Regulation of blood-brain barrier Na,K,2Cl-cotransporter through phosphorylation during in vitro stroke conditions and nicotine exposure

Nicotine, a major constituent of tobacco smoke, has important effects on brain recovery after focal ischemia (Wang et al., 1997). The purpose of this work is to systematically test the effects of nicotine during stroke conditions on blood-brain barrier (BBB) potassium transport, protein expression of the Na,K,2Cl-cotransporter (NKCC), and cell signaling pathways that control NKCC activity at the BBB. Confluent bovine brain microvessel endothelial cells (BBMECs) were exposed to both a hypoxic/aglycemic (H/A) environment to model BBB function during stroke conditions and nicotine and cotinine (N/C) to model plasma levels seen in smokers. BBMECs exhibit both Na,K-ATPase and NKCC activity (60 and 34 nmol/min/g, respectively) that contribute to 98% of the K(+) uptake in cultured endothelial cells. An adaptive up-regulation of NKCC activity was identified to occur on the basolateral surface of the BBB after in vitro stroke conditions. Twenty-four hours of N/C exposure, at doses equivalent to plasma levels of smokers, combined with 6 h of H/A, reduced NKCC protein expression and total NKCC activity (shown by bumetanide-sensitive (86)RB uptake) compared with 6 h of H/A alone (P < 0.01). Basolateral K(+) transport was found to be modulated by nicotinic acetylcholine receptors expressed at the BBB. NKCC activity on the basolateral surface of the BBB is controlled by an ongoing phosphorylation/dephosphorylation processes. We have identified a potential mechanism in altered BBB response to stroke conditions with prior N/C exposure directly implicating damage to brain-to-blood K(+) transport mediated at the BBB and perhaps neuronal recovery after stroke.     

Nicotine, cotinine, and anabasine inhibit aromatase in human trophoblast in vitro.

Epidemiologic studies suggest that women who smoke have lower endogenous estrogen than nonsmokers. To explore the possible link between cigarette smoking and decreased endogenous estrogens, we have examined the effects of constituents of tobacco on estrogen production in human choriocarcinoma cells and term placental microsomes. In choriocarcinoma cell cultures, nicotine, cotinine (a major metabolite of nicotine), and anabasine (a minor component of cigarette tobacco) all inhibited androstenedione conversion to estrogen in a dose-dependent fashion. Removal of nicotine, cotinine, and anabasine from the culture medium resulted in the complete reversal of the inhibition of aromatase. In the choriocarcinoma cell cultures, a supraphysiologic concentration of androstenedione (73 microM) in the culture medium blocked the inhibition of aromatase caused by nicotine, cotinine, and anabasine. In preparations of term placental microsomes, nicotine, cotinine, and anabasine inhibited the conversion of testosterone to estrogen. Kinetic analysis demonstrated the inhibition to be competitive with respect to the substrate. These findings suggest that some nicotinic alkaloids directly inhibit aromatase. This mechanism may explain, in part, the decreased estrogen observed in women who smoke.     

Mentholated cigarette smoking inhibits nicotine metabolism

Smoking mentholated cigarettes has been suggested to convey a greater cancer risk compared with smoking nonmentholated cigarettes. Two of the possible mechanisms by which mentholated cigarette smoking could increase risk are by increasing systemic exposure to tobacco smoke toxins and by affecting the metabolism of nicotine or tobacco smoke carcinogens. To examine these possibilities, we performed a crossover study in 14 healthy smokers, one-half of whom were African-Americans and one-half whites. Subjects were randomly assigned to smoke mentholated or nonmentholated cigarettes for 1 week, then to cross over to the other type of cigarettes for another week. Subjects were confined to a Clinical Research Center for 3 days of each week, during which time blood levels of nicotine and carbon monoxide were measured throughout the day and an intravenous infusion of deuterium-labeled nicotine and cotinine was administered to determine the rate and pathways of nicotine metabolism. The systemic intake of nicotine and carbon monoxide was, on average, not affected by mentholation of cigarettes. Mentholated cigarette smoking did significantly inhibit the metabolism of nicotine (clearance: 1289 versus 1431 ml/min, two sided, p = 0.02). Inhibition of nicotine metabolism occurred both by slower oxidative metabolism to cotinine and by slower glucuronide conjugation. Our data do not support the hypothesis that mentholated cigarette smoking results in a greater absorption of tobacco smoke toxins. Our finding of impaired metabolism of nicotine while mentholated cigarette smoking suggests that mentholated cigarette smoking enhances systemic nicotine exposure.     

Evaluation of methods to estimate cigarette smoke uptake

Exposure to tobacco smoke is measured by a variety of invasive and noninvasive techniques. Our purpose was to examine how well some of these measures correlated when obtained simultaneously from the same subjects. On three occasions, six subjects were studied while they were smoking a single cigarette after 24 hr of abstinence. There were positive correlations between increases in heart rate and plasma nicotine concentrations and between percentage carboxyhemoglobin and exhaled carbon monoxide. Although residual cotinine was readily detected in samples of plasma before the subjects smoked, there was an increase in mean levels, with a peak approximately 1 hr after smoking. Urinary concentrations of nicotine, cotinine, and nicotine-1'-N-oxide and thiocyanate levels in plasma and saliva were essentially unchanged by smoking a single cigarette. Data on smoke generation and nicotine retention in cigarette butts correlated poorly with all other measures of smoke uptake.     

Measuring infant exposure to environmental tobacco smoke

Methods to measure infant exposure to environmental tobacco smoke (ETS) are needed to identify infants at highest risk for ETS-related health problems. The purpose of this study was to validate measures sensitive to changes in levels of infant exposure to ETS and to develop a predictive model of infant exposure to ETS. Fifteen infants of smoking mothers were followed from birth to 6 weeks of age. Exposure to ETS was measured by using a smoking habits questionnaire, cigarette "butt" collection, infant urine nicotine and cotinine levels, and ambient nicotine (personal air monitors). The 24-hour cigarette butt collection was the best predictor of acute (adjusted r2 = .83) and chronic exposure (adjusted r2 = .47) measured by infant urinary nicotine and cotinine levels when the infants were 2 weeks of age. Including scores on the smoking habits questionnaire and ambient nicotine levels increased the adjusted r2 to .88 and .61, respectively.     

Cigarette brand-switching: effects on smoke exposure and smoking behavior

This study examined the effects of cigarette yield (Federal Trade Commission-determined deliveries of nicotine, tar and CO) on both biological exposure to smoke constituents and smoking behaviors. Smokers (N = 10) of high-yield cigarettes were switched in random order among five different commercially available cigarette brands with nicotine yields of 0.1, 0.4, 0.7, 1.1 (altered brand) and 1.0 (usual brand) mg and smoked each cigarette type for 5 days while a wide variety of assessments were performed. Steady-state cotinine and CO levels were substantially lower after 5 days of smoking ultra-low yield cigarettes (cotinine, 152 ng/ml; CO, 25 ppm) than when smoking usual-brand high-yield cigarettes (cotinine, 252 ng/ml; CO, 38 ppm). Both CO and nicotine boost (acute exposure) were related to yield. However, relative between-yield differences in all nicotine and CO exposure measures were smaller than predicted from Federal Trade Commission yield ratings. Substantial yield-related alterations were observed in smoking behavior. Subjects smoked more cigarettes and took larger and more closely spaced puffs when smoking low- as compared with high-yield cigarettes. The amount of tobacco burned per day was similar across all yield conditions. However, filter vent-blocking of ultra-low yield cigarettes did not appear to occur on a consistent basis. Subjective reports indicated poor acceptability of lower-yield cigarettes. We conclude that switching to lower-yield cigarettes brings about substantial alterations in smoking behavior which are at least partially responsible for the observed biological compensation associated with these cigarettes.     

Disposition of nicotine and eight metabolites in smokers and nonsmokers: identification in smokers of two metabolites that are longer lived than cotinine

The disposition of a single intravenous dose of 14C-nicotine was investigated in six cigarette smokers and six nonsmokers. Plasma and urinary elimination of both nicotine and cotinine was faster in smokers than in nonsmokers. In the urine of both smokers and nonsmokers, we identified nicotine and eight metabolites, including two new metabolites: metabolite A (3-hydroxycotinine glucuronide) and metabolite G (demethylcotinine delta 2',3'-enamine). Metabolites A and G were of particular interest because, in smokers, they both persisted longer than cotinine. This property renders them more sensitive than cotinine as potential indicators of passive exposure to cigarette smoke.     

Simulation and evaluation of nicotine intake during passive smoking: cotinine measurements in body fluids of nonsmokers given intravenous infusions of nicotine

The technique of monitoring cotinine concentrations in body fluids as a means of measuring nicotine intake during passive smoking has been evaluated in two studies, both of which used intravenous infusion to stimulate nicotine intake. In the first study, nicotine and cotinine were given separately, for 1 hour in four different intravenous doses (3.2, 15.4, 30.9, and 61.7 nmol/min) to each nonsmoker. In the second study, nicotine and cotinine were infused for 4 hours; each subject received five different doses of nicotine (1.5, 3.1, 6.2, 10.8, and 15.4 nmol/min) and one of cotinine (10.8 nmol/min). The concentration of cotinine was constant in both plasma and saliva from 1 to 4 hours after the nicotine infusion; the plateau levels of cotinine were found to be linearly and directly related to the nicotine intake. The ratio of salivary to plasma cotinine was 1:1.27. A linear relationship was also found between nicotine and cotinine infusion rates and the AUC values for cotinine. The fraction metabolized to cotinine was found to be about 0.5. The results from these studies show that: (1) there is a linear relationship between the plateau concentration of cotinine and the amount of nicotine infused over a period of 1 up to 4 hours; (2) salivary cotinine provides the same information on nicotine intake as does plasma cotinine; and (3) single measurements of either plasma or salivary cotinine concentrations at 1 to 4 hours after the exposure could be used to predict the nicotine intake during 1 to 4 hours of environmental tobacco smoke exposure.     

A sensitive enzyme immunoassay for measuring cotinine in passive smokers

BackgroundBoth active smoking and passive exposure to tobacco smoke are major risk factors for cardiovascular, pulmonary, and oncological diseases. The serum level of cotinine, a major proximate metabolite of nicotine, reflects active or passive exposure to tobacco smoke. However, currently available enzyme-linked immunosorbent assays (ELISAs) for cotinine have limited sensitivity, and a high-throughput quantification of the severity of passive exposure to tobacco smoke has not been possible thus far.MethodsWe generated a phage display of combinatorial antibody library, from which we selected a recombinant antibody against cotinine, developed a sensitive ELISA using this antibody, and evaluated the method in a clinical setting and an animal model.ResultsThe limits of detection and the lower limit of quantification were 31 pg/mL and 1 ng/mL cotinine, respectively. The intra- and inter-assay precisions based on three quality control samples were 3.8–13.5% and 14.0–15.0%, respectively. No significant interference from nicotine, trans-3'-hydroxy cotinine, tobacco alkaloids, or other serum components was found. When we applied our ELISA to serum samples from 36 volunteers, the serum cotinine levels were clustered into two groups, which exactly corresponded to their smoking behavior and this ELISA yielded reproducible and accurate results, which were comparable to those of LC/MS in a split assay. In animal studies, we were able to distinguish between rats injected with a nicotine dose equivalent to that of passive exposure to tobacco and rats without exposure.ConclusionThe competitive ELISA described here is useful for the detection and quantification of the severity of risk of passive smoking.     

Brain uptake kinetics of nicotine and cotinine after chronic nicotine exposure

Blood-brain barrier (BBB) nicotine transfer has been well documented in view of the fact that this alkaloid is a cerebral blood flow marker. However, limited data are available that describe BBB penetration of the major tobacco alkaloids after chronic nicotine exposure. This question needs to be addressed, given long-term nicotine exposure alters both BBB function and morphology. In contrast to nicotine, it has been reported that cotinine (the major nicotine metabolite) does not penetrate the BBB, yet cotinine brain distribution has been well documented after nicotine exposure. Surprisingly, therefore, the literature indirectly suggests that central nervous system cotinine distribution occurs secondarily to nicotine brain metabolism. The aims of the current report are to define BBB transfer of nicotine and cotinine in naive and nicotine-exposed animals. Using an in situ brain perfusion model, we assessed the BBB uptake of [3H]nicotine and [3H]cotinine in naive animals and in animals exposed chronically to S-(-)nicotine (4.5 mg/kg/day) through osmotic minipump infusion. Our data demonstrate that 1) [3H]nicotine BBB uptake is not altered in the in situ perfusion model after chronic nicotine exposure, 2) [3H]cotinine penetrates the BBB, and 3) similar to [3H]nicotine, [3H]cotinine BBB transfer is not altered by chronic nicotine exposure. To our knowledge, this is the first report detailing the uptake of nicotine and cotinine after chronic nicotine exposure and quantifying the rate of BBB penetration by cotinine.     

Exacerbation of acute platelet thrombus formation in stenosed dog coronary arteries with smoke from a non-tobacco-burning cigarette

The tobacco industry proposes to release a "new" cigarette with a burning charcoal tip, which heats the tobacco as air is drawn through it and into the lungs of the smoker. It is claimed to be safer because the tobacco does not burn. Using our established canine model of coronary artery stenosis with moderate intimal damage, we compared this new cigarette (0.4 mg nicotine) with a regular cigarette by ventilating dogs (n = 10) with room air and then either new or regular smoke (two cigarettes over about 10 minutes). In our dog model, periodic acute platelet thrombus formation followed by embolization produced cyclical flow reductions in coronary blood flow where the rate of flow decline was proportional to the degree of in vivo platelet-vessel wall interaction. The rate of flow decline increased from -4.81 +/- 1.29 ml/min2 to -9.60 +/- 2.29 ml/min2 after ventilation with the new-type smoke (p less than 0.01). Similarly, the rate of flow decline increased from -5.43 +/- 1.28 ml/min2 to -9.28 +/- 2.31 ml/min2 after ventilation with regular cigarette smoke (p less than 0.01). Despite the lack of a clear causal link between cigarette smoking and the acute manifestations of cardiovascular disease, the data presented here indicate that the new cigarette is just as potent as regular cigarettes at increasing in vivo platelet activity and exacerbating acute platelet thrombus formation in the dog. We conclude that the new proposed non-tobacco-burning cigarette has deleterious effects similar to those of conventional cigarettes, and thus does not eliminate smoking as a risk factor in cardiovascular disease in human beings.     

Perinatal nicotine exposure eliminates peak in nicotinic acetylcholine receptor response in adolescent rats

Maternal smoking is a risk factor associated with nicotine abuse, so the effect of perinatal nicotine exposure was studied on the responsiveness to nicotine across adolescence in the rat. Pregnant Sprague-Dawley rats were implanted with s.c. Alzet osmotic minipumps delivering nicotine (L-nicotine hydrogen tartrate, 2 mg/kg/day free base) or vehicle (0.9% saline) on gestational day 7. There was no effect of nicotine on dam weight gain, food consumption, or water consumption or on the number of live pups or weights at the time of birth. Pups were cross-fostered to obtain the following prenatal/postnatal exposure groups: control/control, nicotine/nicotine, nicotine/control, and control/nicotine. On postnatal days 28, 35, 49, and 63, nicotine-stimulated (86)Rb(+) efflux was measured in synaptosomes prepared from the frontal cortex, hippocampus, striatum (STR), and thalamus (THL), using a previously developed method. Significant effects of treatment and concentration were detected in all four brain regions, and significant effects of age were observed in the STR and THL. Significant interactions of age and treatment were observed in each of the four brain regions. Nicotine-stimulated (86)Rb(+) efflux peaked during adolescence in control rats. However, perinatal exposure to nicotine eliminated this peak during adolescence. These results are consistent with recent behavioral and receptor binding results from other laboratories and are the first direct evidence at the cellular level that the nicotinic acetylcholine receptor response varies during adolescence and is affected by perinatal nicotine exposure.     

Urinary biomarkers for secondhand smoke and heated tobacco products exposure

Concerns have recently grown about the health effects of secondhand smoke exposure and heated tobacco products. The analysis of tobacco smoke biomarkers is critical to assess the health effects of tobacco smoke exposure. For this purpose, the simultaneous determinations of exposure markers and health effect markers would provide a better evaluation of smoke exposure. In this study, nicotine metabolites (nicotine, cotinine, trans-3''-hydroxycotinine) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine were analyzed as exposure markers. The DNA damage markers, 7-methylguanine and 8-hydroxy-2''-deoxyguanosine, were simultaneously measured as health effect markers. The results revealed significant levels of urinary nicotine metabolites and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in the subjects exposed to secondhand smoke and heated tobacco products. In addition, the urinary levels of 7-methylguanine and 8-hydroxy-2''-deoxyguanosine tended to be high for secondhand smoke and heated tobacco products exposures, as compared to those of non-smokers. These biomarkers will be useful for evaluating tobacco smoke exposure.     

Reference interval and subject variation in excretion of urinary metabolites of nicotine from non-smoking healthy subjects in Denmark

BACKGROUND: Passive smoking has been found to be a respiratory health hazard in humans. The present study describes the calculation of a reference interval for urinary nicotine metabolites calculated as cotinine equivalents on the basis of 72 non-smokers exposed to tobacco smoke less than 25% of the day. METHODS: Twenty subjects (passive smokers) exposed to tobacco smoke more than 25% of the day (subjectively assessed) and 32 smokers were used to validate the estimated reference interval. Urine samples were collected three times during the day approximately at 06.30, 17.00 and 22.45 h. RESULTS: Within-subject variation was found to be 89.4, 72.6, and 79.2% and between-subject variation was found to be 64.5, 64.2, and 36.1%. No gender difference could be demonstrated. In general all subjects showed increased concentrations in the afternoon and evening samples compared to the morning samples. Parametric reference interval for excretion of nicotine metabolites in urine from non-smokers was established according to International Union of Pure and Applied Chemistry (IUPAC) and International Federation for Clinical Chemistry (IFCC) for use of risk assessment of exposure to tobacco smoke. The reference interval for urinary cotinine was estimated to be 1.1-90.0 micromol/mol creatinine in morning samples from non-smokers. An intercomparison between the radioimmunoassay (RIA) method used for determination of nicotine metabolites and a gas chromatography-mass spectrometry (GC-MS) method for determination of cotinine was carried out on 27 samples from non-smokers and smokers. Results obtained from the RIA method showed 2.84 [confidence interval (CI): 2.50; 3.18] times higher results compared to the GC-MS method. A linear correlation between the two methods was demonstrated (rho=0.96). CONCLUSION: The RIA method is rapid and adequate for clinical use in the assessment of exposure to tobacco smoke, i.e. ratio between CV(a)/CV(ti) was<0.50.     

Rat hepatic CYP2E1 is induced by very low nicotine doses: an investigation of induction,time course,dose response,and mechanism

CYP2E1 is an ethanol- and drug-metabolizing enzyme that can also activate procarcinogens and hepatotoxicants and generate reactive oxygen species; it has been implicated in the pathogenesis of liver diseases and cancer. Cigarette smoke increases CYP2E1 activity in rodents and in humans and we have shown that nicotine (0.1-1.0 mg/kg s.c. x 7 days) increases CYP2E1 protein and activity in the rat liver. In the current study, we have shown that the induction peaks at 4 h postnicotine (1 mg/kg s.c. x 7 days) treatment and recovers within 24 h. No induction was observed after a single injection, and 18 days of treatment did not increase the levels beyond that found at 7 days. We found that CYP2E1 is induced by very low doses of chronic (x 7 days) nicotine with an ED50 value of 0.01 mg/kg s.c.; 0.01 mg/kg in a rat model results in peak cotinine levels (nicotine metabolite) similar to those found in people exposed to environmental tobacco smoke (passive smokers; 2-7 ng/ml). Previously, we have shown no change in CYP2E1 mRNA, and our current mechanistic study indicates that nicotine does not regulate CYP2E1 expression by protein stabilization. We postulated that a nicotine metabolite could be causing the induction but found that cotinine (1 mg/kg x 7 days) did not increase CYP2E1. Our findings indicate that nicotine increases CYP2E1 at very low doses and may enhance CYP2E1-related toxicity in smokers, passive smokers, and people treated with nicotine (e.g., smokers, patients with Alzheimer's disease, ulcerative colitis or Parkinson's disease).     

Effects of cigarette smoking and carbon monoxide on nicotine and cotinine metabolism

OBJECTIVES: To examine the effects of cigarette smoking on the disposition kinetics of nicotine and cotinine, to determine the effects of cigarette smoking on pathways of nicotine and cotinine metabolism, and to test the hypothesis that carbon monoxide inhibits the metabolism of nicotine. STUDY DESIGN: Twelve cigarette smokers were studied in three treatment conditions, each lasting 7 days, during which they smoked cigarettes, breathed carbon monoxide to achieve carboxyhemoglobin levels similar to cigarette smoking, or breathed air. In each treatment condition, subjects received a combined infusion of deuterium-labeled nicotine (d2) and cotinine (d4), with measurement of disposition kinetics and urine metabolite profile. RESULTS: Cigarette smoking significantly inhibited the metabolism of nicotine but had no effect on cotinine metabolism. Cigarette smoking markedly induced the O-glucuronidation of trans-3'-hydroxycotinine but had no effect on the N-glucuronidation of nicotine or cotinine. Carbon monoxide had no effect on nicotine or cotinine kinetics or metabolic profile. CONCLUSIONS: This study confirms previous observations that cigarette smoking inhibits nicotine metabolism but disproves the hypothesis that this effect is due to carbon monoxide. Induction of glucuronidation must be considered in understanding the effects of cigarette smoking on drug metabolism.     

Simultaneous analysis of nicotine,nicotine metabolites,and tobacco alkaloids in serum or urine by tandem mass spectrometry,with clinically relevant metabolic profiles

BACKGROUND: Assessment of nicotine metabolism and disposition has become an integral part of nicotine dependency treatment programs. Serum nicotine concentrations or urine cotinine concentrations can be used to guide nicotine patch dose to achieve biological concentrations adequate to provide the patient with immediate relief from nicotine withdrawal symptoms, an important factor in nicotine withdrawal success. Absence of nicotine metabolites and anabasine can be used to document abstinence from tobacco products, an indicator of treatment success. METHODS: The procedure was designed to quantify nicotine, cotinine, trans-3'-hydroxycotinine, anabasine, and nornicotine in human serum or urine. The technique required simple extraction of the sample with quantification by HPLC-tandem mass spectrometry. RESULTS: The procedure for simultaneous analysis of nicotine, its metabolites, and tobacco alkaloids simultaneously quantified five different analytes. Test limit of quantification, linearity, imprecision, and accuracy were adequate for clinical evaluation of patients undergoing treatment for tobacco dependency. The test readily distinguished individuals who had no exposure to tobacco products from individuals who were either passively exposed or were abstinent past-tobacco users from those who were actively using a tobacco or nicotine product. CONCLUSIONS: Nicotine, cotinine, trans-3'-hydroxycotinine, nornicotine, and anabasine can be simultaneously and accurately quantified in either serum or urine by HPLC-tandem mass spectrometry with imprecision <10% at physiologic concentrations and limits of quantification ranging from 0.5 to 5 micro g/L. Knowledge of serum or urine concentrations of these analytes can be used to guide nicotine replacement therapy or to assess tobacco abstinence in nicotine dependency treatment. These measurements are now an integral part of the clinical treatment and management of patients who wish to overcome tobacco dependence.     

Urinary biomarkers for secondhand smoke

The use of a biomarker is mandatory for quantitative analysis of exposure to secondhand smoke (SHS). This article summarizes urinary biomarkers of smoke exposure which can be now quantified. The most reliable urinary biomarkers to assess the exposure to SHS are NNAL 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol and NNAL‐Glucuronides, which is metabolites of tobacco‐specific nitrosamine. These substances were detected even in 50% of children who had undetectable level of cotinine (<0.5 ng/ml). Urinary cotinine, which is determined by a highly sensitive competing enzyme immunoassay, is also a useful biomarker. However, individual variability of CYP2A6 allele,in which nicotine is catalyzed to cotinine, affects the level of urinary cotinine. Approximately 20% of Japanese subjects have homozygotes or heterozygotes of the CYP2A6*4 allele, which has impaired nicotine metabolism and subsequently may underestimate the actual exposure to SHS. In assessing the exposure to SHS, therefore, individual variability of CYP2A6 gene polymorphism should be taken into consideration. The combination of urinary cotinine measurement and self‐report of parents' smoking seems to be accurate to assess the exposure to SHS in mass screening. J. Clin. Lab. Anal. 25:354–358, 2011. © 2011 Wiley‐Liss, Inc.     

Nicotine intake and dose response when smoking reduced-nicotine content cigarettes

OBJECTIVES: The progressive reduction of the nicotine content of cigarettes has been suggested as a way to wean smokers from nicotine and tobacco. As a first step in evaluating this strategy, we studied smokers smoking cigarettes containing tobacco with differing nicotine content. METHODS: Twelve healthy smokers participated in a semiblinded, within-subject, crossover study. Subjects were asked to smoke 1 of their usual brand of cigarette and then on 5 subsequent occasions to smoke a research cigarette, each with differing nicotine content. The research cigarettes contained 0.6 to 10.1 mg nicotine per cigarette. Plasma nicotine and blood carboxyhemoglobin levels, as well as subjective and cardiovascular responses, were measured after smoking. Systemic nicotine intake per cigarette was estimated by use of plasma nicotine concentrations over time and clearance data from the general population. RESULTS: Systemic nicotine intake (0.26-1.47 mg per cigarette) varied with nicotine content of the cigarette (r = 0.82, P < .001). Compensation when smoking single low-nicotine content cigarettes ranged from -1% (95% confidence interval, -23% to 21%) to 34% (95% confidence interval, -39% to 107%) for 1-mg to 8-mg research cigarettes. Carbon monoxide intake and estimated tar exposure were similar across cigarettes. Low-nicotine content cigarettes were rated as being of lower quality and less satisfying than the 12-mg research cigarette or the usual brand (P < .05 for both comparisons). Cigarette smoking increased heart rate and decreased skin temperature, but the nicotine dose-response curve flattened at higher doses, with a maximal response being observed in cigarettes at a nicotine content level of about 8 mg. CONCLUSIONS: Our study suggests that reduced-nicotine content cigarettes are reasonable candidates for trying to reduce the level of nicotine addiction in smokers. The flat nicotine dose-cardiovascular response curve is consistent with other studies demonstrating tolerance to the cardiovascular effects of nicotine.     

Effects of cigarette smoke on immune response: chronic exposure to cigarette smoke impairs antigen-mediated signaling in T cells and depletes IP3-sensitive Ca(2+) stores

Chronic exposure of mice and rats to cigarette smoke affects T-cell responsiveness that may account for the decreased T-cell proliferative and T-dependent antibody responses in humans and animals exposed to cigarette smoke. However, the mechanism by which cigarette smoke affects the T cell function is not clearly understood. Our laboratory has shown that chronic exposure of rats to nicotine inhibits the antibody-forming cell response, impairs the antigen-mediated signaling in T cells, and induces T cell anergy. To determine the mechanism of cigarette smoke-induced immunosuppression and to compare it with chronic nicotine exposure, rats were exposed to diluted, mainstream cigarette smoke for up to 30 months or to nicotine (1 mg/kg b.wt./24 h) via miniosmotic pumps for 4 weeks, and evaluated for immunological function in vivo and in vitro. This article presents evidence suggesting that T cells from long-term cigarette smoke-exposed rats exhibit decreased antigen-mediated proliferation and constitutive activation of protein tyrosine kinase and phospholipase C-gamma1 activities. Moreover, spleen cells from smoke-exposed and nicotine-treated animals have depleted inositol-1, 4,5-trisphosphate-sensitive Ca(2+) stores and a decreased ability to raise intracellular Ca(2+) levels in response to T cell antigen receptor ligation. These results suggest that chronic smoking causes T cell anergy by impairing the antigen receptor-mediated signal transduction pathways and depleting the inositol-1,4, 5-trisphosphate-sensitive Ca(2+) stores. Moreover, nicotine may account for or contribute to the immunosuppressive properties of cigarette smoke.