A 90-day subchronic exposure to heated tobacco product aerosol caused differences in intestinal inflammation and microbiome dysregulation in rats.

INTRODUCTION: Smoking is one of the most important predisposing factors of intestinal inflammatory diseases. Heated tobacco product (HTP) is a novel tobacco category that is claimed to deliver reduced chemicals to human those reported in combustible cigarette smoke (CS). However, the effect of HTP on intestine is still unknown. METHODS: In the framework of Organization for Economic Co-operation and Development guidelines 413 guidelines, Sprague-Dawley rats were exposed to HTP aerosol and CS for 13 weeks. The atmosphere was characterized and oxidative stress and inflammation of intestine were investigated after exposure. Furthermore, the faeces we performed with 16S sequencing and metabolomics analysis. RESULTS: HTP aerosol and CS led to obvious intestinal damage evidenced by increased intestinal pro-inflammatory cytokines and oxidative stress in male and female rats After HTP and CS exposure, the abundance that obviously changed were Lactobacillus and Turiciacter in male rats and Lactobacillus and Prevotella in female rats. HTP mainly induced the metabolism of amino acids and fatty acyls such as short-chain fatty acids and tryptophan, while CS involved into the main metabolism of bile acids, especially indole and derivatives. Although different metabolic pathways in the gut mediated by HTP and CS, both to inflammation and oxidative stress were ultimately induced. CONCLUSIONS: HTP aerosol and CS induced intestinal damage mediated by different gut microbiota and metabolites, while both lead to inflammation and oxidative stress. IMPLICATIONS: The concentration of various harmful components in heated tobacco product aerosol is reported lower than that of traditional cigarette smoke, however, its health risk impact on consumers remains to be studied. Our research findings indicate that heated tobacco product and cigarette smoke inhalation induced intestinal damage through different metabolic pathways mediated by gut microbiome, indicating the health risk of heated tobacco product in intestine.

The Role of Nicotine Metabolic Rate on Nicotine Dependence and Rewarding: Nicotine Metabolism in Chinese Male Smokers and Male Mice.

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.

Liver toxicity in rats after subchronic exposure to HTP aerosol and cigarette smoke.

Background: Heated tobacco product (HTP) considered to be a novel tobacco product which was reported safer than traditional cigarettes evidenced by lower potential harmful components released. Liver is an important detoxification organ of the body, the chemical components in aerosols are metabolized in the liver after absorbed, so it is necessary to explore the effect of HTP on the liver. Materials and Methods: The potential effect of HTP and cigarette smoke (CS) on SD rats was explored according to OECD 413 subchronic inhalation. The rats were randomly divided into Sham (air), different dosage of HTP groups (HTP_10, 23 and 50 µg nicotine/L aerosol) and Cig_23 (23 µg nicotine/L aerosol) group. After exposure, the clinical pathology, inflammation and oxidative stress were measured. Results: The clinical pathology results showed that both HTP_50 and Cig_23 led to abnormality of ALT for male rats. CS and HTP exposure reduced the expression of IL-1ß, IL-6 and TNF-α and mitochondrial medicated oxidative stress. In addition, the ATP production was reduced in Cig_23 group. Although inflammation and oxidative stress were displayed, no apoptosis were observed by TUNEL assay and these existed obvious pathological changes only in HTP_50 group, while in CS group with equivalent nicotine, hepatocytes swelling were observed in liver. Conclusion: CS exposure induced liver damage through mitochondrial mediated oxidative stress and inflammation, which was also observed in high concentration of HTP exposure group. For the same equivalent nicotine, HTP may show lower toxic effect on liver than CS.

Evaluation of toxicity of heated tobacco products aerosol and cigarette smoke to BEAS-2B cells based on 3D biomimetic chip model.

It is still a controversial topic about evaluating whether heated tobacco products (HTP) really reduce harm, which involves the choice of an experimental model. Here, a three-dimensional (3D) biomimetic chip model was used to evaluate the toxicity of aerosols came from HTP and smoke produced by cigarettes (Cig). Based on cell-related experiments, we found that the toxicity of Cig smoke extract diluted four times was also much higher than that of undiluted HTP, showing higher oxidative stress response and cause mitochondrial dysfunction. Meanwhile, both tobacco products all affect the tricarboxylic acid cycle (TCA), which is manifested by a significant decrease in the mRNA expression of TCA key rate-limiting enzymes. Summarily, 3D Biomimetic chip technology can be used as an ideal model to evaluate HTP. It can provide important data for tobacco risk assessment when 3D chip model was used. Our experimental results showed that HTP may be less harmful than tobacco cigarettes, but it does show significant cytotoxicity with the increase of dose. Therefore, the potential clinical effects of HTP on targeted organs such as lung should be further studied.

Testicular tissue response following a 90-day subchronic exposure to HTP aerosols and cigarette smoke in rats.

Background: Researches have shown that chronic inhalation of cigarette smoke (CS) disrupts male reproductive system, but it is unclear about the mechanisms behind reproductive damages by tobacco toxicants in male rats. This study was designed to explore the effects of heated tobacco products (HTP) aerosols and CS exposure on the testicular health of rats. Materials and Methods: Experiments were performed on male SD rats exposed to filtered air, HTP aerosols at 10 µg/L, 23 µg/L, and 50 µg/L nicotine-equivalent contents, and also CS at 23 µg/L nicotine-equivalent content for 90 days in five exposure groups (coded as sham, HTP_10, HTP_23, HTP_50 and Cig_23). The expression of serum testosterone, testicular tissue inflammatory cytokines (IL-1ß, IL-6, IL-10, TNF-α), reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA), NLRP3 inflammasome-related mRNAs and proteins (NLRP3, ASC, and Caspase-1), the degree of pyroptosis and histopathology were investigated. Results: The results demonstrated that HTP_50 and Cig_23 caused varying degrees of oxidative damage to rat testis, resulting in a decrease of sperm quantity and serum testosterone contents, an increase in the deformity rate, expression levels of proinflammatory cytokines, and NLRP3 inflammasome-related mRNA, and an increase in the NLRP3, ASC, and Caspase-1-immunopositive cells, pyroptosis cell indices, and histopathological damage in the testes of rats. Responses from the HTP_10 and HTP_23 groups were less than those found in the above two exposure groups. Conclusion: These findings indicate that HTP_50 and Cig_23 induced oxidative stress in rat testes, induced inflammation and pyroptosis through the ROS/NLRP3/Caspase-1 pathway, and destroyed the integrity of thetesticular tissue structure.

Tracing the geographical origin of tobacco at two spatial scales by stable isotope and element analyses with chemometrics.

Tobacco is a widely cultivated cash crop, but it is often smuggled and sold illegally. Unfortunately, there is currently no way to verify the origin of tobacco in China. In an effort to address this issue, we conducted a study using stable isotopes and elements from 176 tobacco samples at both provincial and municipal scales. Our findings revealed significant differences in δ13C, K, Cs, and 208/206Pb at the provincial-level, and Sr, Se, and Pb at the municipal level. We created a heat map at the municipal level, which showed a similar cluster classification to geographic grouping and provided an initial assessment of tobacco origins. Using OPLS-DA modeling, we achieved a 98.3% accuracy rate for the provincial scale and 97.6% for the municipal scale. It is worth noting that the importance of rankings of variables varied depending on the spatial scale of the evaluation. This study offers the first traceability fingerprint dataset of tobacco and has the potential to combat mislabeling and fraudulent conduct by identifying the geographical origin of tobacco.

Pharmacokinetics of freebase nicotine and nicotine salts following subcutaneous administration in male rats.

Nicotine lactate, nicotine tartrate, nicotine benzoate, and freebase nicotine (FBN) are four forms of nicotine salt systems that are present in tobacco products. However, few in vivo studies have compared their pharmacological (pK) efficacies, which are important for understanding their roles in the addiction and abuse of tobacco and nicotine products. In this work, the pK of the above nicotine salt systems was studied by subcutaneously injecting their aqueous solutions in rats and obtaining blood samples from the jugular vein. Nicotine levels in the blood were analyzed by LC-MS/MS. The results demonstrated that rapid nicotine absorption occurred in all nicotine systems. Of them, NB had the smallest Tmax , while FBN had the largest Tmax . The nicotine metabolic rate and clearance decreased for FBN, indicating that nicotine retention in the body was higher than for the other three salt-based systems. Compared with nicotine salts, FBN could reach and maintain a higher concentration in the animal model. Additionally, as the benzoic acid ratios increased, the Cmax of the nicotine benzoate (NB) in the plasma decreased. This indicates that the lower the pH, the lower the Cmax . When different concentrations of NB were used, the higher the NB concentration, the greater the Cmax and AUC(0-t) . These results demonstrate that nicotine adsorption by NB in the animal model depended on both pH and concentration. This baseline information could be used to explain different clinical pharmacological observations in humans, though this study only considered the effects of nicotine on pharmacokinetics in vivo.

Stable isotope characterization of tobacco products: A determination of synthetic or natural nicotine authenticity.

RATIONALE: "Tobacco-free" or synthetic nicotine products have appeared in some markets, increasing potential health risks and regulatory compliance challenges. Currently, there are few reliable methods for the determination of authenticity of natural and synthetic nicotine. Analytical techniques based on stable isotopes have broad application prospects in the traceability and identification of agricultural products. METHODS: Tobacco leaves from four main tobacco production regions in China and different types of tobacco products were extracted with n-hexane and 5% sodium hydroxide to obtain nicotine extracts. Subsequent stable isotope mass spectrometry was performed by analyzing δ2 H, δ13 C, and δ15 N values of nicotine. RESULTS: Firstly, results from a batch of 233 samples indicated stable isotopes were closely related to climate and geographical locations and provide a basis for a determination of the origin of tobacco leaves. In addition, the δ2 H values had significant differences between natural and synthetic nicotine and the results indicate a δ2 H value of -163.0‰ could be the threshold for assessing synthetic and natural nicotine. Finally, a total of 239 results further validated the δ2 H value as a metric for source authentication of commercial tobacco products. CONCLUSIONS: Synthetic (S)-(-)-nicotine could be accurately and quickly identified using the method developed by measuring δ2 H values in a qualitative manner. To our knowledge, this is the first time a stable isotope mass spectrometry technique has been used for distinguishing the source of nicotine. This technique will aid in the accurate identification, labelling, and regulation of synthetic nicotine-based tobacco products.

Non-nicotine constituents in cigarette smoke extract enhance nicotine addiction through monoamine oxidase A inhibition.

Tobacco addiction has been largely attributed to nicotine, a component in tobacco leaves and smoke. However, extensive evidence suggests that some non-nicotine components of smoke should not be overlooked when considering tobacco dependence. Yet, their individual effect and synergistic effect on nicotine reinforcement remain poorly understood. The study herein focused on the role of non-nicotine constituents in promoting the effects of nicotine and their independent reinforcing effects. Denicotinized cigarettes were prepared by chemical extracting of cut tobacco, and the cigarette smoke extracts (CSE, used as a proxy for non-nicotine ingredients) were obtained by machine-smoking the cigarettes and DMSO extraction. The compositions of harmful components, nicotine, and other minor alkaloids in both cut tobacco and the CSE of experimental denicotinized cigarettes were examined by GC-MS, and compared with 3R4F reference cigarettes. individually and in synergy with nicotine were determined by conditioned place preference (CPP), dopamine (DA) level detection, the open field test (OFT), and the elevated plus maze (EPM). Finally, the potential enhancement mechanism of non-nicotinic constituents was investigated by nicotine metabolism and monoamine oxidase A (MAOA) activity inhibition in the striatum of mice and human recombinant MAOA. Thenicotine content in smoke from the experimental denicotinized cigarettes (under ISO machine-smoking conditions) was reduced by 95.1% and retained most minor alkaloids, relative to the 3R4F reference cigarettes. It was found that non-nicotine constituents increased acute locomotor activities. This was especially pronounced for DA levels in NAc and CPP scores, decreased the time in center zone. There were no differences in these metrics with DNC group when compared to the NS group. Non-nicotine constituents alone did not show reinforcing effects in CPP or striatum DA levels in mice. However, in the presence of nicotine, non-nicotine constituents further increased the reinforcing effects. Furthermore, non-nicotine constituents may enhance nicotine's reinforcing effects by inhibiting striatum MAOA activity rather than affecting nicotine metabolism or total striatum DA content in mice. These findings expand our knowledge of the effect on smoking reinforcement of non-nicotine constituents found in tobacco products.

A self-administered questionnaire to measure Chinese smokers' cigarette dependence.

A self-administered questionnaire for screening cigarette dependence was developed based on a set of 6335 Chinese adult smokers (termed the China Cigarette Dependence Test, CCDT). Both a 20-item version (CCDT-20) and a 7-item of the questionnaire (CCDT-7) were developed following 2-round of tests on their construct validity, test-retest reliability and internal consistency, covering seven dimensions (cigarettes per day, tolerance, withdrawal symptoms, craving, loss of control, regularity, and stereotypy). The results showed that the CCDT-20 and CCDT-7 scores were higher in daily smokers than in occasional smokers, and both were associated with self-rated nicotine dependence, exhaled carbon monoxide (CO), saliva cotinine, and DSM-V. The CCDT-20 and CCDT-7 scales were found to be easier to use by smokers in China and provided a more reliable measure of their cigarette dependence.

Simultaneous quantification of nicotine salts in e-liquids by LC-MS/MS and GC-MS.

Nicotine salts, formed by nicotine and organic acids, are commonly added to electronic cigarette liquids for their ability to provide desirable sensory effects. Analytical strategies have been developed to detect the types of organic acids and nicotine levels, but methods for directly measuring nicotine salts are still desirable. Herein, a novel approach is presented for the simultaneous quantification of non-volatile and volatile nicotine salts via liquid chromatography/tandem mass spectroscopy (LC-MS/MS) and gas chromatography/mass spectroscopy (GC-MS). This approach was validated with recovery experiments, which yielded recovery values between 92.0% and 110.8%. This method is the first technique for quantifying multiple nicotine salts that could be present in commercial e-liquids. Without using derivatization steps, different nicotine salts could be detected rapidly and conveniently. This new method was demonstrated with 10 e-cigarette liquid samples, providing satisfactory outcomes. It could be used to study organic acids and protonated nicotine in e-liquids and the release behaviour of nicotine salts in electronic cigarettes.

Effects of Smoking on Inflammatory-Related Cytokine Levels in Human Serum.

Cardiovascular and respiratory diseases, and several cancers resulting from tobacco smoking, are initially characterized by chronic systemic inflammation. Cytokine imbalances can result in inflammation, making it important to understand the pathological mechanisms behind cytokine production. In this study, we collected blood samples from 78 healthy male volunteers, including non-smokers (n = 30), current smokers (n = 30), and ex-smokers (n = 18), and utilized the liquid suspension chip technique to investigate and compare the expression levels of 17 cytokines and chemokines in the human serum of these volunteers. The results demonstrated that the expression levels of CXCL9/MIG and sIL-6R significantly increased after smoking, and continued to increase after quitting smoking. The expression levels of TARC, ITAC, and sVEGFR-3 increased after smoking but decreased after quitting smoking; the expression level of SAA significantly decreased after smoking and showed an upward trend after quitting smoking. Seven cytokines (IL-1ß, BCA-1, TNF-α, CRP, ENA-78, MDC, and TNFRII) did not vary between the three groups, while four cytokines (IL-1α, IL-6, IL-8, and SCF) were not detected in any serum sample. In conclusion, this study assessed the physiological production of cytokines and chemokines, highlighting the differences in each due to smoking status. Our results could help evaluate the early development of smoking-related chronic diseases and cancers.

Evaluation of inhalation toxicology after a 90-day xylitol aerosol exposure in Sprague-Dawley rats.

Xylitol is a hygroscopic compound known to protect nasal cavity against bacteria. It has also been developed into nasal spray and evaluated as a potential candidate drug for respiratory diseases. Consequently, it is necessary to study its inhalation toxicity. Based on our previous study on its subacute inhalation toxicity, this study aimed to investigate the safety of xylitol inhalation for long-term use. According to the OECD Test Guideline 413, Sprague-Dawley rats were randomly divided into six groups and exposed with different concentrations of xylitol aerosol or air. After exposure for 90-day, the recovery groups were continued to observe for a recovery period of 28-day. No significant changes in body weight were observed between sham and xylitol groups. Several significant differences in hematological, clinical chemistry, bronchoalveolar lavage fluid were observed, which either had no dose-effect relationship for both male and female rats or were restored during the recovery period. Finally, except for high dose group of xylitol, two rats showed a small amount of inflammatory exudate in alveolar and bronchial cavities, which was restored in the recovery period. The rest of rats showed no obvious difference. For the recovery groups, no significant difference was observed between these two groups. In conclusion, the no observable adverse effect level (NOAEL) of xylitol in our subchronic inhalation toxicological experiments was 2.9 mg/L, which indicated that xylitol for rats' long-time inhalation is tolerant and safe.

In Vitro Toxicological Investigation and Risk Assessment of E-Cigarette Aerosols Based on a Novel Solvent-Free Extraction Method.

Cigarettes, potentially safer alternatives to combustible cigarettes, have been reported to increase the health risk for long-term users, so accumulating information about their potential toxicity is of great concern. However, toxicological evaluations of e-cigarette aerosols are limited, which may be attributed to the lack of a simple and efficient extraction method. Here, we developed a high-speed centrifugal method for extracting e-cigarette aerosol collected mass (ACM) and prepared ACM samples of 26 representative e-cigarettes, and 10 samples were further selected based on their cytotoxicity for systematic toxicological assessments. The average extraction efficiency of ACM, primary aerosol components, and typical carbonyls exceeded 85%. The toxicological evaluation showed that the IC50 value range of e-cigarettes for cytotoxicity was 2-52 mg/mL ACM, all e-cigarettes can induce the risk of DNA damage, mitochondrial depolarization, and c-Jun-related signal disturbances; most e-cigarettes significantly caused disturbance of oxidative stress balance. E-cigarettes with higher cytotoxicity appeared to cause a higher degree of damage, while no e-cigarette promoted mutagenicity and cytochrome c release. The toxicity difference among e-cigarettes using nicotine equivalent was significantly lower than that of ACM. This study provides a novel extraction method and a comprehensive in vitro toxicity risk profile of e-cigarette aerosols.

Enhancement of Biocontrol Efficacy of Pichia kudriavzevii Induced by Ca Ascorbate against Botrytis cinerea in Cherry Tomato Fruit and the Possible Mechanisms of Action.

This study investigated the effect of Ca ascorbate on the biocontrol efficacy of Pichia kudriavzevii and the possible mechanisms. The results indicated that the biocontrol activity of P. kudriavzevii was significantly enhanced by 0.15 g L-1 of Ca ascorbate, with higher growth rates of yeast cells in vitro and in vivo. The antioxidant enzyme activity in P. kudriavzevii, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), were improved by Ca ascorbate and reached the maximum at 96 h, 96 h, and 72 h, respectively. The expression of the antioxidant enzyme-related genes CAT1 (8.55-fold) and SOD2 (7.26-fold) peaked at 96 h, while PRXIID (2.8-fold) peaked at 48 h, which were similar to the trends of enzyme activities. Compared with the control, 0.15 g L-1 of Ca ascorbate and CaCl2 increased the activity of succinate dehydrogenase in P. kudriavzevii, thereby enhancing the utilization of nutrients by yeast cells, and calcium ascorbate had the strongest effect. The expressions of HXT5, ADH6, PET100p, and Pga62 were significantly higher in the Ca ascorbate treatment than the other groups, and the CaCl2 treatment was also significantly higher than the control. These results indicated that Ca ascorbate can effectively improve the energy metabolism and cell wall synthesis and slow down the senescence of yeast cells. In general, Ca ascorbate can improve the environmental adaptability of P. kudriavzevii and thus improve the biocontrol effect, which is associated with inducing antioxidant enzymes in yeast cells and enhancing energy metabolism and nutrient utilization efficiency to increase nutrient competition with pathogens. IMPORTANCE Antagonistic yeast is a promising way to control postharvest fruit decay because of its safety and broad-spectrum resistance. However, the biocontrol efficacy of yeast is limited by environmental stress, such as oxidative stress. Therefore, the improvement of antioxidant capacity has become a research hot spot in improving the biocontrol efficacy of yeast. The induction of Ca ascorbate on the antioxidant capacity and physiological activity of yeast was studied. The results showed better induction of antioxidant enzyme and physiological activity in yeast by Ca ascorbate for better antioxidant capacity, and Ca2+ also played a synergistic promotion effect, which improved the biocontrol efficacy. These results provide an approach for the research and application of improving the environmental adaptability and biocontrol effectiveness of yeast.

Toxicity evaluation in rats following 28 days of inhalation exposure to xylitol aerosol.

Xylitol has reported to decrease gingival inflammation and nasopharyngeal pneumonia, which indicated that xylitol may have potential application in respiratory diseases. Although some studies have reported the inhalation toxicity of xylitol, however, the longest period tested was only for 14 days. The inhalation toxicity of xylitol is insufficient. This work investigated the potential subacute toxicity of xylitol according to the OECD TG 412. Rats were randomly divided into a control group and different dosage groups (2 g/m3, 3 g/m3, 5 g/m3), and exposed for 6 hours/day, 5 days/week for 28 days. At the end of the exposure or recovery period, clinical signs, mortality, body weight, food consumption, hematology, blood biochemistry, gross pathology, organ weight, and histopathology were examined. Compared with the control group, rats of both sexes in the exposure groups exhibited no significant changes in body weight, organ mass, and food uptake. After the xylitol exposure, aspartate aminotransferase activity in the xylitol group (3 g/m3) was significantly higher than that in the control group, while other blood indicators and pathological changes of liver and the analysis of the recovery group showed no changes, suggesting that xylitol exerted no observable toxic effect on the liver. Finally, other observations including the histopathology of target organs and hematology also showed no alterations. These results indicated that xylitol had no significant inhalation toxicity at doses up to 5 g/m3. These subacute inhalation toxicity results of xylitol showed that its no-observed-adverse-effect concentration (NOAEC) in rats was determined to 5 g/m3.

Novel Solvent-Free Extraction Method for Analyzing Tobacco Heating Product Aerosols: An Analytical and In Vitro Toxicological Five-Way Product Comparison.

Harmful and potentially harmful constituents (HPHCs) in tobacco smoke are thought to be responsible for the increased health risks. Tobacco heating products (THPs) heat tobacco instead of burning it to achieve significantly fewer toxicants than conventional cigarettes. To assess the toxicity of THP aerosols, it is often desirable to extract the main constituents using a solvent method. In this study, we developed a high-speed centrifugal method for extracting the total particulate matter (TPM) from THPs to quantitatively compare the toxicity of different THPs and conventional cigarettes. Its TPM extraction efficiency exceeded 85%, and the primary aerosol components and typical HPHCs were comparable to those of the solvent method. The TPMs extracted from five THPs were subjected to 14 in vitro toxicology assessments, and the results were compared with those of a 3R4F reference cigarette. Physical separation can improve biases from solvent selectivity and potential interactions between solvent and aerosol constituents. By eliminating solvent influence, the extraction method could achieve high-dose exposures, enabling the toxicity comparison of different THPs. The relative toxicity of the THPs differed under different dosage units, including the TPM concentration, nicotine equivalent, and puff number.

Simultaneous extraction and surface enhanced Raman spectroscopy detection for the rapid and reliable identification of nicotine released from snus products.

Surface enhanced Raman spectroscopy (SERS) is a highly sensitive analytical detection technique that provides unique chemical and structural information on target molecules. Here, simultaneous extraction and SERS detection of nicotine for the rapid and reliable identification of nicotine released from snus products were performed based on a nano-Au assembly hierarchy structure in the capillary. Based on this strategy, the time evolution of the concentrations of nicotine released from the snus products was measured. Through comparison of the intensities of the spectral peaks of the symmetrical breathing of the pyridine moiety of nicotine molecules, with the prolongation of time, the concentration of nicotine released decreased significantly, which is helpful for establishing a method for the rapid evaluation of the processing and selection of excipients of snus products, and provides a new idea for further study of the production of snus pouches and related tobacco products. Moreover, based on data fitting, it can be calculated that the concentration of nicotine in the extraction presented an obvious quadratic relationship with time, and the release of most of the nicotine in the snus pouch, which is held through the gums and palate, was basically completed after ∼15 min. Such destruction-free simultaneous measurements of snus products are opening up new perspectives for further research about the impact of nicotinoids on smokers' health and cessation programs.

Decreased mitochondrial DNA copy number in nerve cells and the hippocampus during nicotine exposure is mediated by autophagy.

Cigarette smoke is a harmful air pollutant and nicotine dependence is the essential cause of the tobacco epidemic. Since mitochondrial abnormalities are associated with substance addiction, in this work we used mitochondrial DNA (mtDNA) copy number as an indicator of mitochondrial function to investigate whether nicotine addicts also exhibit mitochondrial abnormalities. We found significantly lower mtDNA copy number in the peripheral blood of healthy nicotine addicts than in non-smokers, indicating that long-term nicotine exposure through smoking has detrimental effects on mitochondria. We also examined the effects of nicotine on mtDNA levels in a rat conditioned place preference (CPP) model of addiction and in cultured neuron cells, which revealed that the mtDNA copy number was significantly reduced in the hippocampus of CPP rats, in human neuroblastoma SH-SY5Y cells, and in rat pheochromocytoma PC12 cells, suggesting that significantly reduced mtDNA copy number is a potential biomarker of nicotine addiction. In SH-SY5Y cells, nicotine treatment induced several mitochondrial defects, such as increased mtDNA damage, increased reactive oxygen species (ROS) levels, decreased mitochondrial membrane potential (△Ψm), and stimulation of autophagic flux via transcriptional up-regulation of several autophagy-related genes and elevated marker protein accumulation, although genes controlling mtDNA replication were unaffected. In addition, pretreatment with the autophagy inhibitor Bafilomycin A1 led to accumulation of microtubule-associated protein 1 light chain 3b-II (LC3B-II) and counteracted the nicotine-induced decrease in mtDNA copy number. These results were recapitulated in PC12 cells, which also showed significant down-regulation of the marker SQSTM1/P62, suggesting that the decrease in mtDNA copy number is mediated by autophagy. This study shows that prolonged nicotine exposure, such as that in nicotine addicts, leads to a decrease of mtDNA copy number in neurons due to enhanced induction of autophagy. CAPSULE: It was found that smoking or nicotine exposure decreased mtDNA copy number based on population, animal, and cell models, and these effects appear to be mediated by autophagy.

In vitro toxicological evaluation of a tobacco heating product THP COO and 3R4F research reference cigarette on human lung cancer cells.

Cigarette smoking increases health risks, such as respiratory diseases and heart diseases. Despite the decline in smoking rates in some countries, millions of adults still choose to smoke cigarettes. The use of next-generation nicotine delivery devices, such as tobacco heating products (THPs), may become a potentially safer alternative to smoking. Here, we report on the development of an electrically heated THP, coded as THP COO, with three different flavored tobacco sticks. The purpose of the study was to measure the levels of a list of harmful and potentially harmful constituents (HPHCs) in the total particulate matter (TPM) generated and to conduct a set of toxicological assessments of THP COO as compared with 3R4F reference cigarette. For all 55 HPHCs identified, the levels generated by the THP tobacco sticks were significantly lower in comparison to those in 3R4F TPM. The rate of reduction of HPHCs was between 68.6% and 99.9% under Health Canada Intense (HCI) smoking regimen. Human lung cancer cells (NCI-H292) exposed to 3R4F TPM showed dose-dependent responses for most of the 15 in vitro toxicity endpoints, whereas those exposed to comparable doses of THP COO TPMs did not. Therefore, exclusive use of the THP COO products may reduce the exposure of those tested HPHCs and thus potentially reduce health risk of smoking.