Dorothy K. Hatsukami

Evaluation of Toxicant and Carcinogen Metabolites in the Urine of E-Cigarette Users Versus Cigarette Smokers

INTRODUCTION Electronic cigarettes (e-cigarettes) are rapidly increasing in popularity but little information is available on their potential toxic or carcinogenic effects. METHODS Twenty-eight e-cigarette smokers who had not smoked tobacco cigarettes for at least 2 months provided urine samples which were analyzed by validated methods for a suite of toxicant and carcinogen metabolites including 1-hydroxypyrene (1-HOP), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), 3-hydroxypropylmercapturic acid (3-HPMA), 2-hydroxypropylmercapturic acid (2-HPMA), 3-hydroxy-1-methylpropylmercapturic acid (HMPMA), S-phenylmercapturic acid (SPMA), nicotine, and cotinine. Levels of these compounds were compared to those found in cigarette smokers from three previous studies. RESULTS Levels of 1-HOP, total NNAL, 3-HPMA, 2-HPMA, HMPMA, and SPMA were significantly lower in the urine of e-cigarette users compared to cigarette smokers. Levels of nicotine and cotinine were significantly lower in e-cigarette users compared to cigarette smokers in one study but not in another. CONCLUSIONS With respect to the compounds analyzed here, e-cigarettes have a more favorable toxicity profile than tobacco cigarettes.

The ratio of a urinary tobacco-specific lung carcinogen metabolite to cotinine is significantly higher in passive than in active smokers

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol plus its glucuronides (total NNAL), metabolites of the lung carcinogen NNK, and total cotinine, metabolites of nicotine, are biomarkers of active and passive cigarette smoking. We calculated the total NNAL:total cotinine (×103) ratio in 408 passive (infants, children, and adults) and 1088 active smokers. The weighted averages were 0.73 (95% confidence interval 0.71, 0.76) for passive smokers and 0.07 (0.06, 0.08) for active smokers (p < 0.0001). These results demonstrate that cotinine measurements may underestimate exposure of passive smokers to the lung carcinogen NNK in second-hand cigarette smoke. The total NNAL:total cotinine (×103) ratio may provide an improved biomarker for evaluating the health effects of passive smoking.

Variants in Nicotinic Receptors and Risk for Nicotine Dependence

OBJECTIVE A recent study provisionally identified numerous genetic variants as risk factors for the transition from smoking to the development of nicotine dependence, including an amino acid change in the alpha5 nicotinic cholinergic receptor (CHRNA5). The purpose of this study was to replicate these findings in an independent data set and more thoroughly investigate the role of genetic variation in the cluster of physically linked nicotinic receptors, CHRNA5-CHRNA3-CHRNB4, and the risk of smoking. METHOD Individuals from 219 European American families (N=2,284) were genotyped across this gene cluster to test the genetic association with smoking. The frequency of the amino acid variant (rs16969968) was studied in 995 individuals from diverse ethnic populations. In vitro studies were performed to directly test whether the amino acid variant in the CHRNA5 influences receptor function. RESULTS A genetic variant marking an amino acid change showed association with the smoking phenotype (p=0.007). This variant is within a highly conserved region across nonhuman species, but its frequency varied across human populations (0% in African populations to 37% in European populations). Furthermore, functional studies demonstrated that the risk allele decreased response to a nicotine agonist. A second independent finding was seen at rs578776 (p=0.003), and the functional significance of this association remains unknown. CONCLUSIONS This study confirms that at least two independent variants in this nicotinic receptor gene cluster contribute to the development of habitual smoking in some populations, and it underscores the importance of multiple genetic variants contributing to the development of common diseases in various populations.

A genome-wide association study of alcohol dependence

Excessive alcohol consumption is one of the leading causes of preventable death in the United States. Approximately 14% of those who use alcohol meet criteria during their lifetime for alcohol dependence, which is characterized by tolerance, withdrawal, inability to stop drinking, and continued drinking despite serious psychological or physiological problems. We explored genetic influences on alcohol dependence among 1,897 European-American and African-American subjects with alcohol dependence compared with 1,932 unrelated, alcohol-exposed, nondependent controls. Constitutional DNA of each subject was genotyped using the Illumina 1M beadchip. Fifteen SNPs yielded P < 10−5, but in two independent replication series, no SNP passed a replication threshold of P < 0.05. Candidate gene GABRA2, which encodes the GABA receptor α2 subunit, was evaluated independently. Five SNPs at GABRA2 yielded nominal (uncorrected) P < 0.05, with odds ratios between 1.11 and 1.16. Further dissection of the alcoholism phenotype, to disentangle the influence of comorbid substance-use disorders, will be a next step in identifying genetic variants associated with alcohol dependence.

Time to First Cigarette in the Morning as an Index of Ability to Quit Smoking: Implications for Nicotine Dependence

An inability to maintain abstinence is a key indicator of tobacco dependence. Unfortunately, little evidence exists regarding the ability of the major tobacco dependence measures to predict smoking cessation outcome. This paper used data from four placebo-controlled smoking cessation trials and one international epidemiological study to determine relations between cessation success and the Fagerström Test for Nicotine Dependence (FTND), the Heaviness of Smoking Index, the Nicotine Dependence Syndrome Scale, and the Wisconsin Inventory of Smoking Dependence Motives. Results showed that much of the predictive validity of the FTND could be attributed to its first item, time to first cigarette in the morning, and this item had greater validity than any other single measure. Thus the time-to-first-cigarette item appears to tap a pattern of heavy, uninterrupted, and automatic smoking and may be a good single-item measure of nicotine dependence.

The CHRNA5-CHRNA3-CHRNB4 Nicotinic Receptor Subunit Gene Cluster Affects Risk for Nicotine Dependence in African-Americans and in European-Americans

Genetic association studies have shown the importance of variants in the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit gene cluster on chromosome 15q24-25.1 for the risk of nicotine dependence, smoking, and lung cancer in populations of European descent. We have carried out a detailed study of this region using dense genotyping in both European-Americans and African-Americans. We genotyped 75 known single nucleotide polymorphisms (SNPs) and one sequencing-discovered SNP in an African-American sample (N = 710) and in a European-American sample (N = 2,062). Cases were nicotine-dependent and controls were nondependent smokers. The nonsynonymous CHRNA5 SNP rs16969968 is the most significant SNP associated with nicotine dependence in the full sample of 2,772 subjects [P = 4.49 x 10(-8); odds ratio (OR), 1.42; 95% confidence interval (CI), 1.25-1.61] as well as in African-Americans only (P = 0.015; OR, 2.04; 1.15-3.62) and in European-Americans only (P = 4.14 x 10(-7); OR, 1.40; 1.23-1.59). Other SNPs that have been shown to affect the mRNA levels of CHRNA5 in European-Americans are associated with nicotine dependence in African-Americans but not in European-Americans. The CHRNA3 SNP rs578776, which has a low correlation with rs16969968, is associated with nicotine dependence in European-Americans but not in African-Americans. Less common SNPs (frequency <or= 5%) are also associated with nicotine dependence. In summary, multiple variants in this gene cluster contribute to nicotine dependence risk, and some are also associated with functional effects on CHRNA5. The nonsynonymous SNP rs16969968, a known risk variant in populations of European-descent, is also significantly associated with risk in African-Americans. Additional SNPs contribute to risk in distinct ways in these two populations.

Tobacco withdrawal. symptoms: An experimental analysis

This study was a prospective examination of tobacco withdrawal symptoms in a controlled environment. Smokers (N=27) were hospitalized for a 7-day period during which a battery of tests was administered. Smokers were assigned to either an experimental group (N=20) or a control group (N=7). Subjects in the experimental group smoked ad libitum for a 3-day baseline period and then underwent 4 days of tobacco deprivation. Subjects in the control group continued to smoke ad libitum throughout the study. Of the 37 measures of tobacco withdrawal employed in this study, nine showed significant changes following tobacco deprivation. These changes include decreased heart rate and increased caloric intake, weight, craving for tobacco, confusion, depression-dejection, number of awakenings, duration of awakenings, and increased poor concentrations as observed by others.

Multiple distinct risk loci for nicotine dependence identified by dense coverage of the complete family of nicotinic receptor subunit (CHRN) genes

Tobacco smoking continues to be a leading cause of preventable death. Recent research has underscored the important role of specific cholinergic nicotinic receptor subunit (CHRN) genes in risk for nicotine dependence and smoking. To detect and characterize the influence of genetic variation on vulnerability to nicotine dependence, we analyzed 226 SNPs covering the complete family of 16 CHRN genes, which encode the nicotinic acetylcholine receptor (nAChR) subunits, in a sample of 1,050 nicotine‐dependent cases and 879 non‐dependent controls of European descent. This expanded SNP coverage has extended and refined the findings of our previous large‐scale genome‐wide association and candidate gene study. After correcting for the multiple tests across this gene family, we found significant association for two distinct loci in the CHRNA5–CHRNA3–CHRNB4 gene cluster, one locus in the CHRNB3–CHRNA6 gene cluster, and a fourth, novel locus in the CHRND–CHRNG gene cluster. The two distinct loci in CHRNA5–CHRNA3–CHRNB4 are represented by the non‐synonymous SNP rs16969968 in CHRNA5 and by rs578776 in CHRNA3, respectively, and joint analyses show that the associations at these two SNPs are statistically independent. Nominally significant single‐SNP association was detected in CHRNA4 and CHRNB1. In summary, this is the most comprehensive study of the CHRN genes for involvement with nicotine dependence to date. Our analysis reveals significant evidence for at least four distinct loci in the nicotinic receptor subunit genes that each influence the transition from smoking to nicotine dependence and may inform the development of improved smoking cessation treatments and prevention initiatives.

Sex and menstrual cycle differences in the subjective effects from smoked cocaine in humans

To investigate sex and menstrual cycle effects in response to cocaine administration, data from existing studies were analyzed. First, responses to a single delivery of 0.4 mg/kg smoked cocaine were investigated. Women reported lower ratings for measures of paranoid/suspicious and heart racing/pounding than did men. In addition, women in the luteal phase reported diminished ratings for a measure of feel high than did both women in the follicular phase of the menstrual cycle and men. Second, responses to up to 6 deliveries of 0.4 mg/kg smoked cocaine were investigated. Women, compared with men, had lower ratings on feel high, heart racing/pounding, and feel stimulated. Results suggest that there are significant sex and menstrual phase differences in the subjective effects of cocaine.

Safety and immunogenicity of a nicotine conjugate vaccine in current smokers

Immunotherapy is a novel potential treatment for nicotine addiction. The aim of this study was to assess the safety and immunogenicity of a nicotine conjugate vaccine, NicVAX, and its effects on smoking behavior. Smokers (N = 68) were recruited for a noncessation treatment study and assigned to 1 of 3 doses of the nicotine vaccine (50, 100, or 200 μg) or placebo. They were injected on days 0, 28, 56, and 182 and monitored for a period of 38 weeks. Results showed that the nicotine vaccine was safe and well tolerated. Vaccine immunogenicity was dose‐related (P<.001), with the highest dose eliciting antibody concentrations within the anticipated range of efficacy. There was no evidence of compensatory smoking or precipitation of nicotine withdrawal with the nicotine vaccine. The 30‐day abstinence rate was significantly different across the 4 doses (P = .02), with the highest rate of abstinence occurring with 200 μg. The nicotine vaccine appears to be a promising medication for tobacco dependence.