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Sex differences exist in the reinforcing effects of nicotine, smoking cessation rates, and in response to nicotine replacement therapies. Sex differences in availability of nicotinic acetylcholine receptors containing the β2 subunit (β2*-nAChRs) may underlie differential nicotine and tobacco smoking effects and related behaviors in women and men.
To examine β2*-nAChR availability between male and female smokers and nonsmokers. To determine relationships between β2*-nAChR availability and tobacco smoking characteristics and female sex steroid hormones.
Male (n=26) and female (n=28) tobacco smokers participated in one [123I]5-IA-85380 ([123I]5-IA) single photon emission computed tomography (SPECT) scan at 7–9 days of abstinence. Age-matched male (n=26) and female (n=30) nonsmokers participated in a single [123I]5-IA SPECT scan. All participants completed 1 magnetic resonance imaging study.
Academic Imaging Center
Tobacco smokers (n=54) and age- and sex-matched nonsmokers (n=56).
[123I]5-IA SPECT images were converted to equilibrium distribution volumes and analyzed using regions-of-interest.
β2*-nAChR availability was significantly higher in male smokers compared to male nonsmokers in striatum, cortex and cerebellum, but female smokers did not have higher β2*-nAChR availability than female nonsmokers in any region. In women, β2*-nAChR availability in the cortex and cerebellum was negatively and significantly correlated with progesterone level on the day of the scan. In female smokers, on the day of the scan, progesterone levels were positively and significantly correlated with depressive symptoms, craving for a cigarette, and nicotine withdrawal.
The regulatory effects of nicotine in the brain, i.e., tobacco-smoking induced upregulation of β2*-nAChRs, appear to be distinctly different between men and women, and female sex hormones likely play a role in this regulation. These findings suggest an underlying neurochemical mechanism for the reported behavioral sex differences. In order to treat female smokers more effectively, it is critical that non-nicotinic mediated medications are explored.
Nicotine replacement therapies (NRTs), e.g., nicotine patch, gum, lozenge, inhaler, as well as the nicotinic partial agonist therapy varenicline, are the most widely used treatments to help people quit smoking. Many studies show that men respond better to NRT than women1, and women often have a more difficult time quitting smoking than men2–6. In a series of studies, Perkins and colleagues7 demonstrated that men are better able to identify nicotine in a nasal spray and to discriminate nicotine from placebo than are women, indicating that men are better able to detect the interoceptive cues of nicotine. Consistently, women experience greater craving relief then men from a denicotinized tobacco inhaler8, highlighting the role sensory cues play in nicotine dependence for women. These results are consistent with findings showing that women are more reinforced by the non-nicotine conditioned stimuli that are strongly associated with smoking than men, while men are more reinforced by the nicotine per se in cigarettes and in NRT than women9.
Preclinical studies generally support the clinical findings showing sex differences in response to nicotine suggesting an underlying biological sexual dimorphism. For example, female mice responded more to the locomotor stimulating10, 11 and conditioned rewarding properties of nicotine12 whereas male mice responded more to pharmacological properties of nicotine by titrating their consumption to available nicotine dose12. The molecular mechanisms underlying these sex differences are not understood, although it is clear that behaviors, as well as the primary reinforcing effects of nicotine are mediated by nicotinic acetylcholine receptors containing the β2 subunit (β2*-nAChRs)13–16.
The primary reinforcing effects of nicotine are mediated by the β2-containing subunit of the nicotinic acetylcholine receptor (β2*-nAChR). The β2*-nAChRs have a high affinity for nicotine and nicotinic agonists and have been consistently shown in preclinical17, 18 and postmortem19 human studies to upregulate, i.e., increase in number, in response to nicotine. We measure β2*-nAChR availability with the radiotracer [123I]5-IA-85380 ([123I]5-IA) and single photon emission computed tomography (SPECT). [123I]5-IA is a high affinity nicotinic agonist radioligand that binds to the nicotine binding site on β2*-nAChRs20 with excellent test-retest reproducibility21. We22, 23 and others24 demonstrated higher β2*-nAChR availability in smokers compared to nonsmokers with SPECT and PET brain imaging, which likely reflects tobacco-smoking induced upregulation of β2*-nAChRs. Sex differences in β2*-nAChR number have also been examined. Preclinical studies in nicotine-naïve rodents are conflicting, with one study demonstrating sex differences in β2*-nAChR number25 and another showing no sex difference in β2*-nAChR number26. We found no differences in β2*-nAChR availability between male and female nonsmokers27. Several preclinical studies report greater nicotine-induced upregulation of nAChRs in male vs. female rodents compared to each groups same-sex controls25, 28; however, this has not been examined in humans. Understanding sex differences in the underlying neurochemistry involved in tobacco smoking dependence may pave the way for development of novel treatment medications.
In the present study we hypothesized that sex differences in β2*-nAChR availability may underlie sex differences in nicotine and tobacco smoking related behaviors. The primary goal of the current study was to examine sex-differences in β2*-nAChR availability in smokers compared to nonsmokers. A secondary goal of the study was to determine associations between β 2*-nAChR availability and behavioral and physiological variables, such as tobacco-smoking characteristics (craving and withdrawal, and depression) and hormone levels (estrogen and progesterone in women), in smokers and nonsmokers.
Fifty-two men (nonsmokers: n=26, 34.8±11.7 y, range=21–58 y; smokers: n=26, 33.8±11.7 y, range=18–57 y) and fifty-eight women (nonsmokers: n=30, 31.3±11.7 y, range=19–56 y; smokers: n=28, 36.0±10.0 y; range=18–50 y) participated in one [123I]5-IA SPECT scan and one magnetic resonance imaging (MRI) study. Smokers were scanned at 7–9 days of abstinence based on previous studies demonstrating 7–9 days of abstinence are necessary for nicotine to clear from the brain so that nicotine will not compete with the radiotracer at the binding site22, 23.
This study was approved by the Yale University School of Medicine Human Investigation Committee, the West Haven Veterans Administration Human Subjects Subcommittee, and the Radiation Safety Committee. Participants provided written informed consent and were recruited by word of mouth, posters, and television and newspaper advertisements. Eligibility was determined as follows: a medical examination including a physical examination, electrocardiogram, serum chemistries, thyroid function studies, complete blood count, urinalysis, and urine toxicology screening. Participants had no history of significant medical illness or major head trauma. The Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders (SCID) was administered to rule out Axis I Disorders except for Nicotine Dependence in tobacco smokers.
Tobacco smokers had to smoke ≥10 cigarettes daily for at least one year, confirmed by plasma cotinine levels >150 ng/mL, urine cotinine levels >100 ng/mL and carbon monoxide levels >11 at intake. Smokers were helped to quit smoking using Clinical Practice Guidelines and contingency management29–31 as described previously23. Subjects were not permitted to use any NRT or medication throughout the study. Nonsmoker status (defined as <100 cigarettes in lifetime, and none in the previous two years) was confirmed by plasma cotinine levels <15 ng/mL, urine cotinine levels <100 ng/mL and carbon monoxide levels <8 ppm on intake and scan day. On scan day, abstinent smokers had plasma cotinine levels <15 ng/mL, urine cotinine levels <100 ng/mL and carbon monoxide levels <8 ppm. Plasma nicotine and cotinine levels were measured as previously described22. Urine cotinine levels were measured with either Accutest NicoMeter™ cotinine test strips (Jant Pharmacal, Encino, CA) or NicAlert™ cotinine test strips (Nymox Pharmaceutical, Hasbrouck Heights, NJ). Women had a negative pregnancy test during screening and prior to radiotracer injection on scan day. Menstrual cycle phase was not controlled and hormonal contraception was not exclusionary. Blood samples were taken on scan day to determine hormone levels including estradiol and progesterone.
In smokers, nicotine dependence severity was assessed at intake with the Fagerström Test for Nicotine Dependence (FTND)32, and craving and nicotine withdrawal symptoms were assessed with the Urge to Smoke Questionnaire (QSU)33 and the Minnesota Nicotine Withdrawal Scale (MNWS)34, respectively, at intake, and on scan day. The QSU yields two factors, the intention/desire to smoke (QSU-Intent) and relief of negative affect and withdrawal (QSU-Relief). In smokers and nonsmokers, symptoms of depression were assessed at intake and on scan day with the Beck Depression Inventory (BDI).
MRI studies were obtained on a 1.5 Tesla Siemens camera in a standard orientation (TE=5–7 ms; TR=24ms; a 256×192 matrix; 1 NEX; FOV 30 cm; 124 contiguous slices with 1.2 mm thickness) and were used for co-registration to the SPECT images.
Subjects received a 0.6-g saturated solution of potassium iodide to protect their thyroid from possible exposure to radioactive iodide prior to radiotracer administration. [123I]5-IA was synthesized as previously described 35 and administered as a bolus to constant infusion at a ratio of 7.0 for 8 hours. Subjects were injected with equivalent doses of a bolus (mean±SD, 147±25 MBq) and constant infusion (mean±SD, 25±5 MBq/h). Three consecutive 30-min emission scans and one 15-min simultaneous transmission and emission scan were obtained between hours 6 and 8 of the [123I]5-IA infusion on a Picker PRISM 3000 XP (Cleveland, OH) SPECT camera. The axial resolution (full width at half maximum) is 12.2 mm, measured with a 123I line source in water in a cylindrical phantom. Blood was drawn prior to injection and at the beginning and end of the emission scans for analysis of plasma total parent and free fraction of parent tracer in plasma (fP, free fraction). The chemical fate of [123I]5-IA post injection was assessed in plasma as previously described35.
Images were reconstructed and analyzed as previously described including a nonuniform attenuation correction 21. MRIs were coregistered to the SPECT images to provide an anatomical guide for placement of the regions of interest using Medx (version 3.4) software (Medical Numerics, Inc., MD). Regions-of-interest were those known to express β2*-nAChRs and included frontal, parietal, anterior cingulate, temporal and occipital cortices, thalamus, striatum (an average of caudate and putamen) and cerebellum.
The outcome measure VT/fP (regional activity divided by free plasma parent between 6 and 8 hours), was used to correct for possible differences in radiotracer metabolism or plasma protein binding between groups and subjects. Specifically, VT/fP equals [123I]5-IA uptake in a region-of-interest (kBq/cc) / free plasma parent (kBq/mL)36. We refer to VT/fP as “β2*-nAChR availability” because we are measuring receptors that are “available” to be bound by radiotracer. Receptors that are already occupied, e.g., by residual nicotine, a pharmacologically active metabolite (cotinine or nornicotine) or by endogenous neurotransmitter (acetylcholine) are not available. VT/fP is proportional to the binding potential (BP, mL/g=Bmax/KD), which is proportional to the receptor number (Bmax) at equilibrium, given the assumptions that there is no change in affinity (KD) and that non-displaceable (non-specific and free) uptake does not differ between subjects. As described previously21, there is no appropriate reference region for this radiotracer due to the wide expression of β2*-nAChRs in brain.
Statistical analyses were conducted using SPSS version 16.0 (SPSS Inc. Headquarters, Chicago, IL). Independent t-tests were used to examine demographic variables between groups. Multivariate analysis of variance (MANOVA) models were first used to examine differences in β2*-nAChR availability (VT/fp) by sex and smoking status in four brain regions (average cortex [average of frontal, parietal, anterior cingulate, temporal and occipital cortices], striatum, thalamus, and cerebellum). The cortical regions were averaged together due to the high intercorrelations between these regions. Based on our previous finding that β2*-nAChR availability decreases with age37, we initially used age as a covariate, but because there was no effect on β2*-nAChR availability, age was dropped from further analysis. To further examine differences between male and female smokers and male and female nonsmokers (4 groups), ANOVAs were conducted for each of the eight brain regions. Correlational analyses for the associations between β2*-nAChR availability and smoking characteristics (cigarettes per day, FTND, years smoked, QSU [desire/relief], MNWS) and depression (BDI) at baseline and on scan day, and hormone levels on scan day (estrogen and progesterone) within each group were assessed with Pearson correlation coefficients. Due to multiple comparisons, statistical significance for the correlations was considered at p≤0.01.
Fifty-two men (nonsmokers: n=26, smokers: n=26) and 58 age-matched women (nonsmokers: n=30, smokers: n=28) participated in the study. Included in this sample are subjects (n=8 male and n=7 female smokers; n=10 male and n=11 female smokers), from our two smaller previous studies22, 23. Male and female smokers were not matched for smoking characteristics. Plasma cotinine and nicotine and carbon monoxide levels were negligible in nonsmokers and smokers on the day of the scan, confirming abstinence from smoking and there were no significant differences between men and women (Table 1). One female smoker had a cotinine level of 328 ng/mL on SPECT scan day which is responsible for the numerically higher cotinine level in female vs. male smokers on scan day; however, this was a decrease in the level of cotinine from intake and her nicotine and CO levels were also reflective of abstinence from tobacco smoking. Additionally, this participant’s brain receptor levels were within the range of other female smokers so she was not excluded. Male and female smokers did not differ significantly on symptoms of depression (BDI), nicotine withdrawal (MNWS) or craving (QSU-Intent, QSU-Desire) at baseline or on scan day, but there were differences within sex between baseline and 1 week of abstinence (Table 2). Specifically, for men, scores on the QSU-Desire (P<0.05) and for women scores on the QSU-Desire (P<0.01) and QSU-Intent (P<0.05) were significantly lower at 1 week of abstinence compared to baseline.
There were no differences between groups in injected dose of [123I]5-IA, bolus to infusion ratio, or concentrations of [123I]5-IA activity in the blood, e.g., total parent, free fraction (fp) or free parent (total parent * fp). Regional β2*-nAChR availability was compared between smokers and nonsmokers as a function of sex using MANOVA (Table 2, FFigure 1 and and2).2). Multivariate analysis of variance demonstrated significant omnibus tests for a main effect of smoking status (4,102 = 60.24; P<.0005) and a sex by smoking status interaction (F4,102 = 2.48; P<.05). Male smokers had significantly higher β2*-nAChR availability than male nonsmokers in parietal cortex (F1,51=9.33; P=.004), frontal cortex (F1,51=7.48; P=.009), anterior cingulate cortex (F1,51=11.01; P=.002), occipital cortex (F1,51=15.09; P<.001), temporal cortex (F1,51=9.26; P=.004), cerebellum (F1,51=12.03; P=.001), striatum (F1,51=7.96; P=.007), but there were not differences in the thalamus (F1,51=1.66; P=.203), consistent with a previous study22. β2*-nAChR availability between female smokers and nonsmokers did not differ significantly in parietal cortex (F1,57=0.15; P=.700), frontal cortex (F1,57=0.09; P=.772), anterior cingulate cortex (F1,57=1.40; P=.242), occipital cortex (F1,57=2.75; P=.103), temporal cortex (F1,57=0.47; P=.495), cerebellum (F1,57=2.98; P=.090), or striatum (F1,57=0.64; P=.428), but paradoxically, women nonsmokers had higher β2*-nAChR availability than smokers in the thalamus (F1,57=4.37; P=.041) (Table 2, Figure 1 and and2).2). Male smokers were not significantly different in β2*-nAChR availability than female smokers in any region. Finally, there was a trend toward higher β2*-nAChR availability in female vs. male nonsmokers across regions as follows: parietal cortex (F1,55=5.74; P=.020), frontal cortex (F1,55=5.74; P=.020), anterior cingulate cortex (F1,55=3.12; P=.083), occipital cortex (F1,55=2.81; P=.100), temporal cortex (F1,55=2.91; P=.094), cerebellum (F1,55=1.99; P=.164), striatum (F1,55=3.08; P=.085), and thalamus (F1,55=4.79; P=.033) (Table 2, Figure 1).
In women, when the sample was restricted to ages 18–40 to exclude potential peri- or post-menopausal women, the findings were consistent. Although we obtained information on reproductive status and hormonal therapy, the subsamples were too small to reliably evaluate β2*-nAChR availability by this breakdown.
There were no significant correlations in any smoker group (male smokers, female smokers, or smokers as a whole) between regional β2*-nAChR availability and smoking characteristics (cigarettes per day, FTND, years smoked, QSU [desire/relief], MNWS) or in any group (male nonsmokers, male smokers, female nonsmokers, female smokers, or smokers or nonsmokers as a whole) between regional β2*-nAChR availability and depression (BDI), at baseline or on scan day. There were no significant correlations in smokers between the change in craving (QSU [desire/relief]), withdrawal (MNWS), or depression (BDI) from baseline to scan day and regional β2*-nAChR availability.
Average estrogen and progesterone levels on scan day did not differ significantly between female smokers (estrogen: 79±58 pg/mL; progesterone: 3±4 ng/mL) and nonsmokers (estrogen: 81±75 pg/mL; progesterone: 5±5 ng/mL); however, smokers exhibited a more restricted range of both hormones (estrogen: 32–203 pg/mL; progesterone: 0.3–12.0 ng/mL) compared to nonsmokers (estrogen: 32–410 pg/mL; progesterone: 0.3–17.0 ng/mL), which is consistent with studies showing lower hormone levels in smoking vs. nonsmoking women38. β2*-nAChR availability was significantly negatively correlated with progesterone (ng/mL) but not estrogen (pg/mL) or the ratio of estrogen to progesterone (pg/mL) on the day of the scan in female smokers and nonsmokers, such that higher progesterone levels were associated with lower β2*-nAChR availability. This was true both when female smokers and nonsmokers were combined and when examined separately. Specifically, progesterone (ng/mL) and β2*-nAChR availability were significantly negatively correlated in the whole group in the cerebellum (r=−.37; P=0.006), and average cortex (r=−0.35; P=0.009), with a trend in the striatum (r=−0.27; P=0.04), and no significant correlations in the thalamus (r=−0.17; P=0.22) (Figure 3). When separated by group, progesterone and β2*-nAChR availability were significantly negatively correlated in nonsmokers in the average cortex (r=−0.47; P=0.010) and there was a trend for a negative correlation in smokers in the cerebellum (r=−0.39; P=0.04).
There were significant correlations between progesterone levels (ng/mL) on scan day and clinical correlates on scan day. Specifically, progesterone was significantly positively correlated with scores on the BDI (r=.55; P<.005), MNWS (r=.52, P<.005), and QSU-Relief (r=.66; P<.001) (Figure 4). Higher progesterone levels were associated with worse self-reported depression, nicotine withdrawal, and craving to smoke a cigarette to relieve withdrawal.
In the present study we examined sex differences in β2*-nAChR availability between smokers and nonsmokers. We demonstrate significant sex-specific differences in β2*-nAChR availability between smokers and nonsmokers. Specifically, male smokers had significantly higher β2*-nAChR availability compared to male nonsmokers in the striatum (16%), cerebellum (17%) and cortical regions (13–17%), but female smokers had similar β2*-nAChR availability compared to female nonsmokers (1–6%) in these areas. This finding is striking given the wealth of literature on tobacco smoking and nicotine-induced upregulation of nAChRs throughout the brain. Specifically, the upregulation, or increased availability, of nAChRs has been widely demonstrated in preclinical rodent studies18, 39, postmortem human studies19, 40, 41, and in living humans with PET24 and SPECT22, 23 imaging. While previous imaging and postmortem studies were underpowered to examine sex differences, two previous preclinical studies examined sex differences in nicotine-induced nAChR upregulation. These studies determined that male rodents exposed to nicotine compared to non-exposed controls had greater increases in nAChR number than females exposed to nicotine compared to non-exposed females25, 28. Consistent with the preclinical studies, our results revealed higher β2*-nAChR availability in the brains of male vs. female tobacco smokers compared to male and female nonsmokers, respectively. Taken together, these data provide a window into the brain chemistry potentially underlying the multitude of findings of sex differences in tobacco smoking and nicotine related behaviors in both clinical and preclinical studies.
The current finding showing a lack of significant upregulation of β2*-nAChR availability in the thalamus in male smokers is consistent with receptor imaging23, 24, postmortem19 and preclinical42, 43 studies showing that the thalamus is differentially regulated by nicotine compared to other regions. The reasons for this remain unclear, but may be due to a ceiling effect. The thalamus has the highest density of β2*-nAChRs in the brain and in healthy subjects the number of β2*-nAChRs in thalamus may already be at a maximal level and not further increased by smoking. In the current study male smokers and nonsmokers had equivalent thalamic β2*-nAChR availability whereas female smokers had significantly lower thalamic β2*-nAChR availability (17%) compared to female nonsmokers. The thalamus was the only region where differences in β2*-nAChR availability were found in female smokers compared to nonsmokers. Thus, not only do female smokers not have higher β2*-nAChR availability throughout the brain, β2*-nAChR availability is significantly lower in the thalamus compared to female nonsmokers. This is critical given that the relay of sensory information via thalamo-cortical connections may be modulated by nicotine exposure44, 45. Therefore, sex differences in the effects of tobacco smoking on thalamic β2*-nAChR availability may underlie sex differences in cue-induced activation in connecting cortical areas46.
In this study there was a trend for higher β2*-nAChR availability in female compared to male nonsmokers. In our previous study27 we did not find sex differences in β2*-nAChR availability between male (n=10) and female (n=19) nonsmokers; however in this larger study of male (n=26) and female (n=30) nonsmokers, women had significantly higher β2*-nAChR availability than men in the parietal and frontal cortices and the thalamus, with a trend for differences in the other regions. It is not clear whether the lack of a difference in β2*-nAChR availability between female smokers and nonsmokers compared to male smokers vs. nonsmokers is because women are beginning at a higher “baseline”. The current study cannot address this question because it is not clear that all women have higher β2*-nAChR availability or whether there are initial differences in β2*-nAChR availability in women who do, and do not, go on to become smokers. One preclinical study reported female rodents had higher whole brain nAChR densities than males25 and another reported no difference26. In keeping, our findings suggest a basal sex difference in nonsmokers, with women having moderately higher β2*-nAChR availability than men.
Nicotine from tobacco smoke and nornicotine47, 48 have been shown to acutely displace β2*-nAChR ligands from the receptor. Cotinine, given acutely (0.06 mg/kg, IV) did not displace [123I]5-IA in baboon brain (Cosgrove, Unpublished data), and based on these data, it seems unlikely that metabolites of nicotine (cotinine, nornicotine) are influencing β2*-nAChR availability at 7–9 days of abstinence. We chose 7–9 days of abstinence based on our previous studies22, 23 suggesting this is adequate time for nicotine and its metabolites to clear from the brain and body.
We have previously reported relationships between β2*-nAChR availability and craving. Specifically, recently abstinent smokers (~1 week) with higher β2*-nAChR availability reported higher craving to smoke to relieve withdrawal symptoms22. Consistent with our previous study, we did not find relationships between β2*-nAChR availability and smoking characteristics such as cigarettes smoked per day, nicotine dependence, or years smoked. But, we also did not find a relationship between β2*-nAChR availability and craving at 1 week of abstinence in the smokers as a whole group, or when broken down by sex. This is likely due to the much larger sample in the current study of smokers (n=54) vs. the previous study (n=16)22. This suggests that β2*-nAChR availability at 1 week of abstinence may not be related to smoking characteristics, craving or depressive symptoms in healthy smokers. In women and men, craving was significantly lower at 7–9 days of abstinence than prior to quitting, in keeping with studies suggesting withdrawal symptoms that drive craving peak within the first week of quitting, and decline by 7–9 days of abstinence49. Additionally, smokers were helped to remain abstinent with contingency management techniques. The provision of daily support and financial rewards for abstinence likely helped smokers cope with early craving and withdrawal symptoms allowing these symptoms to resolve with longer abstinence thereby attenuating relationships between receptor availability and tobacco smoking characteristics. Relationships between β2*-nAChR availability and clinical correlates may play a more prominent role in smokers with a significant psychiatric comorbidity50 or during more acute or prolonged abstinence as we have shown previously23.
In women, sex-steroid hormones such as progesterone have been shown to play a critical role in tobacco smoking behaviors, including quit attempts51. For example, women had a longer time to relapse when they made their quit attempt in the luteal phase of their menstrual cycle, when levels of progesterone are much higher than estrogen, than in the follicular phase, when estrogen levels are higher and progesterone levels are minimal52, 53, and phase of cycle may influence medication efficacy53. In a previous study with a smaller sample of nonsmokers27, we did not find relationships between menstrual cycle phase or hormone levels on scan day with β2*-nAChR availability. However, in this current larger study, progesterone is significantly negatively associated with β2*-nAChR availability in both female smokers and nonsmokers. Specifically, higher levels of progesterone on scan day were associated with lower β2*-nAChR availability. This suggests that progesterone may be an allosteric modulater of the β2*-nAChR and is consistent with preclinical literature suggesting that progesterone54–56 and its Aring metabolites55 inhibit nAChRs noncompetitively. Progesterone57 and the neurosteroid pregnenolone58 also have been shown to block nicotinic receptor function, and progesterone has reduces the urge to smoke59, 60. Taken together, these findings suggest that progesterone treatment for smoking cessation should be explored. Additionally, progesterone may have inhibitory actions directly or indirectly on the β2*-nAChR and these actions may play a role in the observed sex differences in β2*-nAChR availability.
There is a large literature establishing a relationship between ovarian hormones and mood regulation and affective disorders. Negative affect61 and depression61, 62 have been identified as contributing factors to relapse in female smokers. In female smokers, on the day of the SPECT scan, i.e., at 7–9 days of abstinence, higher progesterone levels were associated with higher self-reported symptoms of depression, worse withdrawal, and greater craving to smoke to relieve withdrawal symptoms. These participants do not have a history or current diagnosis of major depression or other major psychiatric disorders suggesting progesterone modulates mood in healthy smokers. Furthermore, while higher progesterone levels were associated with lower β2*-nAChR availability in female smokers, there was no relationship between depressive symptoms, withdrawal, or craving and β2*-nAChR availability in female smokers. Thus progesterone’s relationship with depressive symptoms, withdrawal and craving is not directly related to β2*-nAChR availability. The finding that higher progesterone during abstinence is associated with higher tobacco smoking craving and higher nicotine withdrawal is somewhat inconsistent with the idea that progesterone may be therapeutically effective. However, this study is limited in that while we obtained hormone levels on scan day, we have included a heterogeneous group of women including women on birth control and menopausal women; thus, this does not preclude the possibility that exogenously administered progesterone will be an effective therapeutic.
There are several limitations to the current study. One limitation is that [123I]5-IA measures β2*-nAChRs in the brain and while it has been traditionally thought that nAChRs containing two α4 and three β2 subunits are the primary receptor subunit composition that upregulates in response to nicotine, there is increasing evidence to suggest that other subunits (α5, α6, β3) may combine with the α4 and β2 subunits and play a role in upregulation of the receptor63. Second, we did not obtain hormone levels in male subjects, therefore the role hormones play in β2*-nAChR availability in men cannot be examined in this study. Third, as previously mentioned we enrolled a heterogeneous group of women, regardless of menopausal status, gynecological surgical history, or current hormonal contraceptive use. Thus, we cannot conclude whether endogenous fluctuating gonadal hormone levels underlie the observed sex difference in β2*-nAChR availability. Fourth, we did not examine whether sex differences in β2*-nAChR availability in smokers are related to differences in time to relapse; we are currently collecting this information as part of an ongoing study. Fifth, although we have demonstrated high test-retest reproducibility of our imaging protocol21, there are inherent limitations in the ability to correct for motion within SPECT scans that may contribute to the variability between subjects.
This study highlights the critical need for sex-specific treatment strategies for tobacco smoking. The vast majority of currently available cessation treatments, e.g., NRTs and varenicline, act at the β2*-nAChR; however, clinical studies consistently demonstrate that men respond more than women to the nicotine in tobacco smoke and that men generally have a better therapeutic response to NRT1 than women. Women also have lower success rates of quitting smoking than men2–6, which underscores the need to determine the biological dimorphisms that may drive these behavioral outcomes. The current findings provide a potential biological rationale for these differences. Specifically, the regulatory effects of nicotine in the brain appear to be distinctly different between men and women. Men are more reinforced by the nicotine per se in tobacco smoke, respond better to NRT1, and have higher β2*-nAChR availability compared to male nonsmokers, i.e., an upregulation of β2*-nAChRs. Female compared to male smokers are more reinforced by the conditioned cues of smoking8, do not respond as well to NRT1 and do not appear, on average, to have higher β2*-nAChR availability compared to female nonsmokers. In order to treat female smokers more effectively, it is critical that non-nicotinic mediated medications are explored.
Funding: This research was supported in part by National Institute of Health grants R01 DA015577 and K02 DA21863 (Staley), K01 DA20651 (Cosgrove), P50 DA13334 and P50 AA15632 (O’Malley). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Alcohol Abuse and Alcoholism, the National Institute on Drug Abuse, or the National Institutes of Health.