.

Facile Fabrication of a Functional Filter Tip for Highly Efficient Reduction of Nicotine Content in Mainstream Smoke.

The nicotine addiction problem is of great concern, particularly in adolescents. Notably, nicotine addiction drives humans to continue smoking. Notably, several diseases and disorders are caused by smoking. To date, various adsorbents have been proposed to develop a functionalization filter tip for reducing nicotine content in mainstream smoke. However, the nicotine adsorption efficiencies of most of the reported functionalization filter tips were not satisfactory, and their preparation process was complex and time-consuming. Herein, we demonstrate a highly active and adsorbing filter tip for cigarettes, fabricated by decorating polydopamine (PDA) on the surface of a commercial filter tip in situ. The PDA coating on the filter tip was obtained by the self-polymerization of dopamine (DA) within 16 h, which was quicker and easier than the preparation processes of other reported functionalized filter tips. Significantly, the PDA-decorated filter tip had a nicotine adsorption efficiency as high as ∼95%, which was much higher than most of the commercial filter tips.

Optimization of MAE for the Separation of Nicotine and Phenolics from Tobacco Waste by Using the Response Surface Methodology Approach.

This study intends to valorize by-products of the industrial processing of tobacco to obtain nicotine and phenolics as value-added compounds. Three influential parameters of the microwave-assisted extraction-MAE (temperature, treatment time, and solvent/solid ratio) were studied for the optimization of the extraction protocol for tobacco leaves and three types of waste-scrap, dust, and midrib, respectively. Nicotine was the dominant bioactive compound in all extracts, ranging from 1.512 to 5.480% in leaves, 1.886 to 3.709% in scrap, 2.628 to 4.840% dust, and 0.867 to 1.783% in midrib extracts. Five phenolic compounds were identified and quantified, predominated by chlorogenic acid and rutin. Additionally, total phenol content and antioxidant activity were determined using spectrophotometric assays. Optimization was performed in two aspects: to obtain a maximum extraction yield with minimum nicotine content and to obtain a maximum extraction yield with maximum nicotine content. These findings demonstrate that tobacco waste is a valuable source of bioactive compounds and MAE can be a promising alternative technique to obtain extracts rich in targeted bioactive compounds, especially nicotine.

Pyridine alkaloids with activity in the central nervous system.

This review discusses all pyridine alkaloids with CNS activity, their therapeutic potential, and the interesting array of sources whence they originate.

Biocompatible SPME fibers for direct monitoring of nicotine and its metabolites at ultra trace concentration in rabbit plasma following the application of smoking cessation formulations.

The ultra-trace determination of nicotine and its 4 major metabolites (cotinine, nornicotine, norcotinine and anabasine) from rabbit plasma was achieved by a newly developed solid phase microextraction-liquid chromatography-tandem mass spectrometry method. Extraction of the target analytes was performed with hydrophilic/lipophilic balance-polyacrylonitrile SPME fibers. Dual fiber extraction was necessary to guarantee improved recovery at parts-per-trillion levels. Liquid chromatographic analysis was achieved in a 6-min run using a C18 (1.9 µm C18, 50 mm x 2.1 mm) column with a mobile phase flow rate of 0.4 mL/min. Tandem mass spectrometry was used for detection and quantification in positive electrospray ionization (ESI+) mode for all the targeted analytes. Two stable isotope-labeled internal standards were used for signal correction and accurate quantification. The mass spectrometer with laminar flow ion flux transport, guaranteed improved signal stability, minimal contamination of the ion guide and reproducibility into the first quadrupole analyzer. The method was validated in line with the Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The results met the acceptance criteria as proposed by the FDA: accuracy was tested at 0.35, 10 and 75 µg L - 1 and ranged between 98.3-112.2% for nicotine, 94.1-101.9% for cotinine, 94.7-107.0% for nornicotine, 81.1-107.2% for norcotinine and 94.3-115.2% for anabasine, with precision up to 14.2%. Stability tests indicated that all the targeted analytes were stable in the desorption solution for at least 1 week. LOQs ranged from 0.05 to 1 µg L-1. The method was successfully applied to analyze plasma samples obtained from rabbits following transdermal application of a smoking cessation formulation loaded with solid lipid nanoparticles containing a nicotine-stearic acid conjugate.

High throughput bar adsorptive microextraction: A simple and effective analytical approach for the determination of nicotine and cotinine in urine samples.

A simple, effective, convenient and environmentally friendly methodology using high throughput bar adsorptive microextraction (HT-BAµE) with microliquid desorption in combination with large volume injection-gas chromatography-mass spectrometry operating in the selected-ion monitoring acquisition mode (LVI-GC-MS(SIM)) was applied for the determination of nicotine and cotinine in urine samples. Under optimized experimental conditions, the developed methodology allowed for linear dynamic ranges between 20.0 and 2000.0 µg L-1 with determination coefficients of 0.9991 and 0.9992, as well as average recovery yields of 61.7-67.5% and 53.9-57.8% for nicotine and cotine, respectively. The developed methodology was applied to monitor urine samples from 86 volunteers having different smoking habits, where nicotine and cotinine were quantified in the range from 23.6 to 2612.6 µg L-1. The target compounds were extracted in a HT-BAµE apparatus, which allows for simultaneous microextraction and subsequent back-extraction of up to 100 samples. This is a major improvement over other microextraction techniques. The data from the proposed methodology were satisfactory and in line with current green analytical chemistry guidelines, and proved to be an effective sample preparation alternative with substantial potential for high throughput bioanalysis.

Dye-Modified Metal-Organic Framework as a Recyclable Luminescent Sensor for Nicotine Determination in Urine Solution and Living Cell.

A water-stable and pH-independent sensor for qualitative and quantitative detection of nicotine in urine solution and living cell was successfully developed. This material, named MB@UiO-66-NH2, can be synthesized by encapsulating methylene blue (MB) with a well-known metal-organic framework (MOF) UiO-66-NH2 through a simple impregnation method. The fluorescence intensity of the system was significantly enhanced when a certain amount of nicotine was added. In the meanwhile, MB is reduced by reductive nicotine to form leucomethylene blue (LB). The proposed sensor displayed excellent selectivity and sensitivity toward nicotine with limit of detection (LOD) of 0.98 µM, which is comparable or even better than that of the electrochemistry detecting methods for nicotine. The obvious enhancement and blue shift of the emission arise from the photoinduced electron transfer (PET) from LB to the UiO-66-NH2. The photophysical properties and the sensing applications of MB@UiO-66-NH2 suggest that this composite can be acted as a sensitive, selective, recyclable, and fluorogenic sensor for nicotine determination in urine solution and living cell.

Nicotinamide adenine dinucleotide immobilized tungsten trioxide nanoparticles for simultaneous sensing of norepinephrine, melatonin and nicotine.

Herein, we report the anionic surfactant, ethylene diamine tetraacetic acid (EDTA), mediated synthesis of WO3 nanoparticles and its subsequent modification through gamma irradiation (GI) and electrochemical immobilization with nicotinamide adenine dinucleotide (NAD). Glassy carbon electrode (GCE) modified with GI-WO3 NPs and the enzyme NAD exhibited strong electro-oxidation of three important biomolecules such as norepinephrine (NEP), melatonin (MEL) and nicotine (NIC) in 0.1 M phosphate buffer saline (PBS) at physiological pH of 7. Square wave voltammetry (SWV) studies exhibited three well-defined peaks at potentials of 120, 570 and 840 mV, corresponding to the oxidation of NEP, MEL and NIC respectively, indicating that simultaneous determination of these compounds is feasible at the NAD/GI EDTA-WO3/GCE. The proposed sensor displayed a wide linear range of 0.010-1000 µM with the lowest detection limit of 1.4 nM for NEP, 2.6 nM for MEL and 1.7 nM for NIC respectively. Furthermore, the modified electrode was successfully applied to detect NEP, MEL and NIC in pharmaceutical and cigarette samples with excellent selectivity and reproducibility.

Comparison of the Conventional and Electroenhanced Direct-Immersion Solid-Phase Microextraction for Sampling of Nicotine in Biological Fluids of the Human Body.

A stainless steel fiber was made porous and adhesive by platinization and then coated by nanostructured polypyrrole (PPy), using an appropriate electrophoretic deposition (EPD) method. The morphological surface structure and functional groups of the PPy-coated fiber were studied using SEM (Scanning electron microscope) instrument. The prepared fiber was used for comparison of direct immersion (DI) and electroenhanced direct immersion solid-phase microextraction (EE-DI-SPME) of nicotine in human plasma and urine samples followed by gas chromatography flame ionization detector (GC-FID) determination. The effects of the influential experimental parameters on the efficiency of the DI-SPME and EE-DI-SPME methods, including the pH and ionic strength of the sample solution, applied Direct current (DC) voltage, extraction temperature and time and stirring rate, were optimized. Under the optimal conditions, the calibration curves for the DI-SPME-GC-FID and EE-DI-SPME-GC-FID methods were linear over the ranges of 0.1⁻10.0 µg mL-1 and 0.001⁻10.0 µg mL-1, respectively. The relative standard deviations (RSDs, n = 6) were found to be 6.1% and 4.6% for the DI and EE strategies, respectively. The LODs (limit of detection) of the DI-SPME-GC-FID and EE-DI-SPME-GC-FID methods were found to be 10 and 0.3 ng mL-1, respectively. The relative recovery values (for the analysis of 1 µg mL-1 nicotine) were found to be 91⁻110% for EE-DI-SPME and 75⁻105% for DI-SPME. The enrichment factors for DI-SPME and EE-DI-SPME sampling were obtained as 38,734 and 50,597, respectively. The results indicated that EE-SPME was more efficient for quantitation of nicotine in biological fluids. The developed procedure was successfully carried out for the extraction and measurement of nicotine in real plasma and urine samples.

Nicotine and methyl vinyl ketone, major components of cigarette smoke extracts, increase protective amyloid-ß peptides in cells harboring amyloid-ß precursor protein.

The increased ratio of longer amyloid-ß (Aß1-42)/shorter amyloid-ß (Aß1-40) peptides, generated from amyloid precursor protein (APP), is known to promote the development of Alzheimer's disease (AD). To investigate the role of smoking in Aß production, we determined the production of Aß species in the presence of nicotine or methyl vinyl ketone (MVK), major components of cigarette smoke extracts, in Flp-In™ T-REx™-293 (T-REx293) cells harboring a single copy of human APP. While treatment with nicotine or MVK did not affect the amount of APP, the levels of Aß1-40 in the culture media were significantly increased. On the other hand, the levels of Aß1-42 were unaltered by nicotine or MVK treatment. The Aß1-42/Aß1-40 ratio was therefore attenuated by cigarette smoke extracts. Similar results were obtained in T-REx293 cells harboring APP of Swedish- or London-type mutation linked to familial AD. T-REx293 cells expressed the nicotinic acetylcholine receptor (nAchR) and tubocurarine, an nAChR antagonist, completely blocked the effects of nicotine. Treatment with nicotine significantly elevated cellular levels of ß-secretase that cleaves APP prior to Aß generation. Taken together, a protective role of nicotine against AD pathology was suggested by enhanced extracellular Aß1-40 production, which may suppress Aß fibrillogenesis.

Effect of nicotine from tobacco root exudates on chemotaxis, growth, biocontrol efficiency, and colonization by Pseudomonas aeruginosa NXHG29.

Accumulated evidence suggests that root exudates have a major role in mediating plant-microbe interactions in the rhizosphere. Here, we characterized tobacco root exudates (TREs) by GC-MS and nicotine, scopoletin, and octadecane were identified as three main components of TREs. Qualitative and quantitative chemotaxis assays revealed that Pseudomonas aeruginosa NXHG29 with antagonistic activity displayed positive chemotactic responses towards TREs and their three main components (nicotine, scopoletin, octadecane) and its enhanced chemotaxis were induced by these substances in a concentration-dependent manner. Furthermore, following GC-MS and chemotaxis analysis, nicotine was selected as the target for evaluation of the effect on NXHG29 regarding antagonism, growth, root colonization and biocontrol efficiency. Results of in vitro studies showed that nicotine as a sole carbon source could enhance growth of NXHG29 and significantly increased the antagonism of NXHG29. We also demonstrated that nicotine exerted enhancing effects on the colonization ability of NXHG29 on tobacco roots by combining CLSM observations with investigation of population level dynamics by selective dilution plating method. Results from greenhouse experiments suggested nicotine exhibited stimulatory effects on the biocontrol efficiency of NXHG29 against bacterial wilt and black shank on tobacco. The stimulatory effect of nicotine was affected by the concentration and timing of nicotine application and further supported by the results of population level of NXHG29 on tobacco roots. This is the first report on the enhancement effect of nicotine from TREs on an antagonistic bacterium for its root colonization, control of soil-borne pathogens, regarding the chemotaxis and in vitro antagonism and growth.

A comprehensive methodology for the chiral separation of 40 tobacco alkaloids and their carcinogenic E/Z-(R,S)-tobacco-specific nitrosamine metabolites.

The predominant enantiomer of nicotine found in nature is (S)-nicotine and its pharmacology has been widely established. However, pharmacologic information concerning individual enantiomers of nicotine-related compounds is limited. Recently, a modified macrocyclic glycopeptide chiral selector was found to be highly stereoselective for most tobacco alkaloids and metabolites. This study examines the semi-synthetic and native known macrocyclic glycopeptides for chiral recognition, separation, and characterization of the largest group of nicotine-related compounds ever reported (tobacco alkaloids, nicotine metabolites and derivatives, and tobacco-specific nitrosamines). The enantioseparation of nicotine is accomplished in less than 20s for example. All liquid chromatography separations are mass spectrometry compatible for the tobacco alkaloids, as well as their metabolites. Ring-closed, cyclized structures were identified and separated from their ring-open, straight chain equilibrium structures. Also, E/Z-tobacco-specific nitrosamines and their enantiomers were directly separated. E/Z isomers also are known to have different physical and chemical properties and biological activities. This study provides optimal separation conditions for the analysis of nicotine-related isomers, which in the past have been reported to be ineffectively separated which can result in inaccurate results. The methodology of this study could be applied to cancer studies, and lead to more information about the role of these isomers in other diseases and as treatment for diseases.

Análisis de aguas residuales con fines epidemiológicos: aplicaciones a la estimación del consumo de sustancias de abuso y en salud pública en general. Red española ESAR-Net / Wastewater-based epidemiology: applications towards the estimation of drugs of abuse consumption and public health in general. The Spanish network ESAR-Net

En este artículo se presenta la metodología de análisis de aguas residuales con fines epidemiológicos (wastewater-based epidemiology, WBE) y su potencial para abordar diversos aspectos relacionados con la salud pública. Esta metodología permite obtener datos a una escala temporal y espacial relativamente pequeña (típicamente datos diarios-semanales sobre un municipio) de hábitos de consumo de sustancias de abuso, ilegales (como la cocaína o el cannabis) o legales (como el alcohol) a través de la determinación de biomarcadores de consumo (el compuesto original no metabolizado o alguno de sus metabolitos) en el agua residual. Aparte de discutir los fundamentos, ventajas y limitaciones de WBE, se comentan los precedentes más relevantes a nivel internacional, y las actividades más destacables en España en este ámbito. Finalmente, se exponen, los objetivos de la Red Española de Análisis de Aguas Residuales con Fines Epidemiológicos (ESAR-Net), una "Red de Excelencia " que agrupa a investigadores españoles con amplia experiencia en el área de WBE, así como las perspectivas de futuro de esta metodología puede tener para mejorar las competencias de la Salud Pública en España

This manuscript introduces Wastewater-Based Epidemiology (WBE) and its potential in the assessment of diverse aspects related to public health. This methodology can provide data in a relatively short temporal and local scale (typically dialy-weekly at the municipal level) on consumption patterns of illicit drugs (e.g. cocaine or cannabis), licit substances of abuse (e.g. alcohol) by measuring their consumption biomarkers (i.e. the original unmetabolized substance or some of its metabolite) in wastewater. Besides discussing the fundaments, advantages and shortcomings of WBE, it reviews some of the main precedents at international level and most remarkable activities that have been taken place in this field in Spain. Finally, the Spanish Network of Wastewater-Based Epidemiology (ESAR-Net) as is presented. ESAR-Net is an Excellence Network that sums up the efforts of the most relevant Spanish researchers in the field of WBE, aiming to investigate future perspectives of this methodology and its impact on Public Health competences in Spain

Enantioseparation of nornicotine in tobacco by ultraperformance convergence chromatography with tandem mass spectrometry.

Nornicotine, an alkaloid constituent of tobacco, is a precursor to the carcinogen N-nitrosonornicotine that is produced during the curing and processing of tobacco. Accumulating evidence reveals that nornicotine enantiomers have different neurochemical and behavioral effects. In the present study, an accurate and rapid method was developed for the enantioseparation of (R)-(+)-nornicotine and (S)-(-)-nornicotine enantiomers in tobacco by ultra-performance convergence chromatography with tandem mass spectrometry. Chromatographic conditions were investigated to achieve the optimal resolution of two enantiomers. Results indicated that (R)-(+)-nornicotine and (S)-(-)-nornicotine could be separated within 5 min when ammonium hydroxide was added into the cosolvent, and the best resolution (Rs  = 4.76) was achieved on a immobilized cellulose tris-(3,5-dichlorophenylcarbamate) chiral stationary phase. The proposed method was validated and was finally applied to analyze the compositions of (R)-(+)-nornicotine and (S)-(-)-nornicotine in three typical types of tobaccos (flue-cured, burley, and oriental). It was found that, enantiomer fraction of nornicotine (the proportion of (S)-(-)-nornicotine in the nornicotine pool) in burley tobacco samples was relatively high and constant compared with flue-cured and oriental tobaccos. The effective and rapid enantioseparation of nornicotine may help the understanding of alkaloid metabolites in different tobacco varieties and may also benefit pharmacological studies of alkaloid enantiomers.

A high area, porous and resistant platinized stainless steel fiber coated by nanostructured polypyrrole for direct HS-SPME of nicotine in biological samples prior to GC-FID quantification.

The surface of a stainless steel fiber was made porous, resistant and cohesive using electrophoretic deposition and coated by the nanostructured polypyrrole using an amended in-situ electropolymerization method. The coated fiber was applied for direct extraction of nicotine in biological samples through a headspace solid-phase microextraction (HS-SPME) method followed by GC-FID determination. The effects of the important experimental variables on the efficiency of the developed HS-SPME-GC-FID method, including pH of sample solution, extraction temperature and time, stirring rate, and ionic strength were evaluated and optimized. Under the optimal experimental conditions, the calibration curve was linear over the range of 0.1-20µgmL-1 and the detection limit was obtained 20ngmL-1. Relative standard deviation (RSD, n=6) was calculated 7.6%. The results demonstrated the superiority of the proposed fiber compared with the most used commercial types. The proposed HS-SPME-GC-FID method was successfully used for the analysis of nicotine in urine and human plasma samples.

E-cigarette liquids: Constancy of content across batches and accuracy of labeling.

AIMS: To assess whether bottles of refill liquids for e-cigarettes were filled true to label, whether their content was constant across two production batches, and whether they contained impurities. METHODS: In 2013, we purchased on the Internet 18 models from 11 brands of e-liquids. We purchased a second sample of the same models 4months later. We analyzed their content in nicotine, anabasine, propylene glycol, glycerol, ethylene glycol and diethylene glycol, and tested their pH. RESULTS: The median difference between the nicotine value on the labels and the nicotine content in the bottles was 0.3mg/mL (range -5.4 to +3.5mg/mL, i.e. -8% to +30%). For 82% of the samples, the actual nicotine content was within 10% of the value on the labels. All models contained glycerol (median 407mg/mL), and all but three models contained propylene glycol (median 650mg/mL). For all samples, levels of anabasine, ethylene glycol and diethylene glycol were below our limits of detection. The pH of all the e-liquids was alkaline (median pH=9.1; range 8.1 to 9.9). The measured content of two batches of the same model varied by a median of 0% across batches for propylene glycol, 1% for glycerol, 0% for pH, and 0.5% for nicotine (range -15% to +21%; 5th and 95th percentiles: -15% and +10%). CONCLUSIONS: The nicotine content of these e-liquids matched the labels on the bottles, and was relatively constant across production batches. The content of propylene glycol and glycerol was also stable across batches, as was the pH.

Combination of Plant Metabolic Modules Yields Synthetic Synergies.

The great potential of pharmacologically active secondary plant metabolites is often limited by low yield and availability of the producing plant. Chemical synthesis of these complex compounds is often too expensive. Plant cell fermentation offers an alternative strategy to overcome these limitations. However, production in batch cell cultures remains often inefficient. One reason might be the fact that different cell types have to interact for metabolite maturation, which is poorly mimicked in suspension cell lines. Using alkaloid metabolism of tobacco, we explore an alternative strategy, where the metabolic interactions of different cell types in a plant tissue are technically mimicked based on different plant-cell based metabolic modules. In this study, we simulate the interaction found between the nicotine secreting cells of the root and the nicotine-converting cells of the senescent leaf, generating the target compound nornicotine in the model cell line tobacco BY-2. When the nicotine demethylase NtomCYP82E4 was overexpressed in tobacco BY-2 cells, nornicotine synthesis was triggered, but only to a minor extent. However, we show here that we can improve the production of nornicotine in this cell line by feeding the precursor, nicotine. Engineering of another cell line overexpressing the key enzyme NtabMPO1 allows to stimulate accumulation and secretion of this precursor. We show that the nornicotine production of NtomCYP82E4 cells can be significantly stimulated by feeding conditioned medium from NtabMPO1 overexpressors without any negative effect on the physiology of the cells. Co-cultivation of NtomCYP82E4 with NtabMPO1 stimulated nornicotine accumulation even further, demonstrating that the physical presence of cells was superior to just feeding the conditioned medium collected from the same cells. These results provide a proof of concept that combination of different metabolic modules can improve the productivity for target compounds in plant cell fermentation.

Fetal exposure to tobacco: nicotine and cotinine concentration in amniotic fluid and maternal saliva.

OBJECTIVE: Fetal exposure to tobacco constituents is a risk factor for negative birth outcomes. We aimed to determine the relationships between nicotine and cotinine concentrations in amniotic fluid and maternal saliva. METHODS: As part of a therapeutic trial, 42 pregnant smokers agreed to sample amniotic fluid (8 samples from amniocentesis, 34 at birth). Their smoking characteristics were collected along with the newborns' birth outcomes. RESULTS: The median concentrations [IQR] in amniotic fluid and saliva were 11 [7-31] and 38 [7-174] µg/L for nicotine and 72 [22-123] µg/L and 55 [17-109] µg/L for cotinine, respectively. Multivariate models showed that saliva cotinine concentration predicted amniotic fluid nicotine and cotinine concentrations (R2 = 0.398, p < 0.0001 and R2 = 0.708, p < 0.0001 respectively). Amniotic fluid nicotine or cotinine concentration was not associated with birth weight. In multivariate analysis, the time elapsed since the last cigarette was the only variable associated with increased birth weight (R2 = 0.237, p = 0.002). CONCLUSIONS: Maternal saliva sampling for the determination of cotinine concentration is of interest to monitor fetal exposure to nicotine of any origin. Nevertheless, the time elapsed since the last cigarette was a better predictor of birth weight than the biomarkers' concentrations in amniotic fluid or maternal saliva.

Biodegradation of lignin and nicotine with white rot fungi for the delignification and detoxification of tobacco stalk.

BACKGROUND: Tobacco stalk is one kind of abundant crop residues in China. The high lignification of tobacco stalk increases its reusing cost and the existing of nicotine will cause serious pollution. The biodegradation of lignocellulosic biomass has been demonstrated to be an environmental and economical approach for the utilization of plant stalk. Meanwhile, many nicotine-degrading microorganisms were found in nature. However, microorganisms which could degraded both nicotine and lignin haven't been reported. Therefore, it's imperative to find some suitable microorganisms to break down lignin and simultaneously remove nicotine in tobacco stalk. RESULTS: The nicotine in tobacco stalk could be degraded effectively by Trametes versicolor, Trametes hirsute and Phanerochaete chrysosporium. The nicotine content in tobacco stalk was lowered to below 500 mg/kg (a safe concentration to environment) after 10 days of fermentation with Phanerochaete chrysosporium and Trametes versicolor, and 15 days with Trametes hirsute. The degradation rate of lignin in the fermented tobacco stalk was 37.70, 51.56 and 53.75% with Trametes versicolor, Trametes hirsute and Phanerochaete chrysosporium, respectively. Meanwhile, 24.28% hemicellulose was degraded by Phanerochaete chrysosporium and 28.19% cellulose was removed by Trametes hirsute. Through the enzyme activity analysis, the main and highest ligninolytic enzymes produced by Phanerochaete chrysosporium, Trametes hirsute and Trametes versicolor were lignin peroxidase (88.62 U · L-1), manganese peroxidase (100.95 U · L-1) and laccase (745.65 U · L-1). Meanwhile, relatively high and stable cellulase activity was also detected during the fermentation with Phanerochaete chrysosporium, and the highest endoglucanase, exoglucanase and filter paper enzyme activities were 0.38 U · mL-1, 0.45 U · mL-1 and 0.35U · mL-1, respectively. Moreover, the products in the fermentation of tobacco stalk with P. chrysosporium were identified with GC-MS, besides the chemicals produced in the degradation of lignin and nicotine, some small molecular valuable chemicals and fatty acid were also detected. CONCLUSIONS: Our study developed a new method for the degradation and detoxification of tobacco stalk by fermentation with white rot fungi Phanerochaete chrysosporium and Trametes hirsute. The different oxidative enzymes and chemical products detected during the degradation indicated a possible pathway for the utilization of tobacco stalk.

Biodegradation and metabolic pathway of nicotine in Rhodococcus sp. Y22.

Nicotine in tobacco is harmful to health and the environment, so there is an environmental requirement to remove nicotine from tobacco and tobacco wastes. In this study, the biotransformation of nicotine by Rhodococcus sp. Y22 was investigated, and three metabolites (NIC1, NIC4 and NIC5) were isolated by column separation, preparative TLC and solid plate's method, respectively. NIC1 was identified as 6-hydoxynicotine based on the results of NMR, MS, HPLC-UV and HRESIMS analysis; NIC4 was a novel compound and identified as 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one based on the results of NMR, MS and UV analysis; NIC5 was identified as nicotine blue based on the results of NMR and MS analysis. Meanwhile, two metabolites NIC2 and NIC3 were identified as 6-hydroxy-N-methylmyosmine and 6-hydroxypseudooxynicotine by HRESIMS analysis, respectively. According to these metabolites, the possible pathway of nicotine degradation by Rhodococcus sp. Y22 was proposed. The nicotine can be transformed to nicotine blue through two pathways (A and B), and 6-hydroxy-N-methylmyosmine is the key compound, which can be converted to 6-hydroxypseudooxynicotine (pathway A) and 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one (pathway B), respectively. Moreover, the encoding gene of nicotine dehydrogenase, ndh, was amplified from Rhodococcus sp. Y22, and its transcriptional level could be up-regulated obviously under nicotine induction. Our studies reported the key metabolites and possible biotransformation pathway of nicotine in Rhodococcus sp. Y22, and provided new insights into the microbial metabolism of nicotine.