Chronic Exposure to E-Cigarettes Elevates CYP2A5 Activity, Protein Expression, and Cotinine-Induced Production of Reactive Oxygen Species in Mice.

Coumarin 7'-hydroxylase activity, a specific marker of CYP2A5 activity, and the protein level were measured in liver microsomes of male mice after chronic exposure to e-cigarettes (e-cigs) (2.4% nicotine). After exposure for 240 minutes per day for 5 days, the activity and the protein level in preproenkephalin (ppENK)-heterozygous [ppENK (+/-)] mice were significantly elevated (P <0.05) compared with the untreated control. This elevation was not due to deletion of the ppENK gene because the activity did not differ among untreated ppENK (+/-), ppENK (-/-), and wild-type ppENK (+/+) controls. Hence, the elevation can reasonably be attributed to nicotine exposure. The production of reactive oxygen species (ROS) upon incubation of the hepatic microsomes of these mice with cotinine was higher in microsomes from the e-cig-treated mice compared with the untreated controls (P < 0.01). Liquid chromatography mass spectrometry assay showed three oxidation products of cotinine, viz trans 3'-hydroxycotinine (3'-HC), 5'-hydroxycotinine (5'-HC), and cotinine N-oxide (CNO) in the plasma of these mice. The result identifies these three oxidation reactions as the source of the observed ROS and also shows that, in nicotine-treated mice, the appropriate "nicotine metabolite ratio" is (3'-HC + 5'-HC + CNO)/cotinine. The results suggest intriguing possibilities that 1) this metabolite ratio may correlate with plasma nicotine clearance and hence impact nicotine's psychoactive effects and 2) chronic e-cig treatment causes ROS-induced oxidative stress, which may play a major role in the regulation of CYP2A5 expression. Our present results clearly show that both the activity and the protein level of CYP2A5 are elevated by repeated exposure to nicotine. SIGNIFICANCE STATEMENT: Nicotine, the psychoactive ingredient of tobacco, is eliminated as the oxidation products of cotinine in reactions catalyzed by the enzymes CYP2A5 in mice and CYP2A6 in humans. This study shows that repeated exposure to e-cigarettes elevates the level of CYP2A5 and the formation of reactive oxygen species. The results suggest an intriguing possibility that CYP2A5 may be upregulated by chronic nicotine exposure due to oxidative stress caused by the oxidation of cotinine in this preclinical model of human smokers.

Inflammatory marker levels in children with tobacco smoke exposure.

BACKGROUND: Tobacco smoke exposure (TSE) has inflammatory and immunosuppressive effects which may be associated with altered levels of inflammatory markers and pediatric illnesses. OBJECTIVE: The primary objective was to examine the associations of cotinine-confirmed and parent-reported child TSE patterns and discharge diagnoses with C-reactive protein (CRP), IL-8, and IL-10 in 0-11-year-old pediatric emergency department (PED) patients who lived with ≥ 1 smoker. METHODS: Saliva samples were obtained from 115 children with a mean (SD) age of 3.5 (3.1) years during the PED visit (T0). Saliva was analyzed for cotinine, CRP, IL-8, and IL-10. Parents self-reported their children's TSE patterns; children's medical records were reviewed to identify and categorize discharge diagnoses. Linear regression models were utilized to find T0 associations of cotinine-confirmed and parent-reported child TSE patterns, and PED diagnoses with each inflammatory marker. All models were adjusted for child race/ethnicity, child sex, annual household income, and housing type. The TSE models also adjusted for child discharge diagnosis. RESULTS: At T0, the geometric mean (GeoM) of cotinine was 4.1 ng/ml [95 %CI = 3.2-5.2]; the GeoMs of CRP, IL-8, and IL-10 were 3,326 pg/ml [95 %CI = 2,696-4,105], 474 pg/ml [95 %CI = 386-583], and 1.1 pg/ml [95 %CI = 0.9-1.3], respectively. Parent-reported child TSE patterns were positively associated with ln-transformed CRP levels, while adjusting for the covariates (ß^ = 0.012 [95 %CI:0.004-0.020], p = 0.037). In the parent-reported child TSE pattern model, there were significant positive associations between the covariate of child age with CRP and IL-8 levels (p = 0.028 and p < 0.001, respectively). Children with a bacterial diagnosis had higher IL-8 levels (p = 0.002) compared to the other diagnosis groups. CONCLUSIONS: Results indicate that parent-reported child TSE increases the expression of CRP in ill children and supports prior work demonstrating that IL-8 is higher in children with TSE who have bacterial infections. These findings should be examined in future research with ill children with and without TSE.

Characterization of cotinine degradation in a newly isolated Gram-negative strain Pseudomonas sp. JH-2.

Cotinine, the primary metabolite of nicotine in the human body, is an emerging pollutant in aquatic environments. It causes environmental problems and is harmful to the health of humans and other mammals; however, the mechanisms of its biodegradation have been elucidated incompletely. In this study, a novel Gram-negative strain that could degrade and utilize cotinine as a sole carbon source was isolated from municipal wastewater samples, and its cotinine degradation characteristics and kinetics were determined. Pseudomonas sp. JH-2 was able to degrade 100 mg/L (0.56 mM) of cotinine with high efficiency within 5 days at 30 ℃, pH 7.0, and 1% NaCl. Two intermediates, 6-hydroxycotinine and 6-hydroxy-3-succinoylpyridine (HSP), were identified by high-performance liquid chromatography and liquid chromatograph mass spectrometer. The draft whole genome sequence of strain JH-2 was obtained and analyzed to determine genomic structure and function. No homologs of proteins predicted in Nocardioides sp. JQ2195 and reported in nicotine degradation Pyrrolidine pathway were found in strain JH-2, suggesting new enzymes that responsible for cotinine catabolism. These findings provide meaningful insights into the biodegradation of cotinine by Gram-negative bacteria.

Affinity of Nicotinoids to a Model Nicotinic Acetylcholine Receptor (nAChR) Binding Pocket in the Human Brain.

The binding affinity of nicotinoids to the binding residues of the α4ß2 variant of the nicotinic acetylcholine receptor (nAChR) was identified as a strong predictor of the nicotinoid's addictive character. Using ab initio calculations for model binding pockets of increasing size composed of 3, 6, and 14 amino acids (3AA, 6AA, and 14AA) that are derived from the crystal structure, the differences in binding affinity of 6 nicotinoids, namely, nicotine (NIC), nornicotine (NOR), anabasine (ANB), anatabine (ANT), myosmine (MYO), and cotinine (COT) were correlated to their previously reported doses required for increases in intracranial self-stimulation (ICSS) thresholds, a metric for their addictive function. By employing the many-body decomposition, the differences in the binding affinities of the various nicotinoids could be attributed mainly to the proton exchange energy between the pyridine and non-pyridine rings of the nicotinoids and the interactions between them and a handful of proximal amino acids, namely Trp156, Trpß57, Tyr100, and Tyr204. Interactions between the guest nicotinoid and the amino acids of the binding pocket were found to be mainly classical in nature, except for those between the nicotinoid and Trp156. The larger pockets were found to model binding structures more accurately and predicted the addictive character of all nicotinoids, while smaller models, which are more computationally feasible, would only predict the addictive character of nicotinoids that are similar to nicotine. The present study identifies the binding affinity of the guest nicotinoid to the host binding pocket as a strong descriptor of the nicotinoid's addiction potential, and as such it can be employed as a fast-screening technique for the potential addiction of nicotine analogs.

Mass spectrometry-based metabolomics study of nicotine exposure in THP-1 monocytes.

The tobacco alkaloid nicotine is known for its activation of neuronal nicotinic acetylcholine receptors. Nicotine is consumed in different ways such as through conventional smoking, e-cigarettes, snuff or nicotine pouches. The use of snuff has been associated with several adverse health effects, such as inflammatory reactions of the oral mucosa and oral cavity cancer. We performed a metabolomic analysis of nicotine-exposed THP-1 human monocytes. Cells were exposed to 5 mM of the alkaloid for up to 4 h, and cell extracts and medium subjected to untargeted liquid chromatography high-resolution mass spectrometry. Raw data processing revealed 17 nicotine biotransformation products. Among these, cotinine and nornicotine were identified as the two major cellular biotransformation products. The application of multi- and univariate statistical analyses resulted in the annotation, up to a certain level of identification, of 12 compounds in the cell extracts and 13 compounds in the medium that were altered by nicotine exposure. Of these, four were verified as methylthioadenosine, cytosine, uric acid, and L-glutamate. Methylthioadenosine levels were affected in both cells and the medium, while cytosine, uric acid, and L-glutamate levels were affected in the medium only. The effects of smoking on the pathways involving these metabolites have been previously demonstrated in humans. Most of the other discriminating compounds, which were merely tentatively or not fully identified, were amino acids or amino acid derivatives. In conclusion, our preliminary data suggest that some of the potentially adverse effects related to smoking may also be expected when nicotine is consumed via snuff or nicotine pouches.

Smoking primes the metabolomic response in trauma.

INTRODUCTION: Smoking is a public health threat because of its well-described link to increased oxidative stress-related diseases including peripheral vascular disease and coronary artery disease. Tobacco use has been linked to risk of inpatient trauma morbidity including acute respiratory distress syndrome; however, its mechanistic effect on comprehensive metabolic heterogeneity has yet to be examined. METHODS: Plasma was obtained on arrival from injured patients at a Level 1 trauma center and analyzed with modern mass spectrometry-based metabolomics. Patients were stratified by nonsmoker, passive smoker, and active smoker by lower, interquartile, and upper quartile ranges of cotinine intensity peaks. Patients were substratified by high injury/high shock (Injury Severity Score, ≥15; base excess, <-6) and compared with healthy controls. p Value of <0.05 following false discovery rate correction of t test was considered significant. RESULTS: Forty-eight patients with high injury/high shock (7 nonsmokers [15%], 25 passive smokers [52%], and 16 active smokers [33%]) and 95 healthy patients who served as controls (30 nonsmokers [32%], 43 passive smokers [45%], and 22 active smokers [23%]) were included. Elevated metabolites in our controls who were active smokers include enrichment in chronic inflammatory and oxidative processes. Elevated metabolites in active smokers in high injury/high shock include enrichment in the malate-aspartate shuttle, tyrosine metabolism, carnitine synthesis, and oxidation of very long-chain fatty acids. CONCLUSION: Smoking promotes a state of oxidative stress leading to mitochondrial dysfunction, which is additive to the inflammatory milieu of trauma. Smoking is associated with impaired mitochondrial substrate utilization of long-chain fatty acids, aspartate, and tyrosine, all of which accentuate oxidative stress following injury. This altered expression represents an ideal target for therapies to reduce oxidative damage toward the goal of personalized treatment of trauma patients. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level IV.

Validity of Expired Carbon Monoxide and Urine Cotinine Using Dipstick Method to Assess Smoking Status / 예방의학회지

OBJECTIVES: We investigated the validity of the dipstick method (Mossman Associates Inc. USA) and the expired CO method to distinguish between smokers and nonsmokers. We also elucidated the related factors of the two methods. METHODS: This study included 244 smokers and 50 exsmokers, recruited from smoking cessation clinics at 4 local public health centers, who had quit for over 4 weeks. We calculated the sensitivity, specificity and Kappa coefficient of each method for validity. We obtained ROC curve, predictive value and agreement to determine the cutoff of expired air CO method. Finally, we elucidated the related factors and compared their effect powers using the standardized regression coefficient. RESULTS: The dipstick method showed a sensitivity of 92.6%, specificity of 96.0% and Kappa coefficient of 0.79. The best cutoff value to distinguish smokers was 5-6ppm. At 5 ppm, the expired CO method showed a sensitivity of 94.3%, specificity of 82.0% and Kappa coefficient of 0.73. And at 6 ppm, sensitivity, specificity and Kappa coefficient were 88.5%, 86.0% and 0.64, respectively. Therefore, the dipstick method had higher sensitivity and specificity than the expired CO method. The dipstick and expired CO methods were significantly increased with increasing smoking amount. With longer time since the last smoking, expired CO showed a rapid decrease after 4 hours, whereas the dipstick method showed relatively stable levels for more than 4 hours. CONCLUSIONS: The dipstick and expired CO methods were both good indicators for assessing smoking status. However, the former showed higher sensitivity and specificity and stable levels over longer hours after smoking, compared to the expired CO method.