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Background: Several studies indicate that smoking is one of the major risk factors for bladder cancer. Nicotine and its metabolites, the main components of tobacco, have been found to be strongly linked to the occurrence and progression of bladder cancer. However, the function of nicotine metabolism-related genes (NRGs) in bladder urothelial carcinoma (BLCA) are still unclear. Methods: NRGs were collected from MSigDB to identify the clusters associated with nicotine metabolism. Prognostic differentially expressed genes (DEGs) were filtered via differentially expression analysis and univariate Cox regression analysis. Integrative machine learning combination based on 10 machine learning algorithms was used for the construction of robust signature. Subsequently, the clinical application of signature in terms of prognosis, tumor microenvironment (TME) as well as immunotherapy was comprehensively evaluated. Finally, the biology function of the signature gene was further verified via CCK-8, transwell migration and colony formation. Results: Three clusters associated with nicotine metabolism were discovered with distinct prognosis and immunological patterns. A four gene-signature was developed by random survival forest (RSF) method with highest average Harrell's concordance index (C-index) of 0.763. The signature exhibited a reliable and accurate performance in prognostic prediction across TCGA-train, TCGA-test and GSE32894 cohorts. Furthermore, the signature showed highly correlation with clinical characteristics, TME and immunotherapy responses. Suppression of MKRN1 was found to reduce the migration and proliferation of bladder cancer cell. In addition, enhanced migration and proliferation caused by nicotine was blocked down by loss of MKRN1. Conclusions: The novel nicotine metabolism-related signature may provide valuable insights into clinical prognosis and potential benefits of immunotherapy in bladder cancer patients. [ABSTRACT FROM AUTHOR]

There are several bioinformatics studies related to lung cancer, but most of them have mainly focused on either microarray data or RNA-Seq data alone. In this study, we have combined both types of data to identify differentially expressed genes (DEGs) specific to lung cancer subtypes. We obtained six microarray datasets from the GEO and also the expression matrix of LUSC and LUAD from TCGA, which were analyzed by GEO2R tool and GEPIA2, respectively. Enrichment analyses of DEGs were performed using the Enrichr database. Protein module identification was done by MCODE plugin in cytoscape software. We identified 30 LUAD-specific, 17 LUSC-specific, and 17 DEGs shared between LUAD and LUSC. Enrichment analyses revealed that LUSC-specific DEGs are involved in lung fibrosis. In addition, DEGs shared between LUAD and LUSC are involved in extracellular matrix (ECM), nicotine metabolism, and lung fibrosis. We identified lung fibrosis-related genes, including SPP1, MMP9, and CXCL2, involved in both LUAD and LUSC, but SERPINA1 and PLAU genes involved only in LUSC. We also found an important module separately for LUAD-specific, LUSC-specific, and shared DEGs between LUSC and LUAD. S100P, GOLM, AGR2, AK1, TMEM125, SLC2A1, COL1A1, and GHR genes were significantly associated with survival. Our findings suggest that different lung fibrosis-related genes may play roles in LUSC and LUAD. Additionally, nicotine metabolism and ECM remodeling were found to be associated with both LUSC and LUAD, regardless of subtype, emphasizing the role of smoking in the development of lung cancer and ECM in the high aggressiveness and mortality of lung cancer. [ABSTRACT FROM AUTHOR]

Cigarette smoking accelerates metabolic disorders and is highly addictive, posing challenges for cessation. Recent studies have proposed that nicotine accumulates in the intestines of smokers, where it interacts with gut microbiota and disrupts systemic metabolism. After smoking, significant alterations in the gut microbiota affect the synthesis of numerous metabolites that are crucial for the development and progression of metabolic disorders. Gut microbiota-mediated nicotine degradation has emerged as a promising therapeutic approach for smoking-related metabolic disorders associated with smoking. Herein, we explore the impact of smoking on the composition and function of the gut microbiota, emphasizing the potential link between smoking-related metabolic disorders and dysbiosis of the gut microbiota and microbial metabolites. Additionally, we discuss the in vivo distribution and metabolism of nicotine along with potential strategies for treating smoking-related metabolic disorders through the microbial degradation of nicotine.

BackgroundSeveral studies indicate that smoking is one of the major risk factors for bladder cancer. Nicotine and its metabolites, the main components of tobacco, have been found to be strongly linked to the occurrence and progression of bladder cancer. However, the function of nicotine metabolism-related genes (NRGs) in bladder urothelial carcinoma (BLCA) are still unclear.MethodsNRGs were collected from MSigDB to identify the clusters associated with nicotine metabolism. Prognostic differentially expressed genes (DEGs) were filtered via differentially expression analysis and univariate Cox regression analysis. Integrative machine learning combination based on 10 machine learning algorithms was used for the construction of robust signature. Subsequently, the clinical application of signature in terms of prognosis, tumor microenvironment (TME) as well as immunotherapy was comprehensively evaluated. Finally, the biology function of the signature gene was further verified via CCK-8, transwell migration and colony formation.ResultsThree clusters associated with nicotine metabolism were discovered with distinct prognosis and immunological patterns. A four gene-signature was developed by random survival forest (RSF) method with highest average Harrell’s concordance index (C-index) of 0.763. The signature exhibited a reliable and accurate performance in prognostic prediction across TCGA-train, TCGA-test and GSE32894 cohorts. Furthermore, the signature showed highly correlation with clinical characteristics, TME and immunotherapy responses. Suppression of MKRN1 was found to reduce the migration and proliferation of bladder cancer cell. In addition, enhanced migration and proliferation caused by nicotine was blocked down by loss of MKRN1.ConclusionsThe novel nicotine metabolism-related signature may provide valuable insights into clinical prognosis and potential benefits of immunotherapy in bladder cancer patients.

Smoking during pregnancy is detrimental to the foetus and the mother, but quitting smoking can be challenging for pregnant women. The nicotine metabolite ratio (NMR), the ratio between 3-hydroxycotinine and cotinine, indicates the rate of nicotine metabolism and is associated with smoking behaviours and cessation success in the general population. Our goal was to establish what is currently known about the rate of nicotine metabolism during pregnancy. A search was conducted from inception to January 2024 using PubMed, EMBASE, CINAHL, PsycINFO, Web of Science, and Medline. Inclusion criteria encompassed pregnant women actively using any tobacco or nicotine-containing products for whom cotinine and 3-hydroxycotinine levels were assessed in maternal saliva, blood, or urine samples. Exclusion criteria comprised non-English articles, passive exposure to smoking, and absence of cotinine and 3-hydroxycotinine measurements. In half of the studies, more than 75% of participants were White. Four studies demonstrated that NMR (i.e. rate of nicotine metabolism) increased during pregnancy. Two studies found opioid use can increase NMR, and higher NMR correlated with greater nicotine dependence during pregnancy. One study found that higher NMR was associated with heavy cigarette use during pregnancy. Similarly, one study revealed pregnant women with higher NMR are less likely to quit smoking. However, one study linked lower NMR to lower birth weight, particularly among African-American populations compared to White populations. NMR tends to increase during pregnancy, complicating smoking cessation. Although race is an important determinant of NMR, the limited range of ethnic backgrounds highlights a need for further research in diverse populations.

Introduction: One of the main ingredients in cigarettes is nicotine, which has a significant impact on a person's dependence on cigarettes. Nicotine can be detected in a smoker's urine as a sign that his body is processing nicotine. The important enzyme CYP2A6 is involved in nicotine metabolism. This study aimed to determine the relationship between CYP2A6 genetic polymorphisms and nicotine metabolism among male smokers in Indonesia. Methods: This study included 100 male smokers who met the inclusion criteria in a cross-sectional design using a consecutive sampling between the ages of 20 and 65 years old. Restrictions fragment length polymorphism (RFLP) of the polymerase chain reaction (PCR) was applied to examine the genetic polymorphism of CYP2A6, and nicotine metabolite levels in urine were examined by high-performance liquid chromatography (HPLC) examination. Results: This study involved one hundred smokers, and 78 tested positive for the CYP2A6 polymorphism. The CYP2A6 genetic polymorphism and nicotine metabolism were not significantly correlated (p-value > 0.05). Allele *1A and genotype 1B/1B were more common in this study population. The majority of study participants had fast metabolic rates. Conclusion: No correlation was seen between the genetic polymorphisms of CYP2A6 and nicotine metabolism in Indonesian male smokers. Consequently, it is crucial to conduct future research in diverse populations with larger samples.

Research on nicotine metabolism has primarily focused on white adults. This study examined associations between nicotine metabolism, tobacco use, and demographic characteristics among Alaska Native adults who smoke cigarettes. Participants (N = 244) were Alaska Native adults who smoked and who provided a plasma sample at baseline (70.1%) or follow-up (29.9%) of a randomized controlled trial of a cardiovascular risk behavior intervention. At baseline, participants self-reported age, sex, Alaska Native heritage, cigarettes per day, time to first cigarette upon wakening, menthol use, perceived difficulty staying quit, tobacco withdrawal symptoms, and past-month tobacco product use, binge drinking, and cannabis use. At 3-, 6-, 12-, and 18-month follow-ups, participants self-reported 7-day point prevalence abstinence from smoking. Height and weight were measured to calculate body mass index (BMI). Participants' nicotine metabolite ratio (NMR), calculated as the ratio of plasma cotinine and trans-3' hydroxycotinine, was log-transformed. The sample (52.0% male, age M = 47.0 years [SD = 13.8], 60.3% of Inupiaq heritage) averaged 12.5 cigarettes per day (SD = 10.5); 64.0% smoked within 30 min of wakening. NMR was not significantly associated with age, sex, Alaska Native heritage, BMI, cigarettes per day, time to first cigarette upon wakening, menthol use, perceived difficulty staying quit, past-month dual tobacco product use, withdrawal symptoms, past-month binge drinking, past-month cannabis use, or abstinence from smoking (all p-values > .050). Characteristics that relate to NMR in Alaska Native adults may differ from those typically identified among white adults. Specifically, results may suggest that Alaska Native adults with slower nicotine metabolism do not titrate their nicotine intake when smoking. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

BackgroundMaternal cigarette smoking is an important modifiable risk factor for low birth weight in the US. We investigated the maternal nicotine metabolite ratio (NMR; trans-3'-hydroxycotinine/cotinine) - a genetically-informed biomarker of nicotine clearance - as a moderator of links between prenatal cigarette use and birth weight. We also explored the role of race in these associations.MethodsParticipants were 454 pregnant women (Mage = 25 years; 11% Black) who smoked cigarettes and their 537 infants from the Collaborative Perinatal Project. Cigarettes smoked per day were assessed at each prenatal visit; maternal NMR was assayed from third trimester serum. Birth weight was obtained from medical records. Generalized estimating equations were used to evaluate associations between cigarette smoking, NMR, race, and birth weight.ResultsNMR moderated continuous associations between cigarettes per day over pregnancy and infant birth weight (p = .025). Among women who smoked at moderate levels (

Abstract Background Paenarthrobacter nicotinovorans ATCC 49919 uses the pyridine-pathway to degrade nicotine and could provide a renewable source of precursors from nicotine-containing waste as well as a model for studying the molecular evolution of catabolic pathways and their spread by horizontal gene transfer via soil bacterial plasmids. Results In the present study, the strain was sequenced using the Illumina NovaSeq 6000 and Oxford Nanopore Technology (ONT) MinION platforms. Following hybrid assembly with Unicycler, the complete genome sequence of the strain was obtained and used as reference for whole-genome-based phylogeny analyses. A total of 64 related genomes were analysed; five Arthrobacter strains showed both digital DNA-DNA hybridization and average nucleotide identity values over the species threshold when compared to P. nicotinovorans ATCC 49919. Five plasmids and two contigs belonging to Arthrobacter and Paenarthrobacter strains were shown to be virtually identical with the pAO1 plasmid of Paenarthrobacter nicotinovorans ATCC 49919. Moreover, a highly syntenic nic-genes cluster was identified on five plasmids, one contig and three chromosomes. The nic-genes cluster contains two major locally collinear blocks that appear to form a putative catabolic transposon. Although the origins of the nic-genes cluster and the putative transposon still elude us, we hypothesise here that the ATCC 49919 strain most probably evolved from Paenarthrobacter sp. YJN-D or a very closely related strain by acquiring the pAO1 megaplasmid and the nicotine degradation pathway. Conclusions The data presented here offers another snapshot into the evolution of plasmids harboured by Arthrobacter and Paenarthrobacter species and their role in the spread of metabolic traits by horizontal gene transfer among related soil bacteria.

Cigarette smoking remains an important health concern and is still a leading cause of preventable mortality. Nicotine is the substance responsible for sustained tobacco use and dependence. Identification of biomarkers underlying nicotine dependence behavior is important to identify people at risk for this dependence. In the present study, we identified biochemical and genetic biomarkers of nicotine dependence detected by the Fagerström Test for Nicotine Dependence (FTDN) in Mexican smokers. The nicotine metabolites nicotine‐N′‐oxide, trans‐3′‐hydroxycotinine‐glucuronide (3HC‐O‐Gluc), and nicotine‐N‐Gluc (Gluc) were useful to differentiate nicotine‐dependent from non‐dependent subjects (p <.0001) with an area under the curve (AUC) of 0.7818. Genetic variants in CYP2A6, FMO3, and UGT2B7 (rs2431413, rs28363545, and rs7439326, respectively) were associated with nicotine dependence (p =.03, p =.01, p =.01, respectively). Variations in the enzymatic activity of CYP2A6 were associated with altered nicotine‐N′‐oxide and 3HC‐O‐Gluc levels. Decreased urinary levels of 3HC‐O‐Gluc and increased nicotine‐N′‐oxide were associated with a decrease in the functional activity of CYP2A6. A strong positive correlation was observed between the ratio of urinary 3HC/cotinine, a measure of CYP2A6 activity, and the levels of 3HC‐O‐Gluc (p <.0001, r =.6835), while a strong negative correlation was observed with nicotine‐N′‐oxide (p <.0001, r =.6522) in nicotine‐dependent subjects. No correlations were observed in non‐nicotine‐dependent subjects. These data suggest that particular urinary nicotine metabolites and genetic variants involved in nicotine metabolism are useful to identify subjects with nicotine dependence in the Mexican population. [ABSTRACT FROM AUTHOR]

Background: Paenarthrobacter nicotinovorans ATCC 49919 uses the pyridine-pathway to degrade nicotine and could provide a renewable source of precursors from nicotine-containing waste as well as a model for studying the molecular evolution of catabolic pathways and their spread by horizontal gene transfer via soil bacterial plasmids. Results: In the present study, the strain was sequenced using the Illumina NovaSeq 6000 and Oxford Nanopore Technology (ONT) MinION platforms. Following hybrid assembly with Unicycler, the complete genome sequence of the strain was obtained and used as reference for whole-genome-based phylogeny analyses. A total of 64 related genomes were analysed; five Arthrobacter strains showed both digital DNA-DNA hybridization and average nucleotide identity values over the species threshold when compared to P. nicotinovorans ATCC 49919. Five plasmids and two contigs belonging to Arthrobacter and Paenarthrobacter strains were shown to be virtually identical with the pAO1 plasmid of Paenarthrobacter nicotinovorans ATCC 49919. Moreover, a highly syntenic nic-genes cluster was identified on five plasmids, one contig and three chromosomes. The nic-genes cluster contains two major locally collinear blocks that appear to form a putative catabolic transposon. Although the origins of the nic-genes cluster and the putative transposon still elude us, we hypothesise here that the ATCC 49919 strain most probably evolved from Paenarthrobacter sp. YJN-D or a very closely related strain by acquiring the pAO1 megaplasmid and the nicotine degradation pathway. Conclusions: The data presented here offers another snapshot into the evolution of plasmids harboured by Arthrobacter and Paenarthrobacter species and their role in the spread of metabolic traits by horizontal gene transfer among related soil bacteria. [ABSTRACT FROM AUTHOR]

BackgroundAdherence to nicotine patches relates to cessation. This is the first study to examine the validity of self-reported nicotine patch adherence relative to saliva cotinine.MethodsWe used data from 198 clinical trial participants who received 11 weeks of nicotine patches, self-reported patch use, had saliva cotinine 1-week after the start of treatment assessed, and were not smoking when saliva was collected (CO < 6). Self-reported patch adherence was defined as: 3-day (before saliva collection), 7-day (before saliva collection), 3-week use (7 days before, and 14 days after, saliva collection), and 11-week use (7 days before, and 10 weeks after, saliva collection). Analyses, including receiver operating characteristic curves, considered differences in nicotine metabolism. Sensitivity, specificity and positive (PPV) and negative predictive value (NPV) assessed optimal cotinine cut-point for adherence.ResultsSelf-reported 7-day (r = 0.13) and 3-week (r = 0.13) patch use marginally correlated with week 1 cotinine (p's = 0.08) but not 3-day or 11-week. Significant area under the curve (AUC) values of 0.67 (95 %CI: 0.55-0.79) and 0.72 (95 %CI: 0.57-0.88) were found using 7-day self-report for the overall sample and for slow metabolizers (p's

The nicotine metabolite ratio (NMR; 3-hydroxycotinine/cotinine) is an index of CYP2A6 activity. CYP2A6 is responsible for nicotine's metabolic inactivation and variation in the NMR/CYP2A6 is associated with several smoking behaviors. Our aim was to integrate established alleles and novel genome-wide association studies (GWAS) signals to create a weighted genetic risk score (wGRS) for the CYP2A6 gene for European-ancestry populations. The wGRS was compared with a previous CYP2A6 gene scoring approach designed for an alternative phenotype (C2/N2; cotinine-d2/(nicotine-d2 + cotinine-d2)). CYP2A6 genotypes and the NMR were assessed in European-ancestry participants. The wGRS training set included N = 933 smokers recruited to the Pharmacogenetics of Nicotine Addiction and Treatment clinical trial [NCT01314001]. The replication cohort included N = 196 smokers recruited to the Quit 2 Live clinical trial [NCT01836276]. Comparisons between the two CYP2A6 phenotypes and with fractional clearance were made in a laboratory-based pharmacokinetic study (N = 92 participants). In both the training and replication sets, the wGRS, which included seven CYP2A6 variants, explained 33.8% (P

Abstract Background There is a need to match characteristics of tobacco users with cessation treatments and risks of tobacco attributable diseases such as lung cancer. The rate in which the body metabolizes nicotine has proven an important predictor of these outcomes. Nicotine metabolism is primarily catalyzed by the enzyme cytochrone P450 (CYP2A6) and CYP2A6 activity can be measured as the ratio of two nicotine metabolites: trans-3’-hydroxycotinine to cotinine (NMR). Measurements of these metabolites are only possible in current tobacco users and vary by biofluid source, timing of collection, and protocols; unfortunately, this has limited their use in clinical practice. The NMR depends highly on genetic variation near CYP2A6 on chromosome 19 as well as ancestry, environmental, and other genetic factors. Thus, we aimed to develop prediction models of nicotine metabolism using genotypes and basic individual characteristics (age, gender, height, and weight). Results We identified four multiethnic studies with nicotine metabolites and DNA samples. We constructed a 263 marker panel from filtering genome-wide association scans of the NMR in each study. We then applied seven machine learning techniques to train models of nicotine metabolism on the largest and most ancestrally diverse dataset (N=2239). The models were then validated using the other three studies (total N=1415). Using cross-validation, we found the correlations between the observed and predicted NMR ranged from 0.69 to 0.97 depending on the model. When predictions were averaged in an ensemble model, the correlation was 0.81. The ensemble model generalizes well in the validation studies across ancestries, despite differences in the measurements of NMR between studies, with correlations of: 0.52 for African ancestry, 0.61 for Asian ancestry, and 0.46 for European ancestry. The most influential predictors of NMR identified in more than two models were rs56113850, rs11878604, and 21 other genetic variants near CYP2A6 as well as age and ancestry. Conclusions We have developed an ensemble of seven models for predicting the NMR across ancestries from genotypes and age, gender and BMI. These models were validated using three datasets and associate with nicotine dosages. The knowledge of how an individual metabolizes nicotine could be used to help select the optimal path to reducing or quitting tobacco use, as well as, evaluating risks of tobacco use.

This study aims to describe the nicotine metabolite ratio among tobacco smokers and electronic cigarette (e-cigarette) users and nonusers. We analyzed pooled data from a longitudinal and a cross-sectional study of the adult population from the city of Barcelona. The final sample included information on 166 smokers, 164 e-cigarettes users with nicotine, 41 e-cigarette users without nicotine, 95 dual users (users of both products), and 508 nonusers. We used log-linear models to control for the potential confounding effect of the daily number of cigarettes smoked. Salivary nicotine metabolic rate assessment included the rate of nicotine metabolism (cotinine/nicotine) and the nicotine metabolite ratio (trans-3′-hydroxycotinine/cotinine). Exclusive users of e-cigarette without nicotine have the lowest rate of nicotine metabolism (Geometric mean: 0.08, p-values < 0.001) while cigarette smokers have the highest (Geometric mean: 2.08, p-values < 0.001). Nonusers have lower nicotine metabolic rate than cigarette smokers (Geometric means: 0.23 vs. 0.18, p-value < 0.05). Younger individuals (18–44 years) have a higher rate of nicotine metabolism than older individuals (45–64 years and 65–89) (Geometric means: 0.53 vs. 0.42 and 0.31, respectively, p-values < 0.01) and individuals with lower body mass index (21–25 kg/m2) have a higher rate of nicotine metabolism than the rest (26–30 kg/m2 and 31–60 kg/m2) (Geometric means: 0.52 vs. 0.35 and 0.36, respectively-values < 0.01). Nicotine metabolic rates are useful biomarkers when reporting smoking status and biological differences between individuals. [ABSTRACT FROM AUTHOR]

Cytoglobin (Cygb) regulates vascular tone by modulating nitric oxide (NO) metabolism in vascular smooth muscle cells (VSMCs). In the presence of its cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism via oxygen-dependent NO dioxygenation. Electronic cigarette (EC) use has been shown to induce vascular dysfunction and decrease NO bioavailability; however, the role of Cygb-mediated NO metabolism in the pathophysiology of this process has not been previously investigated. Therefore, we utilized aortic VSMCs with EC vape extract (ECE) exposure to elucidate the effects of EC vape constituents on NO degradation and alterations in the process of Cygb-mediated NO metabolism. VSMCs were exposed to ECE, either nicotine-free (ECEV) or nicotine-containing (ECEN), for various durations. NO decay rates were measured along with cellular expression of Cygb and its B5/B5R reducing system. Exposure to ECEV led to a much higher rate of NO consumption by VSMCs, with an even larger effect following ECEN exposure. With 4 h of exposure, a modest increase in NO decay rate occurred that was followed by much higher increases with exposure times of 24-48 h. This effect was paralleled by upregulation of Cygb and B5/B5R expression. siRNA-mediated knock-down of Cygb expression largely reversed this ECE-induced increase in NO metabolism rate. Thus, ECE exposure led to increased Cygb-mediated NO metabolism in VSMCs with diminished NO bioavailability, which in turn can play a key role in EC-induced vascular dysfunction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plants produce an immense diversity of defensive specialized metabolites. However, despite extensive functional characterization, the relative importance of different defensive compounds is rarely examined in natural settings. Here, we compare the efficacy of three Nicotiana benthamiana defensive compounds, nicotine, acylsugars and a serine protease inhibitor, by growing plants with combinations of knockout mutations in a natural setting, quantifying invertebrate interactions and comparing relative plant performance. Among the three tested compounds, acylsugars had the greatest defensive capacity, affecting aphids, leafhoppers, spiders and flies. Nicotine mutants displayed increased leafhopper feeding and aphid colonization. Plants lacking both nicotine and acylsugars were more susceptible to flea beetles and thrips. By contrast, knockout of the serine protease inhibitor did not affect insect herbivory in the field. Complementary experiments under controlled laboratory conditions with caterpillars, grasshoppers and aphids confirmed results obtained in a natural setting. We conclude that the three metabolite groups collectively provide broad-spectrum protection to N. benthamiana. However, there is a gradient in their effects on the interacting invertebrates present in the field. Furthermore, we demonstrate that, even if individual metabolites do not have a measurable defensive benefit on their own, they can have an additive effect when combined with other defensive compounds., (© 2024 John Wiley & Sons Ltd.)

Nicotiana benthamiana has long served as a crucial plant material extensively used in plant physiology research, particularly in the field of plant pathology, because of its high susceptibility to plant viruses. Additionally, it serves as a production platform to test vaccines and other valuable substances. Among its approximately 3.1 Gb genome, 57 583 genes have been annotated within a 61 Mb region. We created a comprehensive and easy-to-use platform to use transcriptomes for modern annotation. These tools allow to visualize gene expression profiles, draw molecular evolutionary phylogenetic trees of gene families, perform functional enrichment analyses, and facilitate output downloads. To demonstrate their utility, we analyzed the gene expression profiles of enzymes within the nicotine biosynthesis pathway, a secondary metabolic pathway characteristic of the Nicotiana genus. Using the developed tool, expression profiles of the nicotine biosynthesis pathway genes were generated. The expression patterns of eight gene groups in the pathway were strongly expressed in the roots and weakly expressed in leaves and flowers of N. benthamiana. The results were consistent with the established gene expression profiles in Nicotiana tabacum and provided insights into gene family composition and expression trends. The compilation of this database tool can facilitate genetic analysis of N. benthamiana in the future., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

American alligators (Alligator mississippiensis) are apex predators and sentinel species in the coastal wetland ecosystem along the Gulf of Mexico. There is concern for alligator exposure and susceptibility to chemical contaminants due to their high trophic level and lower metabolic capability. At present, their hepatic biotransformation capacity to metabolize or detoxify contaminants has not been comprehensively determined. In this study, the hepatic biotransformation capability of juvenile American alligators to metabolize two commonly found environmental pharmaceuticals: carbamazepine (CBZ) or nicotine (NCT) was evaluated. The formation of their respective primary metabolites, i.e., carbamazepine-10,11-epoxide (CBZ-E) and cotinine (CTN), was evaluated at 10 μM (within the human therapeutic range). The in vitro S9 and a novel in situ liver perfusion assays were used to characterize and compare metabolic ability in isolated hepatic enzymes vs. whole organ (liver). For CBZ, the perfused livers exhibited only 30% of intrinsic formation clearance (CL f,int ) relative to the S9 assay. The metabolism of NCT was not detectable in the S9 assay and was only observed in the perfused liver assay. Compared to the corresponding rat models (S9 or perfused livers),alligators' CL f,int was 2060% for CBZ and 50% for NCT of rats. Additionally, NCT exposure increased lactate levels in perfused livers indicating metabolic stress. This study provides insight into the hepatic capability of alligators to metabolize CBZ and NCT using an established in vitro (S9) system and a newly developed in situ liver perfusion system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nicotine is a toxic and addictive alkaloid found in tobacco and tobacco products that is harmful to human health and is an environmental pollutant. Nicotine-degrading microorganisms are unique microorganisms with the ability to use nicotine molecules as their sole source of nitrogen and carbon needed for growth. They are capable of degrading nicotine into less toxic or non-toxic metabolites. This review describes the environment's primary nicotine contamination sources and potential hazards. It also summarizes various types of nicotine-degrading microorganisms, their optimal culture conditions, and degradation efficiency. Four different catabolic pathways of nicotine in microorganisms are discussed, and the applications of nicotine-degrading microorganisms in different fields, such as the tobacco, pharmaceutical, and environmental protection industries, are outlined. This review describes the hazards of nicotine and the current research and application of nicotine-degrading microorganisms. It provides a theoretical reference for future research on nicotine-degrading microorganisms and their applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The quality of cigar tobacco leaves is profoundly affected by the timing of their harvest, with both early and late collections resulting in inferior characteristics. While the relationship between maturity and physiological metabolic processes is acknowledged, a comprehensive understanding of the physiological behavior of cigar leaves harvested at different stages remains elusive. This research investigated the physiological and metabolomic profiles of the cigar tobacco variety CX-014, grown in Danjiangkou City, Hubei Province, with leaves sampled at 35 (T1), 42 (T2), 49 (T3), and 56 (T4) days post-inflorescence removal. Leaf color transitioned from green to yellow, accompanied by the appearance of white mature spots. Notable increases in photosynthetic pigments and gas exchange parameters occurred between T1 and T2, followed by decline at T3 and T4. The optimal sugar-to-nicotine and potassium-to-chlorine ratios, critical determinants of smoking quality and tobacco combustibility, were observed at T3, indicating a superior chemical balance in leaves harvested at this stage. Metabolomic analysis revealed 2153 distinct metabolites, with the most significant changes occurring between T2 and T3, highlighting critical physiological transformations during this interval. Pathway enrichment analysis via KEGG pinpointed notable shifts in amino acid synthesis pathways, particularly those involving tryptophan, alanine, and aspartate. Tryptophan metabolism and zeatin biosynthesis were substantially altered, with compounds like indolepyruvic acid, N-formylpurine nucleotide, isopentenyladenine nucleotide, and dihydrozeatin showing marked reductions at T3. This study also explored how the timing of lower leaf harvest influences the physiological processes of middle leaves, finding that a plethora of metabolites associated with the breakdown of arachidonic acid-a primitive metazoan signaler implicated in plant stress and defense networks-were abundant in T3 leaves when lower leaves were harvested 43-38 days prior. Overall, these findings elucidates the complex physiological dynamics of cigar leaves during maturation, highlighting critical metabolites involved in essential metabolic pathways., Competing Interests: Competing interests: This study was funded by the China National Tobacco Corporation Science and Technology Major Project (110202001039, XJ-01). This funder also provided the site and part of the analytical instruments for the study. All authors declare no potential financial or non-financial conflict of interest., (© 2024. The Author(s).)

To investigate the structural and functional similarities of microbial communities in burnt-sweetness alcoholized tobacco as a function of distance from the equator and their effects on tobacco quality, we sampled alcoholized tobacco from Chenzhou, Hunan Province, China and from Brazil and Zimbabwe, which are also burnt-sweetness-type tobacco producing regions, and performed high-throughput sequencing of tobacco bacterial and fungal communities along with an analysis of the main chemical constituents of the tobacco to analyze differences in the quality of the tobacco and similarities in the structure of the microbial communities. The total nitrogen, nicotine and starch contents of Chenzhou tobacco were greater than those of Brazilian and Zimbabwean tobacco, and the total sugar and reducing sugar contents of the Brazilian and Zimbabwean tobacco were greater than those of the Chenzhou tobacco (P < 0.05). The alpha diversity indices of the bacterial communities in Chenzhou tobacco were lower than those in the Brazilian and Zimbabwean tobacco, and the alpha diversity indices of the fungal communities in Chenzhou tobacco were greater than those in the Brazilian and Zimbabwean tobacco (P < 0.05). In the ecological networks, bacterial-fungal interactions in the Brazilian and Zimbabwean tobacco were more complex than those in the Chenzhou tobacco, and the microbial ecological networks of the burnt-sweetness-type tobacco from three different regions were dominated by competitive relationships. The microbial community composition of Chenzhou tobacco was similar to that of Brazilian tobacco at the bacterial genus and fungal phylum level, with Sphingomonas being a significantly enriched genus in Brazilian tobacco and a key genus in the Chenzhou network that is able to participate in the degradation of polyphenols and aromatic compounds. Functional microbes related to aromatic compounds and cellulose degradation were significantly more abundant in the Brazilian and Zimbabwean tobacco than in Chenzhou tobacco, and the related degradation of tobacco substances was responsible for the better quality of the Brazilian and Zimbabwean tobacco. In conclusion, there are similarities in the structure, composition and functional flora of microbial communities in tobacco from Chenzhou and Brazil because these regions have similar latitudinal distributions. This study provides theoretical support for selecting cultivation regions for the burnt-sweetness-type alcoholized tobacco and for the alcoholization of tobacco leaves., Competing Interests: Declarations. Competing interests: Yes, the authors have competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper., (© 2024. The Author(s).)

In plants, cytochrome P450s are monooxygenase that play key roles in the synthesis and degradation of intracellular substances. In tobacco, the majority of studies examining the P450 superfamily have concentrated on the CYP82E subfamily, where multiple family members function as demethylases, facilitating the synthesis of nornicotine. In this study, NtCYP82C4, a tobacco P450 superfamily member, was identified from a gene-edited tobacco mutant that nicotine biosynthesis in tobacco leaves is evidently reduced. Compared to the wild-type plants, the knockout of NtCYP82C4 resulted in a significantly lower nicotine content and biomass in tobacco leaves. Transcriptome and metabolome analyses indicated that the knockout of NtCYP82C4 inhibites secondary metabolic processes in tobacco plants, leading to the accumulation of some important precursors in the nicotine synthesis process, including aspartic acid and nicotinic acid, and increases nitrogen partitioning associated with those processes such as amino acid synthesis and utilization. It is speculated that NtCYP82C4 may function as an important catalase downstream of the nicotine synthesis. Currently, most of the steps and enzymes involved in the nicotine biosynthesis process in tobacco have been elucidated. Here, our study deepens the current understanding of nicotine biosynthesis process and provides new enzyme targets for nicotine synthesis in tobacco plants., Competing Interests: Declaration of competing interest The authors declare neither competing interests nor conflict of financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Genetic studies of smoking cessation have been limited by short-term follow-up or cross-sectional design. Within seven genes (CHRNA3, CHRNA5, CHRNB2, CHRNB4, DRD2, DBH and CYP2A6) influencing biological mechanisms relevant to smoking, this study aimed to identify single nucleotide polymorphisms (SNPs) associated with smoking cessation throughout up to 38-years of follow-up., Methods: Participants were from two all-female cohort studies, Nurses' Health Study (NHS) (n = 10,017) and NHS-2 (n = 2,793). For 132 SNPs providing coverage of these genes, genotype associations with the probability of quitting smoking over time were evaluated using generalized estimating equations models. For SNPs reaching nominal statistical significance (p < 0.05) within NHS, NHS-2 was used as the replication cohort to control for multiple testing (false discovery rate (FDR) < 0.05). SNP genotype by smoking intensity (lifetime light versus non-light smoking) interactions were also evaluated., Results: Five SNPs identified in NHS were replicated in NHS-2 with FDR < 0.05. Women with the minor alleles of CHRNA5 SNPs rs637137 [OR = 1.21] and rs503464 [OR = 1.24] had increased odds of cessation. Women with the minor alleles of CYP2A6 SNPs rs56113850 [OR = 0.81] and rs56267346 [OR = 0.82] and DBH SNP rs6479643 [OR = 0.78] had lower odds of cessation throughout adulthood. An interaction with smoking intensity was indicated for three SNPs, CHRNB4 rs4887074, CHRNA3 SNP rs77438700, and CHRNA5 SNP rs76474922., Conclusion: Genetic associations with smoking cessation over decades of follow-up were observed and may guide targeted approaches for smokers most at risk for long-term relapse., Implications: This study identified single nucleotide polymorphisms (SNPs) within CHRNA5-A3-B4, CYP2A6, and DBH that were associated with smoking cessation in women over decades of follow-up. This study is the first to examine these genetic associations over years of follow-up. Some associations were novel while others replicated previous findings from short-term studies for the first time. Potential differences in some associations between light and non-light smokers were also observed. Genetic factors associated with long-term smoking behavior may help inform interventions modeled on long-term chronic disease management approaches; specifically, targeted maintenance interventions to sustain abstinence could be implemented among high-risk smokers., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: There is a need to match characteristics of tobacco users with cessation treatments and risks of tobacco attributable diseases such as lung cancer. The rate in which the body metabolizes nicotine has proven an important predictor of these outcomes. Nicotine metabolism is primarily catalyzed by the enzyme cytochrone P450 (CYP2A6) and CYP2A6 activity can be measured as the ratio of two nicotine metabolites: trans-3'-hydroxycotinine to cotinine (NMR). Measurements of these metabolites are only possible in current tobacco users and vary by biofluid source, timing of collection, and protocols; unfortunately, this has limited their use in clinical practice. The NMR depends highly on genetic variation near CYP2A6 on chromosome 19 as well as ancestry, environmental, and other genetic factors. Thus, we aimed to develop prediction models of nicotine metabolism using genotypes and basic individual characteristics (age, gender, height, and weight). Results: We identified four multiethnic studies with nicotine metabolites and DNA samples. We constructed a 263 marker panel from filtering genome-wide association scans of the NMR in each study. We then applied seven machine learning techniques to train models of nicotine metabolism on the largest and most ancestrally diverse dataset (N=2239). The models were then validated using the other three studies (total N=1415). Using cross-validation, we found the correlations between the observed and predicted NMR ranged from 0.69 to 0.97 depending on the model. When predictions were averaged in an ensemble model, the correlation was 0.81. The ensemble model generalizes well in the validation studies across ancestries, despite differences in the measurements of NMR between studies, with correlations of: 0.52 for African ancestry, 0.61 for Asian ancestry, and 0.46 for European ancestry. The most influential predictors of NMR identified in more than two models were rs56113850, rs11878604, and 21 other genetic variants near CYP2A6 as well as age and ancestry. Conclusions: We have developed an ensemble of seven models for predicting the NMR across ancestries from genotypes and age, gender and BMI. These models were validated using three datasets and associate with nicotine dosages. The knowledge of how an individual metabolizes nicotine could be used to help select the optimal path to reducing or quitting tobacco use, as well as, evaluating risks of tobacco use. [ABSTRACT FROM AUTHOR]

Introduction. To find genetic variants in the DRD4 and HTR2A genes' promoter regions and exons that are associated with tobacco smoking and nicotine addiction in Egyptian university students as well as to study the association between personality traits and smoking status. Methods. A self-administered questionnaire about cigarette smoking and personality attributes (Big Five Inventory, ESPAD Questionnaire on Substance Abuse, and Buss-Perry Aggression Questionnaire). The participants in the study were 90 nonsmokers (NS) and 88 current smokers (CS), who were divided into two groups depending on their cigarette consumption per day (cpd): 55 heavy smokers (HS, >20 cpd) and 33 light smokers (LS, 1–10 cpd). Four and eight single nucleotide polymorphisms (SNPs) in the DRD4, HTR2A, and MOA genes, respectively, were genotyped. Results. Smokers scored lower on neuroticism, agreeableness, conscientiousness, openness, and extraversion than nonsmokers, but higher on aggression. Furthermore, the C allele of rs1800955 in DRD4 was associated with cigarette smoking in the HS vs. NS and LS vs. NS studies. The T allele of the HTR2A rs6313 gene was discovered to be strongly associated with cigarette smoking. There was no link discovered between MOA rs1137070 and MOA rs1137070. Conclusions. Using a comprehensive personality model (FFM), this study repeats and extends earlier research. Personality and genetic studies may aid in the development of a more complete and conclusive understanding of cigarette smoking, as well as more precise policies and guidelines for smoking cessation and quitting. [ABSTRACT FROM AUTHOR]

As the main effect substances of tobacco products, the physiological effects of nicotine have attracted the attention of researchers, especially in recent years, the discussion on the benefits and harms of nicotine (or tobacco products) has become increasingly fierce. In this review, the structure, distribution and physiological effects of nicotinic acetylcholine receptor (nAchR) are summarized. The absorption, distribution, metabolism and excretion of nicotine in the body were introduced. Further, the positive effects of low-dose and short-term nicotine exposure on mitochondrial function regulation, stem cell proliferation and differentiation, nervous system protection and analgesia were elucidated. At the same time, it is also discussed that high-dose and long-term nicotine exposure can activate the oxidative stress effect, mediate abnormal epigenetic modification, and enhance the immune inflammatory response, and then produce negative effects on the body. To sum up, this review suggests that there is a "double-edged sword" effect of nicotine, which on the one hand helps people to understand the physiological effects of nicotine more comprehensively and carefully, and on the other hand provides some theoretical basis for the rational use of nicotine and the innovative development of related products. The "double-edged sword effect" of nicotine. [Display omitted] [ABSTRACT FROM AUTHOR]

Smoking is a public health concern, and even though smoking cessation methods exist, nicotine replacement therapy (NRT) is often ineffective. Smoking behavior is related to the nicotine metabolizing enzyme (NME) P450 2A6 (mouse 2A5) polymorphisms. Accordingly, fast metabolizers are nicotine dependent, and have low quitting rates compared to slow metabolizers. In this study we examined the ability of Ginkgo biloba L (GB) and its constituents to inhibit the NME, using mouse liver microsomes containing the 2A5 enzyme. Our results indicate that GB can inhibit 2A5 (25% inhibition at 5%v/v), with the flavonoids quercetin, isorhamnetin, and kaempferol being responsible for this inhibition (23.5%, 10.7%, 25.2% inhibition at 60 ng/μL, respectively). Importantly, the flavonoids inhibited 2A5 via mechanism based inhibition (for quercetin 30 ng/μl inhibition increased from 20.8% to 26.9% within 15 minutes). Our results suggest that GB if consumed on a regular basis can help NRT enhancement particularly in fast nicotine metabolizers. [ABSTRACT FROM AUTHOR]

Aim. Plasma values of nicotine and its metabolites are highly variable, and this variability has a strong genetic influence. In our study, we analysed the impact of common polymorphisms associated with smoking on the plasma values of nicotine, nicotine metabolites and their ratios and investigated the potential effect of these polymorphisms and nicotine metabolite ratios on the successful treatment of tobacco dependence. Methods. Five variants (rs16969968, rs6474412, rs578776, rs4105144 and rs3733829) were genotyped in a group of highly dependent adult smokers (n=103). All smokers underwent intensive treatment for tobacco dependence; 33 smokers were still abstinent at the 12-month follow-up. Results. The rs4105144 (CYP2A6, P

Plant species can accumulate secondary metabolites in optically pure form or, occasionally, as enantiomeric mixtures. Interestingly, enantiomers of the same molecule can confer different biological activities. In tobacco (Nicotiana tabacum L.), the pyridine alkaloids nicotine, nornicotine, anatabine, and anabasine naturally exist as scalemic mixtures of (R)- or (S)-enantiomers, with the (S)-isoforms predominating. The mechanisms by which tobacco alkaloid enantiomers accumulate remain largely unknown. Experiments were carried out involving tobacco genotypes possessing induced deleterious mutations in three genes coding for nicotine demethylase (NND) enzymes and three genes coding for Berberine Bridge Like (BBL) enzymes that act near the end of the nicotine, anatabine, and anabasine biosynthetic pathways. Data indicate that (R)-nicotine is naturally produced at appreciable levels but is observed in only small amounts due to preferential demethylation by NND enzymes. Data further suggest that BBL-a and BBL-b are preferentially involved in the biosynthesis of (S)-alkaloid enantiomers, while BBL-c is preferentially involved in the biosynthesis of (R)-enantiomers. Gene duplication followed by genetic divergence thus played a role in the evolution of scalemic alkaloid accumulation in tobacco. Through a combination of mutation breeding and transgene overexpression, tobacco genotypes were generated in which the predominant alkaloid enantiomers were reversed from the (S)- to the (R)-isoforms. These results shed light on the genetic control of alkaloid accumulation in N. tabacum and on mechanisms of scalemic mixture formation of secondary metabolites in plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Rate of nicotine metabolism has been identified as an important factor influencing nicotine intake and can be estimated using the nicotine metabolite ratio (NMR), a validated biomarker of CYP2A6 enzyme activity. Individuals who metabolize nicotine faster (higher NMR) may alter their smoking behavior to titrate their nicotine intake in order to maintain similar levels of nicotine in the body compared to slower nicotine metabolizers. There are known racial differences in the rate of nicotine metabolism with African Americans on average having a slower rate of nicotine metabolism compared to Whites. The goal of this study was to determine if there are racial differences in the relationship between rate of nicotine metabolism and measures of nicotine intake assessed using multiple biomarkers of nicotine and tobacco smoke exposure. Using secondary analyses of the screening data collected in a recently completed clinical trial, treatment-seeking African American and White daily smokers (10 or more cigarettes per day) were grouped into NMR quartiles so that the races could be compared at the same NMR, even though the distribution of NMR within race differed. The results indicated that rate of nicotine metabolism was a more important factor influencing nicotine intake in White smokers. Specifically, Whites were more likely to titrate their nicotine intake based on the rate at which they metabolize nicotine. However, this relationship was not found in African Americans. Overall there was a greater step-down, linear type relationship between NMR groups and cotinine or cotinine/cigarette in African Americans, which is consistent with the idea that differences in blood cotinine levels between the African American NMR groups were primarily due to differences in CYP2A6 enzyme activity without titration of nicotine intake among faster nicotine metabolizers.

Tobacco smoke contains several electrophilic constituents which are capable of forming adducts with nucleophilic sites in DNA and proteins like hemoglobin (Hb) and albumin. New nicotine and tobacco products are discussed as less harmful forms of tobacco use compared to smoking combustible cigarettes (CC) due to reduced exposure to harmful constituents. Hence, the adduct profile in users of various tobacco/nicotine products is expected to differ characteristically. In this article, we present a novel nontargeted screening strategy using GC-MS/MS for Hb adducts based on the analysis of the respective derivatized N-terminal valine adducts after modified Edman degradation. We analyzed blood samples from a clinical study with habitual users of CCs, electronic cigarettes, heated tobacco products (HTPs), oral tobacco, nicotine replacement therapy products and nonusers of any tobacco/nicotine products. Our nontargeted approach revealed significant differences in the Hb adduct profiles of the investigated tobacco/nicotine product user groups. Adduct identification was performed by means of an internal database, retention time estimations based on the theoretical boiling points, as well as in-house synthesized reference compounds. Several chemicals that form adducts with Hb could be identified: methylating and ethylating agents, ethylene oxide, acrylonitrile, acrylamide, glycidamide and 4-hydroxybenzaldehyde. Levels were elevated in smokers compared to all other groups for Hb adducts from methylating agents, ethylene oxide, acrylonitrile, acrylamide and glycidamide. Our approach revealed higher concentrations of Hb adducts formed by ethylation, acrylamide and glycidamide in users of HTPs compared to nonusers. However, concentrations for the latter two were still lower than in smokers. Due to their long half-lives, Hb adducts related to acrylonitrile, acrylamide (glycidamide), and ethylene oxide exposure may be useful for the biochemical verification of subjects̀ compliance in longitudinal and cross-sectional studies with respect to smoking and HTP use/abstinence.

Nicotine acts as an angiogenic factor by stimulating endogenous cholinergic pathways. Several subtypes of nicotinic acetylcholine receptors (nAChRs) have been demonstrated to be closely correlated to the formation and progression of different types of cancers. Recently, several studies have found that nicotinic acetylcholine receptors α9 (α9-nAChRs) are highly expressed in breast tumors, especially in tumors derived from patients diagnosed at advanced stages. In vitro studies have demonstrated that activation of α9-nAChRs is associated with increased proliferation and migration of breast cancer. To study the tumor-promoting role of α9-nAChRs in breast cancers, we generated a novel anti-α9-nAChR and methoxy-polyethylene glycol (mPEG) bispecific antibody (α9 BsAb) for dissecting the molecular mechanism on α9-nAChR-mediated tumor progression. Unexpectedly, we discovered the angiogenic role of α9-nAChR in nicotine-induced neovascularization of tumors. It revealed α9 BsAbs reduced nicotine-induced endothelial cell tube formation, blood vessel development in Matrigel plug assay and angiogenesis in microtube array membrane murine model (MTAMs). To unbraid the molecular mechanism of α9-nAChR in nicotine-mediated angiogenesis, the α9 BsAbs were applied and revealed the inhibitory roles in nicotine-induced production of hypoxia-inducible factor-2 alpha (HIF-2α), vascular endothelial growth factor A (VEGF-A), phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2), vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase-9 (MMP9) from triple-negative breast cancer cells (MDA-MB-231), suggesting α9-nAChRs played an important role in nicotine-induced angiogenesis. To confirm our results, the shRNA targeting α9-nAChRs was designed and used to silence α9-nAChR expression and then evaluated the angiogenic role of α9-nAChRs. The results showed α9 shRNA also played an inhibitory effect in blocking the nicotine-induced angiogenic signaling. Taken together, α9-nAChR played a critical role in nicotine-induced angiogenesis and this bispecific antibody (α9 BsAb) may serve as a potential therapeutic candidate for treatments of the α9 positive cancers., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Tobacco alkaloids in tobacco-cultivated soils pose potential risks for succeeding crops, due to their allelopathy and toxicity. Effects of biochar on the dissipation of tobacco alkaloids in soil-crop systems remain poorly understood. In this study, a 40-day pot experiment was conducted to explore the effect of cow dung biochar (CDBC) and maize straw biochar (MSBC) on the uptake of nicotine and nornicotine by pea (Pisum sativum L.) and their dissipation in an agricultural soil. The results revealed that the bioaccumulation of nicotine and nornicotine by pea shoots in the soils added with CDBC and MSBC at 1.5% and 3.0% significantly decreased by 46.97-79.13% and 33.64-71.59%, respectively. CDBC more effectively decreased the uptake and bioaccumulation of nicotine and nornicotine by pea shoots than MSBC due to the higher soil pH and nutrient content. In addition, the enhanced relative abundances of soil nicotine-degrading bacteria belonging to the genera Arthrobacter and Gemmatimonas also contributed to the decreasing uptake of nicotine by pea plants. The decreased bioavailability in the soils due to the increased adsorption was the key factor for the reduced bioaccumulation of tobacco alkaloids. This study provides guidance to protect subsequent crops in tobacco-cultivated soil from tobacco alkaloids with biochar., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Enzymatic therapy with nicotine-degrading enzyme is a new strategy in treating nicotine addiction, which can reduce nicotine concentrations and weaken withdrawal in the rat model. However, when O 2 is used as the electron acceptor, no satisfactory performance has been achieved with one of the most commonly studied and efficient nicotine-catabolizing enzymes, NicA2. To obtain more efficient nicotine-degrading enzyme, we rationally designed and engineered a flavoenzyme Pnao, which shares high structural similarity with NicA2 (RMSD = 1.143 Å) and efficiently catalyze pseudooxynicotine into 3-succinoyl-semialdehyde pyridine using O 2 . Through amino acid alterations with NicA2, five Pnao mutants were generated, which can degrade nicotine in Tris-HCl buffer and retain catabolic activity on its natural substrate. Nicotine-1'- N -oxide was identified as one of the reaction products. Four of the derivative mutants showed activity in rat serum and Trp220 and Asn224 were found critical for enzyme specificity. Our findings offer a novel avenue for research into aerobic nicotine catabolism and provide a promising method of generating additional nicotine-catalytic enzymes., Importance: Nicotine, the main active substance in tobacco, results in cigarette addiction and various diseases. There have been some attempts at using nicotine oxidoreductase, NicA2, as a therapeutic for nicotine cessation. However, it uses cytochrome c as it is electron acceptor, which is impractical for therapeutic use compared with using O2 as an oxidant. Thus, amino acid alteration was performed on Pnao using NicA2 as model. Five of the mutants generated degraded nicotine at a rate similar to NicA2, and one of the catabolic compounds was identified as nicotine-1'-N-oxide. Our research highlights a new direction in developing enzymes that efficiently catabolize nicotine without co-enzymes and suggests that structure-similar human original MAOA (or B) may assist with nicotine cessation after being engineered., Competing Interests: The authors declare no conflict of interest.

Nicotine exhibits obvious heterosis, which can be used to create Nicotiana tabacum L. (tobacco) varieties with varying nicotine content. However, the reasons for the formation of nicotine heterosis and its relationship to nicotine transport and accumulation remain unknown. This study conducted a comprehensive analysis of six tobacco hybrids with varying heterosis levels and their parent materials from various aspects, such as phenotype, physiology, and transcriptomics. The results showed that the direct path coefficient of transport heterosis to nicotine heterosis was highest in hybrids, at 0.98, and a highly significant positive correlation between the two. The plant height, thick stalk circumference, large flow of tissue fluid in the stalk, and high nicotine concentration of tobacco were the underlying factors that led to the strong nicotine transport capacity of hybrids. The formation of nicotine transport heterosis in hybrids was mainly influenced by non-additive gene effects (accounting for 89.93%), with over-dominant effects playing a dominant role (accounting for 58.79%). Among non-additive expression DEGs, nicotine transporter related multi antimicrobial extrusion protein, drug/metabolite transporter, ABC family transporter, and glutathione S-transferase were significantly upregulated in hybrid strains. The RT-qPCR results indicated that these genes related nicotine transport also exhibited heterosis at the expression level. Our results revealed that the formation of nicotine heterosis is mainly achieved by enhancing the nicotine transport capacity in hybrids. The results are not only beneficial for promoting the theoretical study of nicotine heterosis in tobacco and the breeding and utilization of hybrids, but are also of great significance for guiding nicotine production and promoting its multipurpose utilization., (© 2024. The Author(s).)

The exact relationship between nicotine metabolism and dependence is not fully understood but is known to be influenced at a molecular level by genetic factors. A sample comprising 274 Chinese adult male smokers was categorized into groups based on their metabolic rates, namely fast, intermediate, and slow metabolizers. We then measured their smoking topography, evaluated their nicotine dependence, and assessed the rewarding effects. Based on these findings, we proposed the hypothesis that the rate of nicotine metabolism could influence the level of dopamine release which in turn had repercussions on the pleasurable and rewarding effects. To test this hypothesis, male mice were selected with different nicotine metabolic rates that closely resembled in the smoker group. We evaluated their nicotine dependence and rewarding effects through conditioned place preference and withdrawal symptom tests, supplemented with dopamine release measurements. In both animal and human, the slow metabolism group (SMG) required less nicotine to maintain a comparable level of dependence than the fast metabolism group (FMG). The SMG could achieve similar rewarding effects to FMG despite consuming less nicotine. Comparable dopamine levels released were therefore critical in setting the nicotine acquisition behavior in this animal model and also for the smokers tested. Our findings suggested that even within the same ethnicity of established smokers (Chinese Han), differences in nicotine metabolism were an important parameter to modulate the degree of nicotine dependence., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Pharmacogene Variation Consortium (PharmVar) provides nomenclature for the human CYP2A gene locus containing the highly polymorphic CYP2A6 gene. CYP2A6 plays a role in the metabolism of nicotine and various drugs. Thus, genetic variation can substantially contribute to the function of this enzyme and associated efficacy and safety. This GeneFocus provides an overview of the clinical significance of CYP2A6, including its genetic variation and function. We also highlight and discuss caveats in the identification and characterization of allelic variation of this complex pharmacogene, a prerequisite for accurate genotype determination and prediction of phenotype status., (© 2024 The Author(s). Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Common flue-cured tobacco (Nicotiana tabacum L.) primarily accumulates nicotine, and its flue-cured leaves exhibit a lemon appearance. In contrast, a spontaneous cherry-red variant (CR60) primarily accumulates nornicotine, accompanied by distinctive red dapples on the cured leaves. In this study, suppression of conversion of nicotine to nornicotine by genome editing resulted in decreased nornicotine and N-acyl nornicotines (NacNNs), and the subsequent disappearance of red dapples in CR60. Conversely, overexpression of CYP82E4 increased nornicotine and NacNNs accumulation, inducing a red dapple phenotype in common tobacco. Notably, nicotine conversion triggered significant alterations in leaf total sugars, alkaloids, and nitrogens. Metabolome analyses using 1352 identified compounds indicated nicotine conversion dramatically affected the entire metabolic network and induced unique metabolic responses across diverse genetic backgrounds. Further WGCNA analysis revealed that nicotine conversion caused substantial contents variation of alkaloids, flavonoids and amino acids and derivatives in cured leaves. Overall, this research provides valuable insights into the mechanisms underlying red dapple formation in cherry-red tobacco, elucidating profound influence of nicotine conversion on entire metabolic network., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: The effects of nicotine metabolism on the kidneys of healthy individuals have not been determined. The nicotine metabolite ratio (NMR) indicates the rate of nicotine metabolism and is linked to smoking behaviors and responses to tobacco treatments. We conducted this study in order to investigated the relationship between nicotine metabolite ratio (NMR) and kidney function., Methods: An analysis of cross-sectional data of adults was conducted using a population survey dataset (National Health and Nutrition Examination Survey Data 2013/2018 of the United States). A weighted multivariate regression analysis was conducted to estimate the correlation between NMR and kidney function. Furthermore, we apply fitting smooth curves to make the relationship between NMR and estimated glomerular filtration rate (eGFR) more visualized., Results: The research included a total of 16153 participants. Weighted multivariate regression analyses adjusted for possible variables showed a negative relationship between NMR and estimated glomerular filtration rate (eGFR).The β (95%CI) of the regression equation between NMR and eGFR was -2.24 (-2.92, -1.55), the trend testing showed consistent results. NMR is positively correlated with urinary albumin creatinine ratio (uACR), but it is not statistically significant. A stratified analysis found a negative correlation between NMR and eGFR in all age, gender and diabetes subgroups, the results were not statistically significant among Mexican Americans and other races. Notably, each unit rise in NMR corresponded to a 4.54 ml/min·1.73m² lower eGFR in diabetic participants and a 6.04 ml/min·1.73m² lower eGFR in those aged 60 and above., Conclusions: Our study shows that nicotine metabolite ratio is negatively associated with kidney function among most adults. It will be necessary to conduct more well-designed prospective clinical trials in order to determine the exact causal interactions between NMR and kidney function. Specific mechanisms also need to be further explored in basic experiments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang and Su.)

Spodoptera litura is a significant agricultural pest, and its glutathione S -transferase (GST) plays a crucial role in insecticide resistance. This study aimed to investigate the relationship between the SlGSTe11 gene of S. litura and resistance to cyantraniliprole and nicotine. Transcriptome analysis revealed that SlGSTe11 is highly expressed mainly in fat bodies, with a significant increase in SlGSTe11 gene expression under induction by cyantraniliprole and nicotine. The ectopic expression of the SlGSTe11 gene in transgenic fruit flies resulted in a 5.22-fold increase in the tolerance to cyantraniliprole. Moreover, compared to the UAS- SlGSTe11 line, the Act5C-UAS> SlGSTe11 line laid more eggs and had a lower mortality after nicotine exposure. RNAi-mediated inhibition of SlGSTe11 gene expression led to a significant increase in the mortality of S. litura under cyantraniliprole exposure. In vitro metabolism experiments demonstrated that the recombinant SlGSTe11 protein efficiently metabolizes cyantraniliprole. Molecular docking results indicated that SlGSTe11 has a strong affinity for both cyantraniliprole and nicotine. These findings suggest that SlGSTe11 is involved in the development of resistance to cyantraniliprole and nicotine in S. litura .

With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) which is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with K m  = 2.7 μM and V max  = 794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in the lungs of control mice with an even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Nicotine constitutes approximately 90% of the total alkaloid content in leaves within the Nicotiana species, rendering it the most prevalent alkaloid. While the majority of genes responsible for nicotine biosynthesis express in root tissue, the influence of light on this process through shoot-to-root mobile ELONGATED HYPOCOTYL 5 (HY5) has been recognized. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), a key regulator of light-associated responses, known for its role in modulating HY5 accumulation, remains largely unexplored in its relationship to light-dependent nicotine accumulation. Here, we identified NtCOP1, a COP1 homolog in Nicotiana tabacum, and demonstrated its ability to complement the cop1-4 mutant in Arabidopsis thaliana at molecular, morphological, and biochemical levels. Through the development of NtCOP1 overexpression (NtCOP1OX) plants, we observed a significant reduction in nicotine and flavonol content, inversely correlated with the down-regulation of nicotine and phenylpropanoid pathway. Conversely, CRISPR/Cas9-based knockout mutant plants (NtCOP1 CR ) exhibited an increase in nicotine levels. Further investigations, including yeast-two hybrid assays, grafting experiments, and Western blot analyses, revealed that NtCOP1 modulates nicotine biosynthesis by targeting NtHY5, thereby impeding its transport from shoot-to-root. We conclude that the interplay between HY5 and COP1 functions antagonistically in the light-dependent regulation of nicotine biosynthesis in tobacco., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

ObjectiveNicotine, the main addictive ingredient in tobacco, is metabolically inactivated to cotinine primarily by the hepatic enzyme CYP2A6. Considerable genetic variation in the CYP2A6 gene results in large variation in the rates of nicotine metabolism, which in turn alters smoking behaviours (e.g. amount of cigarettes smoked, risk for dependence and success in smoking cessation). The aim of this study was to identify and characterize novel variants in CYP2A6.Materials and methodsThe CYP2A6 gene from African American phenotypically slow nicotine metabolizers was sequenced and seven novel variants were identified [CYP2A6*39 (V68M), CYP2A6*40 (I149M), CYP2A6*41 (R265Q), CYP2A6*42 (I268T), CYP2A6*43 (T303I), CYP2A6*44 (E390K), CYP2A6*44 (L462P)]. Variants were introduced into a bi-cistronic cDNA expression construct containing CYP2A6 and P450 oxidoreductase and assessed for protein expression, enzymatic activity and stability as evaluated using western blotting and nicotine metabolism. Genotyping assays were developed and allelic frequencies were assessed in 534 African Americans.ResultsThe variants showed significantly lower protein expression (P

Gram-negative bacteria employ secretion systems to translocate proteinaceous effectors from the cytoplasm to the extracellular milieu, thus interacting with the surrounding environment or microniche. It is known that bacteria can benefit from the type VI secretion system (T6SS) by transporting ions to combat reactive oxygen species (ROS). Here, we report that T6SS activities conferred tolerance to nicotine-induced oxidative stress in Pseudomonas sp. strain JY-Q, a highly active nicotine degradation strain isolated from tobacco waste extract. AA098_13375 was identified to encode a dual-functional effector with antimicrobial and anti-ROS activities. Wild-type strain JY-Q grew better than the AA098_13375 deletion mutant in nicotinecontaining medium by antagonizing increased intracellular ROS levels. It was, therefore, tentatively designated TseN (type VI secretion system effector for nicotine tolerance), homologs of which were observed to be broadly ubiquitous in Pseudomonas species. TseN was identified as a Tse6-like bacteriostatic toxin via monitoring intracellular NAD+. TseN presented potential antagonism against ROS to fine tune the heavy traffic of nicotine metabolism in strain JY-Q. It is feasible that the dynamic tuning of NAD+ driven by TseN could satisfy demands from nicotine degradation with less cytotoxicity. In this scenario, T6SS involves a fascinating accommodation cascade that prompts constitutive biotransformation of N-heterocyclic aromatics by improving bacterial robustness/growth. In summary, the T6SS in JY-Q mediated resistance to oxidative stress and promoted bacterial fitness via a contact-independent growth competitive advantage, in addition to the well-studied T6SS-dependent antimicrobial activities. [ABSTRACT FROM AUTHOR]

Reducing cigarette nicotine content to nonaddictive levels facilitates smoking cessation; however, very low nicotine content cigarettes (VLNCs) may not be equally effective across heterogeneous smokers. We evaluated the impact of acute VLNC smoking versus control (sham puffs) on craving, withdrawal and smoking lapse behaviour and whether genetically influenced differences in nicotine metabolism and individual differences in nicotine dependence moderate observed effects. Thirty-three overnight-abstinent smokers (15 slow vs. 17 normal nicotine metabolizers; 17 low vs. 16 high nicotine dependence) smoked a 0.05-mg nicotine VLNC during one session and took sham VLNC puffs during another session, in a counterbalanced order. Craving and withdrawal were assessed before and after smoking and sham puffing. Next, participants completed the McKee Smoking Lapse Task, which measures ability to resist smoking and quantity of ad libitum smoking. VLNC (vs. sham) reduced craving and withdrawal, increased ability to resist smoking and reduced ad libitum smoking. VLNC-induced reduction in craving for positive reinforcement was greater in slow (vs. normal) metabolizers. Nicotine metabolism did not moderate any other VLNC responses. High-dependence (vs. low-dependence) participants engaged in greater ad lib smoking across VLNC and sham conditions. Nicotine dependence did not moderate VLNC responses. VLNC reduced craving, withdrawal and smoking lapse behaviour. Individual differences in nicotine metabolism and dependence had a minimal impact on VLNC responses; however, VLNCs were less effective at reducing craving for positive reinforcement among normal (vs. slow) metabolizers. These findings suggest that desirable VLNC effects may extend across heterogeneous groups of smokers. [ABSTRACT FROM AUTHOR]

Aim. Plasma values of nicotine and its metabolites are highly variable, and this variability has a strong genetic influence. In our study, we analysed the impact of common polymorphisms associated with smoking on the plasma values of nicotine, nicotine metabolites and their ratios and investigated the potential effect of these polymorphisms and nicotine metabolite ratios on the successful treatment of tobacco dependence. Methods. Five variants (rs16969968, rs6474412, rs578776, rs4105144 and rs3733829) were genotyped in a group of highly dependent adult smokers (n=103). All smokers underwent intensive treatment for tobacco dependence; 33 smokers were still abstinent at the 12-month follow-up. Results. The rs4105144 (CYP2A6, P<0.005) and rs3733829 (EGLN2, P<0.05) variants were significantly associated with plasma concentrations of 3OH-cotinine and with 3OH-cotinine: cotinine ratios. Similarly, the unweighted gene score was a significant (P<0.05) predictor of both cotinine:nicotine and 3OH-cotinine:cotinine ratios. No associations between the analysed polymorphisms or nicotine metabolite ratios and nicotine abstinence rate were observed. Conclusion. Although CYP2A6 and EGLN2 polymorphisms were associated with nicotine metabolism ratios, neither these polymorphisms nor the ratios were associated with abstinence rates. [ABSTRACT FROM AUTHOR]

Background: Lung is the largest mucosal area of the human body and directly connected to the external environment, facing microbial exposure and environmental stimuli. Therefore, studying the internal microorganisms of the lung is crucial for a deeper understanding of the relationship between microorganisms and the occurrence and progression of lung cancer., Methods: Tumor and adjacent nontumor tissues were collected from 38 lung adenocarcinoma patients and used nanopore sequencing technology to sequence the 16s full-length sequence of bacteria, and combining bioinformatics methods to identify and quantitatively analyze microorganisms in tissues, as well as to enrich the metabolic pathways of microorganisms., Results: the microbial composition in lung adenocarcinoma tissues is highly similar to that in adjacent tissues, but the alpha diversity is significantly lower than that in adjacent tissues. The difference analysis results show that the bacterial communities of Streptococcaceae , Lactobacillaceae , and Neisseriales were significantly enriched in cancer tissues. The results of metabolic pathway analysis indicate that pathways related to cellular communication, transcription, and protein synthesis were significantly enriched in cancer tissue. In addition, clinical staging analysis of nicotine exposure and lung cancer found that Haemophilus , paralinfluenzae , Streptococcus gordonii were significantly enriched in the nicotine exposure group, while the microbiota of Cardiobactereae and Cardiobacterales were significantly enriched in stage II tumors. The microbiota significantly enriched in IA-II stages were Neisseriaeae , Enterobacteriales , and Cardiobacterales , respectively., Conclusion: Nanopore sequencing technology was performed on the full length 16s sequence, which preliminarily depicted the microbial changes and enrichment of microbial metabolic pathways in tumor and adjacent nontumor tissues. The relationship between nicotine exposure, tumor progression, and microorganisms was explored, providing a theoretical basis for the treatment of lung cancer through microbial targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yang, Wang, Ding, Zhang, Zhu, Wang, Pan, Li, Wang and Yu.)