To our knowledge, this study represents the first test of the association between genetic variation and initial sensitivity to acute nicotine responses in humans (i.e. in those essentially naïve to nicotine). Results should be considered very preliminary, given the relatively small sample and the number of genes and comparisons examined. However, our findings suggest that genetic variants purported to influence dopamine receptor function, particularly DRD4, were associated with initial sensitivity to mood and other effects of acute nicotine administration, and these effects often differed as a function of subject sex.
For DRD4, compared with those without the 7 allele, presence of the 7 allele was associated with increased sensitivity to mood (buzzed, decreased vigor and positive affect), nicotine perception (“feel effects”), and startle responses, but only in men. Regardless of sex, presence of the DRD4 7 allele was also associated with increased cortisol response and impaired rapid information processing performance. Together with the observed lower nicotine choice behavior, these findings suggest that presence of the DRD4 7 allele is associated with greater aversive responses to nicotine. Two prior studies in adult smokers have shown that the 7 allele is associated with increased risk of relapse in smoking cessation treatment (Shields et al., 1998
; David et al., in press
). Thus, our data suggest that greater aversive responses in the DRD4 7 group may reflect increased sensitivity to nicotine’s effects, which may be protective with regard to smoking onset in teens. However, among those who become smokers, those carrying the DRD4 7 repeat allele may become tolerant to these initially aversive responses, fostering greater subsequent risk of nicotine dependence. This notion is consistent with the “sensitivity” model (Pomerleau 1995
) and with preclinical studies suggesting that rodent strains most sensitive to nicotine upon initial exposure tend to show the greatest and most rapid tolerance to such effects (Marks et al. 1991
). Yet, far more research is needed before the “sensitivity” model, and DRD4 gene associations with sensitivity, can be adequately evaluated.
Associations of nicotine sensitivity with other genes believed related to dopamine function were less commonly found. In other analyses, we examined two putative functional SNPs related to the D2 dopamine receptor, DRD2 C957T and DRD2/ANKK1, and polymorphisms in the dopamine transporter, SLC6A3. Similar to DRD4, the DRD2 C957T variant was associated with anger and fatigue responses, as well as nicotine perception, across nicotine doses but only in men. The C957 allele has been associated with increased mRNA stability in vitro (Duan et al., 2003
), while the T957 allele has been associated with increased receptor binding potential in vivo (Hirvonen et al. 2004
). Thus, our findings appear more consistent with prior in vivo data than prior in vitro data. The second SNP (DRD2/ANKK1), located about 10kb upstream of DRD2 in the kinase gene ANKK1, was related only to startle (in men) and PPI responses to nicotine. Similarly, the dopamine transporter SLC6A3 variant was related only to PPI response.
Very few significant associations with nicotine sensitivity were seen for the genes not believed to be linked to dopamine function. The serotonin transporter 5HTTLPR variant was associated with nicotine reward (liking) in men but not in women. Similarly, in an exploratory comparison, among those homozygous for the 5HTTLPR long allele, men tended to choose nicotine more than did women, while there was no sex difference among those with the short allele. Finally, none of the genetic factors was associated with cardiovascular responses to nicotine, and none of the phenotypes examined was associated with the OPRM1 A118G SNP. While the absence of a finding for OPRM1 A118G may appear inconsistent with prior associations with smoking behavior and cessation (Lerman et al., 2003
; Ray et al., 2006
), these previous studies focused on nicotine dependent adults while the present study focused on nicotine sensitivity in nicotine-naïve subjects. Thus, as with our findings regarding DRD4, discussed previously, the role of these genes may be dependent on the particular smoking phenotype examined, such as vulnerability to onset of dependence versus smoking persistence after the establishment of dependence.
Our results may have implications for understanding sex differences in the discriminative stimulus effects and perhaps other effects of nicotine, as some research with dependent smokers indicates such effects are less pronounced in women compared to men (Perkins 1999
; in press
). We found that DRD2 and DRD4 variants influenced nicotine perception (“feel effects”), as well as some mood (“anger”, “fatigue”) responses, but only in men and not women. These observations suggest that, rather than being due simply to sex per se, many differences in nicotine responses between men and women may be mediated, or at least moderated, by particular genes. Although other research suggests that sex differences in nicotine responses of smokers may be moderated by genes (Yudkin et al. 2004
; Ray et al. 2006
), our results go beyond those findings to indicate that genes may moderate sex differences in responses among nicotine naïve individuals, prior to the onset of dependence.
Aside from the novelty of our study focus, genetic associations with initial nicotine sensitivity in never-smokers, many of the methods of this study were novel and constitute key strengths. One strength was our conservative lifetime tobacco use cutoff for inclusion (<10 uses), as well as exclusion of anyone with any use in the prior 3 years, to minimize the possibility of chronic adaptation to nicotine effects (i.e. tolerance or sensitization; Perkins 2002
), which would alter subjects’ sensitivity across doses. Our criterion is more stringent than that used in most other research, which typically defines “ever-smoker” as those with at least 100 lifetime uses (e.g. Giovino 2002
), while those with less than 100 are non- or never-smokers. Another strength of the study was the prospective assessment of nicotine responses, which included standardized measures of responses to nicotine other than self-report, as opposed to the retrospective self-report of early responses to nicotine via smoking in past studies of initial sensitivity (e.g. Pomerleau et al. 1998
). The broad array of responses was also a strength, allowing us to determine whether genetic associations may be specific to some but not other nicotine effects, although the large number of responses also raises concerns about the number of comparisons (see below). Other strengths of this study included the controlled dosing of nicotine and correction of doses for body weight, inclusion of placebo and more than one dose of nicotine, and use of nasal sensory irritation as a covariate to rule out differential irritation during testing as an explanation for differences in nicotine sensitivity.
On the other hand, this study had several clear limitations. One major limitation was the relatively small sample size of 101, which produced small genetic subgroups, although this sample is larger than past research assessing responses to nicotine in nonsmokers (see Perkins 2002
). This problem reflects the practical conflict between studying a limited amount of data from a large sample versus extensive phenotyping of a smaller sample (e.g., prospectively assessing many different responses to multiple nicotine doses across sessions, as in this study). As such, we may have had inadequate power to detect other genetic influences on acute nicotine responses. Somewhat in contrast, another major limitation is that some of the findings could have occurred by chance, given that we examined 6 genes, multiplying the number of comparisons for each measure of interest. To reduce the likelihood of chance findings, we focused solely on just two effects for each gene, the interactions of gene x dose and gene x dose x sex, given our interest in determining the association of genes with response to nicotine dose and how sex may moderate that association. We also limited the number of comparisons for mood and performance measures by employing multivariate analyses, conducting analyses of individual mood and performance responses only when the overall multivariate results were significant for either of these interactions involving gene x dose. The fact that many of the significant interactions involved DRD4 suggests that results for that gene were not due to chance, although the few significant effects for the other genes could have been due to chance. Given the complete lack of prior research on genetic associations with prospectively assessed initial nicotine sensitivity, this study was not intended to be definitive but rather exploratory and heuristic in nature. This study should be replicated with larger samples to verify the findings, particularly those for DRD4.
In addition, despite its advantages, our nicotine administration procedure was also a limitation of the study. We chose this nicotine nasal spray procedure to provide a fairly rapid method of delivery of controlled nicotine doses. However, sensitivity to nicotine via nasal spray may not relate to sensitivity to nicotine via cigarette smoking, although one study with smokers suggested generally comparable acute mood responses to nicotine between these methods (Perkins et al. 1994b
). Even more rapid uptake of nicotine by smoking, or stimuli accompanying smoking (e.g. taste, smell) but not nasal spray, may influence genetic associations with initial sensitivity to nicotine. Thus, although our method may have allowed for assessment of initial sensitivity to nicotine per se, sensitivity to nicotine via cigarette smoke inhalation may be affected differently by genetic factors. In addition, the doses used were fairly low, and greater influences of genetic factors on nicotine sensitivity may be more apparent in acute testing with larger doses of nicotine. Finally, we recruited young adults, rather than adolescents, due to ethical and practical concerns about giving nicotine to naïve adolescents. Adolescence is the age at which those who become smokers usually experience initial nicotine exposure, and genetic factors may have more influence on nicotine sensitivity at this age.
In summary, although these findings are preliminary and require replication, they suggest that initial sensitivity to some acute responses to nicotine is associated with specific genetic variants thought to be related to dopamine function, especially in men. In particular, these findings suggest that presence of the DRD4 7 allele is associated with increased sensitivity to nicotine’s aversive effects. Gene variants thought to be linked to the function of the dopamine D2 receptor showed some association with other nicotine responses, but most of the genes examined here were not consistently associated with initial nicotine sensitivity. Also, these genetic associations may be specific to only this phenotype, initial sensitivity to nicotine, and may not be seen in other phenotypes related to tobacco exposure (e.g. persistence of smoking). Future research should examine whether these genetic factors influence initial sensitivity to nicotine via smoking, if possible to do ethically and practically in a naïve sample, and should explore whether these genes are related to risk of nicotine dependence in teens with any past exposure to tobacco. Such findings may demonstrate that variability in initial sensitivity to nicotine is a mechanism by which genetic factors promote vulnerability to onset of nicotine dependence.