Genome-wide association study across European and African American ancestries identifies a SNP in DNMT3B contributing to nicotine dependence.

Cigarette smoking is a leading cause of preventable mortality worldwide. Nicotine dependence, which reduces the likelihood of quitting smoking, is a heritable trait with firmly established associations with sequence variants in nicotine acetylcholine receptor genes and at other loci. To search for additional loci, we conducted a genome-wide association study (GWAS) meta-analysis of nicotine dependence, totaling 38,602 smokers (28,677 Europeans/European Americans and 9925 African Americans) across 15 studies. In this largest-ever GWAS meta-analysis for nicotine dependence and the largest-ever cross-ancestry GWAS meta-analysis for any smoking phenotype, we reconfirmed the well-known CHRNA5-CHRNA3-CHRNB4 genes and further yielded a novel association in the DNA methyltransferase gene DNMT3B. The intronic DNMT3B rs910083-C allele (frequency=44-77%) was associated with increased risk of nicotine dependence at P=3.7 × 10-8 (odds ratio (OR)=1.06 and 95% confidence interval (CI)=1.04-1.07 for severe vs mild dependence). The association was independently confirmed in the UK Biobank (N=48,931) using heavy vs never smoking as a proxy phenotype (P=3.6 × 10-4, OR=1.05, and 95% CI=1.02-1.08). Rs910083-C is also associated with increased risk of squamous cell lung carcinoma in the International Lung Cancer Consortium (N=60,586, meta-analysis P=0.0095, OR=1.05, and 95% CI=1.01-1.09). Moreover, rs910083-C was implicated as a cis-methylation quantitative trait locus (QTL) variant associated with higher DNMT3B methylation in fetal brain (N=166, P=2.3 × 10-26) and a cis-expression QTL variant associated with higher DNMT3B expression in adult cerebellum from the Genotype-Tissue Expression project (N=103, P=3.0 × 10-6) and the independent Brain eQTL Almanac (N=134, P=0.028). This novel DNMT3B cis-acting QTL variant highlights the importance of genetically influenced regulation in brain on the risks of nicotine dependence, heavy smoking and consequent lung cancer.

Nicotine dependence is associated with functional variation in FMO3, an enzyme that metabolizes nicotine in the brain.

A common haplotype of the flavin-containing monooxygenase gene FMO3 is associated with aberrant mRNA splicing, a twofold reduction in in vivo nicotine N-oxidation and reduced nicotine dependence. Tobacco remains the largest cause of preventable mortality worldwide. CYP2A6, the primary hepatic nicotine metabolism gene, is robustly associated with cigarette consumption but other enzymes contribute to nicotine metabolism. We determined the effects of common variants in FMO3 on plasma levels of nicotine-N-oxide in 170 European Americans administered deuterated nicotine. The polymorphism rs2266780 (E308G) was associated with N-oxidation of both orally administered and ad libitum smoked nicotine (P⩽3.3 × 10-5 controlling for CYP2A6 genotype). In vitro, the FMO3 G308 variant was not associated with reduced activity, but rs2266780 was strongly associated with aberrant FMO3 mRNA splicing in both liver and brain (P⩽6.5 × 10-9). Surprisingly, in treatment-seeking European American smokers (n=1558) this allele was associated with reduced nicotine dependence, specifically with a longer time to first cigarette (P=9.0 × 10-4), but not with reduced cigarette consumption. As N-oxidation accounts for only a small percentage of hepatic nicotine metabolism we hypothesized that FMO3 genotype affects nicotine metabolism in the brain (unlike CYP2A6, FMO3 is expressed in human brain) or that nicotine-N-oxide itself has pharmacological activity. We demonstrate for the first time nicotine N-oxidation in human brain, mediated by FMO3 and FMO1, and show that nicotine-N-oxide modulates human α4ß2 nicotinic receptor activity in vitro. These results indicate possible mechanisms for associations between FMO3 genotype and smoking behaviors, and suggest nicotine N-oxidation as a novel target to enhance smoking cessation.

Genome-wide meta-analysis reveals common splice site acceptor variant in CHRNA4 associated with nicotine dependence.

We conducted a 1000 Genomes-imputed genome-wide association study (GWAS) meta-analysis for nicotine dependence, defined by the Fagerström Test for Nicotine Dependence in 17 074 ever smokers from five European-ancestry samples. We followed up novel variants in 7469 ever smokers from five independent European-ancestry samples. We identified genome-wide significant association in the alpha-4 nicotinic receptor subunit (CHRNA4) gene on chromosome 20q13: lowest P=8.0 × 10(-9) across all the samples for rs2273500-C (frequency=0.15; odds ratio=1.12 and 95% confidence interval=1.08-1.17 for severe vs mild dependence). rs2273500-C, a splice site acceptor variant resulting in an alternate CHRNA4 transcript predicted to be targeted for nonsense-mediated decay, was associated with decreased CHRNA4 expression in physiologically normal human brains (lowest P=7.3 × 10(-4)). Importantly, rs2273500-C was associated with increased lung cancer risk (N=28 998, odds ratio=1.06 and 95% confidence interval=1.00-1.12), likely through its effect on smoking, as rs2273500-C was no longer associated with lung cancer after adjustment for smoking. Using criteria for smoking behavior that encompass more than the single 'cigarettes per day' item, we identified a common CHRNA4 variant with important regulatory properties that contributes to nicotine dependence and smoking-related consequences.

Smoking status as the new vital sign: effect on assessment and intervention in patients who smoke.

OBJECTIVE: To assess the effect of expanding the vital signs to include smoking status. DESIGN: We prospectively conducted exit interviews with patients at a general internal medicine clinic in Madison, Wisconsin, during a 16-month period from 1991 to 1993. METHODS: Patients were surveyed briefly before (N = 870) and after (N = 994) the implementation of a simple institutional change in clinical practice. This change involved training the staff in how to use progress notepaper with a vital sign stamp that included smoking status (current, former, or never) along with the traditional vital signs. Included in the survey were questions about whether the patient smoked, whether the patient was asked that day about smoking status (by a clinician or other staff), and, for smokers, whether they were urged to quit smoking and given specific advice on how to do so. RESULTS: After expansion of the vital signs, patients were much more likely to report inquiries about their smoking status on the day of a clinic visit (an increase from approximately 58% at baseline to 81% at intervention; P < 0.0001). The vital sign intervention was associated with significant increases in the percentage of smokers who reported that their clinician advised them that day to quit smoking (from approximately 49% at baseline to 70% during the intervention; P < 0.01) and in the percentage who reported that their clinician gave them specific advice that day on how to stop smoking (from approximately 24% at baseline to 43% during the intervention; P < 0.01). CONCLUSION: Expanding the vital signs to include smoking status was associated with a dramatic increase in the rate of identifying patients who smoke and of intervening to encourage and assist with smoking cessation. This simple, low-cost intervention may effectively prompt clinicians to inquire about use of tobacco and offer recommendations to smokers.

Two studies of the clinical effectiveness of the nicotine patch with different counseling treatments.

OBJECTIVE: To assess the effectiveness of transdermal nicotine therapy for smoking cessation and suppression of withdrawal severity in conjunction with two different adjuvant counseling treatments. DESIGN: Two independent randomized placebo-controlled double-blind trials. SETTING: Smoking cessation clinic. SUBJECTS: Eighty-eight (study 1) and 112 (study 2) adult volunteers motivated to quit smoking. INTERVENTIONS: Eight weeks of 22-mg transdermal nicotine therapy with group counseling (study 1); 4 weeks of 22 mg followed by 2 weeks of 11-mg transdermal nicotine therapy with brief individual counseling (study 2). MAIN OUTCOME MEASURES: Modified point prevalence (7 consecutive days of nonsmoking) at the end of patch treatment and 6 months after treatment initiation was assessed by self-report and biochemically confirmed; survival analyses were also conducted for both studies to compare treatment efficacy. Also, we examined the impact of the nicotine patch on specific withdrawal symptoms (anger, anxiety, awakening, difficulty concentrating, depression, hunger, impatience, and craving). RESULTS: Transdermal nicotine treatment produced higher cessation rates at the end of treatment than did placebo with both adjuvant counseling interventions: 59 percent vs 40 percent (p < 0.05 in study 1) and 37 percent vs 20 percent (p < 0.05 in study 2), respectively. Smoking cessation efficacy was maintained 6 months after initiation of treatment: 34 percent vs 21 percent (p = 0.08 in study 1) and 18 percent vs 7 percent (p = 0.05 in study 2). Survival analyses also revealed significant group differences in efficacy in both studies. Nicotine patches also suppressed a variety of withdrawal symptoms, including craving in the first weeks after patients quit smoking. CONCLUSION: Transdermal nicotine effectively augments smoking cessation rates with two different types of counseling treatment. Overall, the nicotine patch approximately doubles the sustained rate of smoking cessation. Additionally, the nicotine patch provides relief from some tobacco withdrawal symptoms.

Influence of stress on morphine-induced hyperthermia: relevance to drug conditioning and tolerance development.

Controversy exists regarding (a) whether rats become tolerant, or sensitized, to morphine-induced hyperthermia and (b) the directionality of the conditioned pyretic effects of morphine. In these studies, stress produced by temperature-assessment procedures affected rats' pyretic response to morphine. Under conditions of high stress, rats first showed diminished, and then enhanced, hyperthermic responding across repeated morphine dosing (5 or 35 mg/kg). The diminished hyperthermia can be attributed to habituation to high levels of assessment stress. Repeated morphine doses delivered under conditions of low stress produced only enhanced hyperthermic responding, which indicates that rats become sensitized to morphine's hyperthermic effects. There was little evidence that morphine supported conditioning of pyretic responses. Finally, the temperature-assessment stress that produced hyperthermia was mediated by opiate peptides, was blocked by naloxone, and enhanced the agonist effects of morphine. The relevance of these findings to theories of drug conditioning and tolerance is discussed.

Characterization of morphine's excitatory effects.

Although the excitatory effects of opiates have assumed greater importance in theories of compulsive drug use, the nature and time course of these effects remains unclear. The authors attempted to characterize the excitatory effects of morphine by administering doses of 5 mg/kg and 20 mg/kg in both warm and cold environments and by undertaking simultaneous assessments of core temperature, locomotor activity, and oxygen consumption. The results argue for a two receptor model to account for morphine's thermic effects: a high-affinity receptor that activates thermogenic systems, and a low-affinity receptor that mediates a poikilothermic response.

Conditioned morphine tolerance in the rat: absence of a compensatory response and cross-tolerance with stress.

In four separate experiments with rats as subjects, strong evidence was obtained that tolerance development to morphine analgesia occurs most rapidly when morphine delivery is paired with salient contextual cues. However, contextual cues previously paired with morphine did not elicit conditioned drug-compensatory responses when presented to nondrugged animals. These results were obtained by different analgesia assessments, with different drug-administration--analgesia-test latencies, and in environments differing with respect to stress level. Stress level did influence nociceptive response, as it was found that the combination of bright illumination, white noise, and a strong odor resulted in antinociception in the absence of drug. Moreover, rats that had a history of receiving morphine in this stressful context were tolerant to this stress-induced antinociception, but only when morphine was present in their systems. In the final two studies, this antinociception, which was cross-tolerant with morphine, was characterized with respect to naloxone reversibility and brain levels of met- and leu-enkephalin as determined by radioimmunoassay.

Potentiation of ethanol withdrawal by prior dependence.

Thirty rats were randomly assigned to three groups. Group 1 was given a 21-day exposure to an ethanol (EtOH) liquid diet, while Groups 2 and 3 were given equivalent amounts of an isocaloric non-EtOH liquid diet. Group 1 rats had withdrawal syndromes following EtOH removal. After a two-week recovery period, Groups 1 and 2 were both exposed to an EtOH diet, while Group 3 again received an isocaloric non-EtOH liquid diet. Groups 1 and 2 were withdrawn after 12 days of EtOH exposure and were rated with a behavioral withdrawal rating scale, for which interobserver reliability estimates were determined. Previously dependent (Group 1) rats showed more severe withdrawal syndromes, including a higher incidence of seizures, than rats undergoing their initial withdrawal (Group 2). Studies that do not agree with this finding are discussed.

Effects of dose on effector mechanisms in morphine-induced hyperthermia and poikilothermia.

The effect of a variety of morphine doses on thermoregulatory effector systems was examined in ambient temperatures of 27.0 degrees C and 4.0 degrees C. Rats were given saline or morphine sulfate (5, 15, or 25 mg/kg); their core temperature, oxygen consumption, and activity were monitored for 4 or 6 h post-injection. The results suggest two distinct actions of morphine, possibly mediated by two opiate receptors. Low doses of morphine produce hyperthermia that is the result of a direct activation of activity and whole body heat production. High doses produce effects dependent on ambient temperature: hypermetabolism and hyperthermia in the 27.0 degrees C environment; hypometabolism, vasodilation, and hypothermia in the 4.0 degrees C environment. The findings suggest limitations in current set-point theories of morphine's thermic actions.

Drug signals enhance morphine tolerance development in hypophysectomized rats.

Hypophysectomized and sham-operated (normal) rats were given morphine (5 mg/kg) either paired or unpaired with distinctive environmental cues. Both hypophysectomized and normal animals developed analgesic tolerance when drug effects were signaled, but little tolerance was evident in either surgery group when drug was unsignaled. Results suggest that the pituitary is not critical to associational tolerance development.

Aversion instead of preference learning indicated by nicotine place conditioning in rats.

Although nicotine is a drug of abuse for millions of smokers, it has been difficult to demonstrate clearly the motivational properties of nicotine with rats using the conditioned place preference (CPP) paradigm. The first experiment attempted to replicate CPPs reported by other researchers using nicotine doses of 0.4, 0.8, and 1.2 mg/kg. There was a trend for all three doses to produce aversions, but it was significant only for the 0.8 mg/kg dose. Exposures to the CS alone extinguished aversions, but a "priming" dose (0.2 mg/kg) of nicotine given after extinction produced aversions only in animals exposed to 1.2 mg/kg. Experiment 2 tested whether preexposure to morphine or nicotine would sensitize animals to nicotine's reinforcing effects. In this experiment, rats were exposed to either six nicotine (0.6 mg/kg) or morphine (1.0 mg/kg) dosings prior to preference conditioning. Neither preferences nor aversions were observed in any group following subsequent conditioning with 0.6 mg/kg nicotine. The results suggest that previous observations of preference effects may have been due to specific procedural factors or may have depended on negative reinforcement due to stress reduction.

Characterization of tobacco withdrawal symptoms: transdermal nicotine reduces hunger and weight gain.

The accurate assessment of both tobacco withdrawal and the impact of the nicotine patch on withdrawal may be compromised by attrition of subjects, or by subjects smoking during withdrawal. To reduce these occurrences, 211 participants were provided with intensive cessation counseling while trying to quit smoking with either nicotine (21 mg) or placebo transdermal patches. Subject attrition was low, with 80.5% of participants continuing through the 5-week study period. Abstinence rates were also high over this period (75% and 61% in active and placebo groups, respectively). In this multisite, double-blind trial, withdrawal severity was assessed using a nine-item daily self-report questionnaire, and abstinence was confirmed via CO monitoring. Abrupt smoking cessation increased multiple tobacco withdrawal symptoms/signs including craving for cigarettes, irritability, anxiety, appetite, sleep disruption, difficulty concentrating, restlessness, depression, and impatience. Treatment with transdermal nicotine reduced craving for cigarettes, anxiety, irritability, and appetite, as well as weight gain (1.85 versus 2.88 kg mean gain over 4 weeks in active and placebo groups, respectively).

Dynorphin (1-13): analgesia, hypothermia, cross-tolerance with morphine and beta-endorphin.

Intracerebroventricular administration of 20, 40 and 60 nmol of dynorphin (1-13) produced analgesia, as assessed by flinch/jump response to footshock, and hypothermia in the rat. Rats developed tolerance to both the analgesic and thermic effects of the 20 nmol dose of dynorphin. Dynorphin and beta-endorphin showed cross-tolerance with respect to their analgesic but not their thermic effects. Dynorphin and morphine also produced cross-tolerant analgesic effects. Naloxone (10 mg/kg, IP) completely blocked the barrel rolling produced by 20 nmol dynorphin but did not alter its analgesic or thermic effects.

Endogenous analgesia in the pregnant rat: an artifact of weight-dependent measures?

It has been reported that pregnancy produces an opioid-mediated, endogenous analgesia in the rat. In an attempt to confirm this finding, we used 5 different analgesic measures to compare the responsiveness of pregnant and non-pregnant female rats to painful stimuli. Pregnant and non-pregnant rats differed only when assessed by measures that were highly correlated with body weight. Furthermore, the reduced pain responsiveness of pregnant rats was not prevented by administration of the opioid antagonists, naloxone or naltrexone. We can find no evidence for an endogenous analgesia of pregnancy; instead, our results suggest that findings of a diminished response to painful stimuli in pregnant rats may be an artifact related to the greater body mass/weight of the pregnant animals.

Effect of preconditioning unconditioned stimulus experience on learned taste aversions.

One taste-aversion study using male Long-Evans rats in which ethanol was the unconditioned stimulus (UCS) and six studies in which lithium chloride (LiCl) was the UCS demonstrate that (a) exposure to the UCS prior to conditioning retards subsequent acquisition of learned taste aversions; (b) a single preconditioning UCS exposure is sufficient to attenuate conditioning; (c) the preconditioning UCS exposure must occur within a limited period prior to conditioning to attenuate learning; (d) repeated conditioning trials will override the effect of prior exposure to the UCS; (e) tolerance to the UCS is not a necessary condition for the attenuation effect to occur; (f) pairing the preconditioning UCS with a novel flavor other than the CS does not remove the preexposure effect, although it may reduce its magnitude; and (g) the degree of disruption is a positive function of preconditioning UCS dosage and an inverse function of conditioning UCS dosage.

Measures of affect and nicotine dependence predict differential response to smoking cessation treatments.

Smokers (N = 126) were randomly assigned to 6-session smoking cessation treatments consisting of 1 of 2 counseling strategies (skills training or support) and 1 of 2 nicotine exposure strategies (nicotine gum or rapid smoking). Counseling and nicotine strategies were completely crossed; all four combinations resulted in equivalent 1-year abstinence rates. Skills training produced higher initial cessation and more coping responses posttreatment than did support. Rapid smoking, but not nicotine gum, produced tachycardia to the taste of cigarettes posttreatment, consistent with cigarette aversion. The treatments were differentially effective among subpopulations of smokers: Subjects high in pretreatment negative affect responded best to support counseling; those low in pretreatment negative affect responded best to skills training. Self-reports of pretreatment craving predicted response to the nicotine exposure treatments.

Tobacco withdrawal and nicotine replacement influence objective measures of sleep.

Research has not adequately characterized the impact of tobacco withdrawal on objectively assessed sleep parameters despite the recent inclusion of insomnia as a nicotine withdrawal sign in the Diagnostic and Statistical Manual of Mental Disorders (4th ed.; American Psychiatric Association, 1994). Moreover, whether 24-hr nicotine replacement aids or interferes with sleep during withdrawal is unknown. In a double-masked, randomized clinical trial, 34 cigarette smokers who were motivated to quit received either active nicotine patches or placebo patches while quitting. Sleep was polysomnographically monitored for 2 precessation nights and 3 postcessation nights. The study demonstrates that among dependent smokers (a) tobacco withdrawal increases objectively assessed sleep disturbance (sleep fragmentation) and (b) nicotine replacement results in postcessation improvements in important polysomnographic measures of sleep quality (sleep fragmentation, Stage 3 and Stage 4 sleep).

Strike while the iron is hot: can stepped-care treatments resurrect relapsing smokers?

The efficacies of 2 group counseling step-up treatments for smoking cessation, cognitive-behavioral/skill training therapy (CBT) and motivational interviewing/supportive (MIS) therapy, were compared with brief intervention (BI) treatment in a sample of 677 smokers. Differential efficacy of the 2 step-up treatments was also tested in smokers at low and high risk for relapse (no smoking vs. any smoking during the first postquit week. respectively). All participants received 8 weeks of nicotine patch therapy. BI consisted of 3 brief individual cessation counseling sessions; CBT and MIS participants received BI treatment and 6 group counseling sessions. Neither CBT nor MIS treatment improved long-term abstinence rates relative to BI. Limited support was found for the hypothesis that high-risk smokers would benefit more from MIS than CBT. Other hypotheses were not supported.

Gender differences in smoking cessation.

Gender differences in smoking quit rates are frequently reported and are the subject of much speculation. This study examined the generalizability of gender differences in abstinence across study sites, treatments, and time of relapse, as well as potential mediators and moderators of gender effects. Participants were smokers who participated in 3 randomized clinical trials of the nicotine patch (N = 632). Men had higher cessation rates than women at all follow-ups. The impact of gender on abstinence was unaffected by controlling for study site, treatment, or time of relapse. There was little evidence for mediation or moderation of this relation by any of a host of predictor variables. The magnitude and consistency of the gender differential, coupled with an inability to account for it, highlights a compelling need for additional research specifically aimed at elucidating the relation between gender and abstinence.