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.

Impact of nicotine and cotinine on macrophage inflammatory plasticity via vesicular modifications in gastrointestinal bacteria.

OBJECTIVES: This study aimed to elucidate mechanistic explanation(s) for compositional changes to enteric microbiota by determining the impacts of continuous nicotine/cotinine exposure on representative gastrointestinal bacteria and how these alterations impact innate immune cell plasticity. METHODS: In vitro cultures of the gastrointestinal bacteria (Bacteroides fragilis 25285, Prevotella bryantii B14, and Acetoanaerobium sticklandii SR) were continuously exposed to nicotine or cotinine. Supernatant samples were collected for fermentation acid analysis. Vesicles were collected and analyzed for physiological changes in number, size, and total protein cargo. Cultured macrophages were stimulated to a tolerogenic phenotype, exposed to control or altered (nicotine or cotinine - exposed) vesicles, and inflammatory plasticity assessed via inflammatory cytokine production. RESULTS: Nicotine/cotinine exposure differentially affected metabolism of all bacteria tested in a Gram (nicotine) and concentration-dependent (cotinine) manner. Physiological studies demonstrated changes in vesiculation number and protein cargo following nicotine/cotinine exposures. Continuous exposure to 1 µM nicotine and 10 µM cotinine concentrations reduced total protein cargo of Gram (-) - 25285 and B14 vesicles, while cotinine generally increased total protein in Gram (+) - SR vesicles. We found that theses physiological changes to the vesicles of 25285 and SR formed under nicotine and cotinine, respectively, challenged the plasticity of tolerogenic macrophages. Tolerogenic macrophages exposed to vesicles from 1 µM nicotine, and 5 or 10 µΜ cotinine cultures produced significantly less IL-12p70, TNFα, or KC/GRO, regardless of macrophage exposure to nicotine/cotinine. CONCLUSIONS: Nicotine/cotinine exposure differentially alters bacterial metabolism and vesicle physiology, ultimately impacting the inflammatory response of tolerogenic macrophages.

The Associations of Trans-3'-Hydroxy Cotinine, Cotinine, and the Nicotine Metabolite Ratio in Pediatric Patients with Tobacco Smoke Exposure.

(1) Background: Trans-3'-hydroxy cotinine (3HC) and cotinine (COT) are tobacco smoke exposure (TSE) biomarkers and the 3HC/COT ratio is a marker of CYP2A6 activity, an enzyme which metabolizes nicotine. The primary objective was to assess the associations of these TSE biomarkers with sociodemographics and TSE patterns in children who lived with ≥1 smoker. (2) Methods: A convenience sample of 288 children (mean age (SD) = 6.42 (4.8) years) was recruited. Multiple linear regression models were built to assess associations of sociodemographics and TSE patterns with urinary biomarker response variables: (1) 3HC, (2) COT, (3) 3HC+COT sum, and (4) 3HC/COT ratio. (3) Results: All children had detectable 3HC (Geometric Mean [GeoM] = 32.03 ng/mL, 95%CI = 26.97, 38.04) and COT (GeoM = 10.24 ng/mL, 95%CI = 8.82, 11.89). Children with higher cumulative TSE had higher 3HC and COT (ß^ = 0.03, 95%CI = 0.01, 0.06, p = 0.015 and ß^ = 0.03, 95%CI = 0.01, 0.05, p = 0.013, respectively). Highest 3HC+COT sum levels were in children who were Black (ß^ = 0.60, 95%CI = 0.04, 1.17, p = 0.039) and who had higher cumulative TSE (ß^ = 0.03, 95%CI = 0.01, 0.06, p = 0.015). Lowest 3HC/COT ratios were in children who were Black (ß^ = -0.42, 95%CI = -0.78, -0.07, p = 0.021) and female (ß^ = -0.32, 95%CI = -0.62, -0.01, p = 0.044). (4) Conclusion: Results indicate that there are racial and age-related differences in TSE, most likely due to slower nicotine metabolism in non-Hispanic Black children and in younger children.

Acute nicotine exposure blocks aromatase in the limbic brain of healthy women: A [11C]cetrozole PET study.

BACKGROUND: Of interest to women's mental health, a wealth of studies suggests sex differences in nicotine addiction and treatment response, but their psychoneuroendocrine underpinnings remain largely unknown. A pathway involving sex steroids could indeed be involved in the behavioural effects of nicotine, as it was found to inhibit aromatase in vitro and in vivo in rodents and non-human primates, respectively. Aromatase regulates the synthesis of oestrogens and, of relevance to addiction, is highly expressed in the limbic brain. METHODS: The present study sought to investigate in vivo aromatase availability in relation to exposure to nicotine in healthy women. Structural magnetic resonance imaging and two [11C]cetrozole positron emission tomography (PET) scans were performed to assess the availability of aromatase before and after administration of nicotine. Gonadal hormones and cotinine levels were measured. Given the region-specific expression of aromatase, a ROI-based approach was employed to assess changes in [11C]cetrozole non-displaceable binding potential. RESULTS: The highest availability of aromatase was found in the right and left thalamus. Upon nicotine exposure, [11C]cetrozole binding in the thalamus was acutely decreased bilaterally (Cohen's d = -0.99). In line, cotinine levels were negatively associated with aromatase availability in the thalamus, although as non-significant trend. CONCLUSIONS: These findings indicate acute blocking of aromatase availability by nicotine in the thalamic area. This suggests a new putative mechanism mediating the effects of nicotine on human behaviour, particularly relevant to sex differences in nicotine addiction.

Quantitation of Ten Urinary Nicotine Metabolites, Including 4-Hydroxy-4-(3-pyridyl) Butanoic Acid, a Product of Nicotine 2'-Oxidation, and CYP2A6 Activity in Japanese Americans, Native Hawaiians, and Whites.

Smoking intensity varies across smokers and is influenced by individual variability in the metabolism of nicotine, the major addictive agent in tobacco. Therefore, lung cancer risk, which varies by racial ethnic group, is influenced by the primary catalyst of nicotine metabolism, cytochrome P450 2A6 (CYP2A6). In smokers, CYP2A6 catalyzes nicotine 5'-oxidation. In vitro, CYP2A6 also catalyzes, to a much lower extent, 2'-oxidation, which leads to the formation of 4-hydroxy-4-(3-pyridyl) butanoic acid (hydroxy acid). The urinary concentration of hydroxy acid has been quantified in only a few small studies of White smokers. To quantitatively assess the importance of nicotine 2'-oxidation in smokers, an LC-MS/MS-based method was developed for the analysis of nicotine and ten metabolites in urine. The concentrations of nicotine and these metabolites were measured in 303 smokers (99 Whites, 99 Native Hawaiians, and 105 Japanese Americans), and the relative metabolism of nicotine by four pathways was determined. Metabolism by these pathways was also compared across quartiles of CYP2A6 activity (measured as the plasma ratio of 3-hydroxycotinine to cotinine). As reported previously and consistent with their average CYP2A6 activity, nicotine 5'-oxidation was highest in Whites and lowest in Japanese Americans. Nicotine N-glucuronidation and N-oxidation increased with decreasing CYP2A6 activity. However, the relative urinary concentration of hydroxy acid (mean, 2.3%; 95% CI, 2.2-2.4%) did not vary by ethnic group or by CYP2A6 activity. In summary, CYP2A6 is not an important catalyst of nicotine 2'-oxidation in smokers, nor does nicotine 2'-oxidation compensate for decreased CYP2A6 activity.

Inhibition of Nicotine Metabolism by Cannabidiol (CBD) and 7-Hydroxycannabidiol (7-OH-CBD).

Cannabis-based products have experienced notable increases in co-usage alongside tobacco products. Several cannabinoids exhibit inhibition of a number of cytochrome P450 (CYP) and UDP glucuronosyltransferase (UGT) enzymes, but few studies have examined their inhibition of enzymes involved in nicotine metabolism. The goal of the present study was to examine potential drug-drug interactions occurring in the nicotine metabolism pathway perpetrated by cannabidiol (CBD) and its active metabolite, 7-hydroxy-CBD (7-OH-CBD). The inhibitory effects of CBD and 7-OH-CBD were tested in microsomes from HEK293 cells overexpressing individual metabolizing enzymes and from human liver tissue. Assays with overexpressing microsomes demonstrated that CBD and 7-OH-CBD inhibited CYP-mediated nicotine metabolism. Binding-corrected IC50,u values for CBD inhibition of nicotine metabolism to cotinine and nornicotine, and cotinine metabolism to trans-3'-hydroxycotinine (3HC), were 0.27 ± 0.060, 0.23 ± 0.14, and 0.21 ± 0.14 µM, respectively, for CYP2A6; and 0.26 ± 0.17 and 0.029 ± 0.0050 µM for cotinine and nornicotine formation, respectively, for CYP2B6. 7-OH-CBD IC50,u values were 0.45 ± 0.18, 0.16 ± 0.08, and 0.78 ± 0.23 µM for cotinine, nornicotine, and 3HC formation, respectively, for CYP2A6, and 1.2 ± 0.44 and 0.11 ± 0.030 µM for cotinine and nornicotine formation, respectively, for CYP2B6. Similar IC50,u values were observed in HLM. Inhibition (IC50,u = 0.37 ± 0.06 µM) of 3HC to 3HC-glucuronide formation by UGT1A9 was demonstrated by CBD. Significant inhibition of nicotine metabolism pathways by CBD and 7-OH-CBD suggests that cannabinoids may inhibit nicotine metabolism, potentially impacting tobacco addiction and cessation.

Prolonged, physiologically relevant nicotine concentrations in the airways of smokers.

Nicotine from cigarette smoke is a biologically active molecule that has pleiotropic effects in the airway, which could play a role in smoking-induced lung disease. However, whether nicotine and its metabolites reach sustained, physiologically relevant concentrations on airway surfaces of smokers is not well defined. To address these issues, concentrations of nicotine, cotinine, and hydroxycotinine were measured by mass spectrometry (MS) in supernatants of induced sputum obtained from participants in the subpopulations and intermediate outcome measures in COPD study (SPIROMICS), an ongoing observational study that included never smokers, former smokers, and current smokers with and without chronic obstructive pulmonary disease (COPD). A total of 980 sputum supernatants were analyzed from 77 healthy never smokers, 494 former smokers (233 with COPD), and 396 active smokers (151 with COPD). Sputum nicotine, cotinine, and hydroxycotinine concentrations corresponded to self-reported smoking status and were strongly correlated to urine measures. A cutoff of ∼8-10 ng/mL of sputum cotinine distinguished never smokers from active smokers. Accounting for sample dilution during processing, active smokers had airway nicotine concentrations in the 70-850 ng/mL (∼0.5-5 µM) range, and concentrations remained elevated even in current smokers who had not smoked within 24 h. This study demonstrates that airway nicotine and its metabolites are readily measured in sputum supernatants and can serve as biological markers of smoke exposure. In current smokers, nicotine is present at physiologically relevant concentrations for prolonged periods, supporting a contribution to cigarette-induced airway disease.

CYP2C19 Plays a Major Role in the Hepatic N-Oxidation of Cotinine.

The primary mode of metabolism of nicotine is via the formation of cotinine by the enzyme CYP2A6. Cotinine undergoes further CYP2A6-mediated metabolism by hydroxylation to 3-hydroxycotinine and norcotinine, but can also form cotinine-N-glucuronide and cotinine-N-oxide (COX). The goal of this study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway. Specific inhibitors of major hepatic cytochrome P450 (P450) enzymes were used in cotinine-N-oxidation reactions using pooled human liver microsomes (HLMs). COX formation was monitored by ultrahigh-pressure liquid chromatography-tandem mass spectrometry and enzyme kinetic analysis was performed using microsomes from P450-overexpressing human embryonic kidney 293 (HEK293) cell lines. Genotype-phenotype analysis was performed in a panel of 113 human liver specimens. Inhibition of COX formation was only observed in HLMs when using inhibitors of CYP2A6, CYP2B6, CYP2C19, CYP2E1, and CYP3A4. Microsomes from cells overexpressing CYP2A6 or CYP2C19 exhibited similar N-oxidation activity against cotinine, with maximum reaction rate over Michaelis constant values (intrinsic clearance) of 4.4 and 4.2 nL/min/mg, respectively. CYP2B6-, CYP2E1-, and CYP3A4-overexpressing microsomes were also active in COX formation. Significant associations (P < 0.05) were observed between COX formation and genetic variants in CYP2C19 (*2 and *17 alleles) in HLMs. These results demonstrate that genetic variants in CYP2C19 are associated with decreased COX formation, potentially affecting the relative levels of cotinine in the plasma or urine of smokers and ultimately affecting recommended smoking cessation therapies. SIGNIFICANCE STATEMENT: This study is the first to elucidate the enzymes responsible for cotinine-N-oxide formation and genetic variants that affect this biological pathway. Genetic variants in CYP2C19 have the potential to modify nicotine metabolic ratio in smokers and could affect pharmacotherapeutic decisions for smoking cessation treatments.

Predicting nicotine metabolism across ancestries using genotypes.

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.

Nicotine and cotinine quantification after a 4-week inhalation of electronic cigarette vapors in male and female mice using UPLC-MS/MS.

OBJECTIVES: To detect the cotinine and nicotine serum concentrations of female and male C57BL/6J mice after a 4-week exposure to electronic (e)-cigarette vapors using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: This experimental study was carried out at an animal facility and laboratories, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, between January and August 2020. A 4-week exposure to e-cigarettes was carried out using male and female mice and serum samples were obtained for cotinine and nicotine quantification using UPLC-MS/MS. The chromatographic procedures involved the use of a BEH HSS T3 C18 column (100 mm x 2.1 mm, 1.7 µm) with acetonitrile as a mobile phase and 0.1% formic acid (2:98 v/v). RESULTS: The applied methodology has highly efficient properties of detection, estimation, and extraction, where the limit of quantification (LOQ) for nicotine was 0.57 ng/mL and limit of detection (LOD) for nicotine was 0.19 ng/mL, while the LOQ for cotinine was 1.11 ng/mL and LOD for cotinine was 0.38 ng/mL. The correlation coefficient was r2>0.99 for both compounds. The average recovery rate was 101.6±1.33 for nicotine and 100.4±0.54 for cotinine, while the precision and accuracy for cotinine and nicotine were less than 6.1. The serum cotinine level was higher in males (433.7±19.55) than females (362.3±16.27). CONCLUSION: This study showed that the gender factor might play a crucial role in nicotine metabolism.

Melatonin prevents oocyte deterioration due to cotinine exposure in mice†.

Levels of cotinine, a major metabolite of nicotine, have been positively correlated with risks of cigarette smoking-related diseases. Melatonin is synthesized by the pineal gland and has been demonstrated to be beneficial to oocyte maturation due to its antioxidative activity. In this study, we investigated the effects of cotinine on mouse oocyte meiosis and the protective roles of melatonin in vitro and in vivo. The results showed that cotinine exposure caused defects in the first polar body extrusion and reduced parthenogenetic activation in in vitro-matured oocytes. Additionally, cotinine exposure increased the level of oxidative stress, which resulted in aberrant actin distribution, abnormal spindle morphology, chromosome misalignment, and even oocyte aneuploidy. Simultaneously, cotinine exposure decreased the mitochondrial membrane potential and antioxidant gene expression and increased apoptosis-related gene expression. However, all these toxic effects of cotinine could be reversed after the addition of melatonin, and the mechanism may be a decrease in reactive oxygen species production. In conclusion, cotinine causes poor oocyte quality, which could be rescued by melatonin supplementation during meiotic maturation in mouse oocytes.

Parahydrogen hyperpolarization of minimally altered urine samples for sensitivity enhanced NMR metabolomics.

Parahydrogen hyperpolarization has been shown to enhance NMR sensitivity in urine analysis by several orders of magnitude if urine samples are prepared by solid phase extraction (SPE). We present a different approach, developed for minimal sample alteration before analysis. Removing SPE from the workflow allows to retain a wider range of metabolites and paves the way towards more universal hyperpolarized NMR metabolomics of low abundance metabolites.

Secondhand smoke exposure in school children in Malta assessed through urinary biomarkers.

School children may be exposed to secondhand smoke (SHS) either at home, in transit or in social gatherings permitting smoking in their presence. Questionnaires about SHS often underestimate prevalence and extent of exposure. A more accurate tool is the use of biomarkers such as cotinine (COT) and trans-3'-hydrocycotinine (3HC) as biomarkers of SHS exposure, alongside 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a reduction product in the body of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), both potent carcinogens. We measured urinary COT, 3HC and total NNAL using sensitive and specific high-performance LC-MS/MS methods. The limit of quantification (LOQ) for each assay were 0.05 ng/mL, 0.1 ng/mL and 0.25 pg/mL respectively. The aim of this study was to evaluate the exposure to SHS of school children (9-11 years), from five public schools in the island of Malta, from questionnaire information about smoking at home and verify it by urinary biomarker data of COT, 3HC and NNAL. These biomarkers were measurable in 99.4%, 95.4% and 98.3% of the participating children respectively. From the children reporting smoking at home, 11% had a history of asthma and had COT, 3HC and NNAL geometric mean concentrations double compared to the non-asthmatic group. In has been confirmed that non-smokers exposed to SHS and THS have a higher NNAL/COT ratio than the group identified as smokers according to specific and defined COT threshold levels (despite the fact that a priori, the entire study group was composed of non-smokers). The implication of high measured levels of urinary NNAL in children should be of concern given its potency. A main effects multifactor ANOVA model was developed and the children's house and school locations and the smoking frequency were statistically significant to predict the levels of the three metabolites. For 3HC only, the status of the employment of the mother was also an important predictor.

Trans-placental transfer of nicotine: Modulation by organic cation transporters.

Nicotine is a highly addictive substance and harmful to the developing foetus. However, few studies have investigated the transporter mechanism responsible for regulating the transfer of nicotine across the blood-placental interface. A multiple in-vivo microdialysis system coupled to ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed to monitor simultaneously nicotine and cotinine in the blood, placenta, foetus, and amniotic fluid of pregnant rats. The pharmacological mechanism of nicotine transfer across the placenta was investigated by co-administering corticosterone, an inhibitor of organic cation transporters (OCTs) that partly mediate the exchange of nicotine across the placenta. The results revealed that intravenously administered nicotine (1 mg/kg) was rapidly metabolised to cotinine with a transformation ratio (AUCcotinine/AUCnicotine) of 0.67 ± 0.08, 0.21 ± 0.05, 0.25 ± 0.12, 0.31 ± 0.05 in maternal blood, placenta, amniotic fluid, and foetus, respectively. The tissue transformation ratios (AUCtissue/AUCblood) were 0.83 ± 0.16, 0.65 ± 0.17, 0.57 ± 0.13 for nicotine, and 0.25 ± 0.06, 0.24 ± 0.12, 0.26 ± 0.04 for cotinine at placenta, amniotic fluid and foetus, respectively. Following the co-administration of corticosterone (2 mg/kg), the tissue transformation ratio of nicotine was significantly reduced in the placenta but was significantly increased in the foetus. Levels of cotinine were not significantly altered by the administration of corticosterone. These findings implicate OCT in mediating the transfer of nicotine across the blood-placenta barrier. Understanding the mechanism of nicotine transfer through the placenta may inform therapeutic strategies to lessen the exposure of the developing foetus to nicotine in the maternal bloodstream.

A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets.

Natural killer (NK) cells are immune effector cells with outstanding features for adoptive immunotherapy. Immune effector cells with chimeric antigen receptors (CARs) are promising targeted therapeutic agents for various diseases. Because tumor cells exhibit heterogeneous antigen expression and lose cell surface antigen expression during malignant progression, many CARs fixed against only one antigen have limited efficacy and are associated with tumor relapse. To expand the utility of CAR-NK cells, we designed a split and universal cotinine-CAR (Cot-CAR) system, comprising a Cot-conjugator and NK92 cells (α-Cot-NK92 cells) engineered with a CAR containing an anti-Cot-specific single-chain variable fragment and intracellular signaling domain. The efficacy of the Cot-CAR system was assessed in vitro using a cytolysis assay against various tumor cells, and its single- or multiple- utility potential was demonstrated using an in vivo lung metastasis model by injecting A549-Red-Fluc cells. The α-Cot-NK92 cells could switch targets, logically respond to multiple antigens, and tune cytolytic activation through the alteration of conjugators without re-engineering. Therefore the universal Cot-CAR system is useful for enhancing specificity and diversity of antigens, combating relapse, and controlling cytolytic activity. In conclusion, this universal Cot-CAR system reveals that multiple availability and controllability can be generated with a single, integrated system.

Cotinine and cytokine levels in the vitreous body and blood serum of smokers and non-smokers - A pilot study.

Tobacco smoking is a risk factor for many ocular diseases. Of the multiple tobacco smoke compounds nicotine and its main metabolite cotinine are likely agents in disease modulation. The interaction of these compounds with exposed tissue is complex and ranges from proinflammatory to potentially neuroprotective properties. We aimed to determine cotinine and cytokines in the vitreous in smokers and non-smokers in this prospective, cross-sectional study at the Department of Ophthalmology, Medical University Graz, Austria. We included 10 smokers and 10 non-smokers. Vitreous and serum samples were analyzed for cotinine and cytokines. The cytokine analysis was performed with multiplex assay and cotinine was quantified with enzyme-linked immunosorbent assay. Cotinine was detectable in smokers only with a mean of 154.0 ng/ml ± 107.3 ng/ml in the vitreous and of 194.1 ng/ml ± 121.3 ng/ml in the serum. The difference between intraocular and systemic levels was statistically significant. There were no statistically significant differences between the cytokine levels of smokers and non-smokers. However, intravitreal VEGF-A was by trend elevated in smokers and correlated positively with intravitreal cotinine (r = 0.59, p = 0.073). In conclusion cotinine is detectable in the vitreous of smokers and is lower than the serum. There is a trend towards elevation of VEGF-A in the vitreous of smokers.

Cotinine Hydroxylase CotA Initiates Biodegradation of Wastewater Micropollutant Cotinine in Nocardioides sp. Strain JQ2195.

Cotinine is a stable toxic contaminant, produced as a by-product of smoking. It is of emerging concern due to its global distribution in aquatic environments. Microorganisms have the potential to degrade cotinine; however, the genetic mechanisms of this process are unknown. Nocardioides sp. strain JQ2195 is a pure-culture strain that has been reported to degrade cotinine at micropollutant concentrations. This strain utilizes cotinine as its sole carbon and nitrogen source. In this study, a 50-kb gene cluster (designated cot), involved in cotinine degradation, was predicted based on genomic and transcriptomic analyses. A novel three-component cotinine hydroxylase gene (designated cotA1A2A3), which initiated cotinine catabolism, was identified and characterized. CotA from Shinella sp. strain HZN7 was heterologously expressed and purified and was shown to convert cotinine into 6-hydroxycotinine. H218O-labeling and electrospray ionization-mass spectrometry (ESI-MS) analysis confirmed that the hydroxyl group incorporated into 6-hydroxycotinine was derived from water. This study provides new molecular insights into the microbial metabolism of heterocyclic chemical pollutants. IMPORTANCE In the human body, cotinine is the major metabolite of nicotine, and 10 to 15% of generated cotinine is excreted in urine. Cotinine is a structural analogue of nicotine and is much more stable than nicotine. Increased tobacco consumption has led to high environmental concentrations of cotinine, which may have detrimental effects on aquatic ecosystems and human health. Nocardioides sp. strain JQ2195 is a unique cotinine-degrading bacterium. However, the underlying genetic and biochemical foundations of cotinine degradation are still unknown. In this study, a 50-kb gene cluster (designated cot) was identified by genomic and transcriptomic analyses as being involved in the degradation of cotinine. A novel three-component cotinine hydroxylase gene (designated cotA1A2A3) catalyzed cotinine to 6-hydroxy-cotinine. This study provides new molecular insights into the microbial degradation and enzymatic transformation of cotinine.

Cotinine Fluctuation in Maternal Saliva During and After Pregnancy: Implications for Perinatal Outcomes.

PURPOSE: The purpose of this study was to determine maternal cotinine levels in saliva at the first (T-1) and third trimesters of pregnancy (T-2), and postpartum (T-3) among women who reported smoking a consistent number of cigarettes. The goal was to generate data to provide suggestions about how nurses can improve patient outcomes for women who smoke. METHODS: Saliva cotinine values obtained from the randomized controlled trial (BabyBEEP) were used for this secondary analysis. We examined variations in saliva cotinine between the first and third trimesters of pregnancy and postpartum among light and heavy smokers. Cotinine values for pregnant smokers who were in the control group (not randomized to any intervention) and reported smoking the same range of cigarettes during and after pregnancy were used for the analyses (n = 43). Descriptive statistics and repeated-measures ANOVA were used for analyses. RESULTS: There were differences in maternal saliva cotinine levels across the different time points and between the groups. There was a significant interaction effect between time and group. Cotinine decreased from T-1 to T-2 in heavy smokers and increased from T-2 to T-3 among both light and heavy smokers. Cotinine at T-3 was higher than T-1 among light smokers. CLINICAL IMPLICATIONS: Findings support accelerated nicotine metabolism during pregnancy and need for smoking cessation efforts throughout pregnancy to help women stay smoke free during postpartum.

Brief Report: Relationship Between Cotinine Levels and Peripheral Endogenous Concentrations of Oxytocin, ß-Endorphin, and Orexin in Individuals With Both Alcohol and Nicotine Use Disorders.

BACKGROUND AND OBJECTIVES: In this secondary analysis of a pilot clinical trial with individuals with alcohol and nicotine use disorders, we investigate the relationship between serum concentrations of oxytocin, ß-endorphin, melatonin, α-melanocyte-stimulating hormone, substance P, and orexin, with objective biomarkers (salivary cotinine and serum γ-glutamyl transferase [GGT]) as well as with self-reported smoking and alcohol drinking. METHODS: Biomarkers for a total of N = 19 participants were analyzed using multiplexed, competitive format immune-assay (peptides) and enzyme competitive immunoassay (saliva). A regression analysis using Pearson's correlation coefficient was utilized to determine correlations. We controlled for multiple comparisons, checked for collinearities, and ran two-sided statistical tests. RESULTS: We found significant positive correlations for cotinine and oxytocin (P = .002), ß-endorphin (P = .008), and orexin (P < .001), but not for either GGT or self-reported smoking or alcohol drinking. CONCLUSION AND SCIENTIFIC SIGNIFICANCE: These preliminary results suggest a relationship between cotinine and oxytocin, ß-endorphin, and orexin, which opens up new potential hypotheses on the potential role of these endocrine pathways in tobacco smokers. (Am J Addict 2021;30:88-91).