Sex differences exist in tobacco smoking. Women become addicted more quickly after exposure (Thorner et al., 2007), experience greater health consequences (US Department of Health Human Services, 2014), are less responsive to the first line of treatment (nicotine replacement therapies) (Fattore et al., 2008), and are more likely to relapse after abstinence (Fattore et al., 2008). Tobacco smoking is driven in part by the reinforcing effects of nicotine, the primary addictive component in cigarettes. In the brain, nicotine binds to and activates nicotinic acetylcholine receptors, facilitating dopamine release in striatal and cortical regions (Benowitz, 2010; Cosgrove et al., 2015) via the mesolimbic and mesocortical dopamine pathways, respectively. The mesolimbic (“reward”) dopamine pathway drives the reinforcing effects of tobacco smoking, while the mesocortical (“goal-directed”) dopamine pathway—including the dlPFC—is critical for inhibitory control (Wu et al., 2013). Dysregulated dopamine D_2/3 receptor (D₂R) signaling in dlPFC is associated with cognitive deficits, such as impairments in attention, learning, working memory, and inhibitory control (Arnsten et al., 2015), that are known to impede quit attempts. Literature has shown that sex steroid hormones, such as estradiol and progesterone, influence drug-taking (including nicotine) behaviors, through dopaminergic actions. This suggests that sex steroid hormone influences reward and cognition may partially explain sex differences in tobacco smoking and the greater vulnerability in women. We have previously shown that women (vs. men) who smoke have greater dlPFC dopamine deficits. However, the relationship between these dopamine deficits and sex steroid hormone levels has not yet been examined.

Cigarette smoking has been associated with adverse reproductive outcomes including infertility, subfecundity, early menopause, and menstrual disorders (Department of Health Human Services, 2001). Cigarette smoke contains known toxicants to the reproductive system (Mattison and Thorgeirsson, 1978) and promotes estrogen deficiency or hypoestrogenism (Baron et al., 1990). Among women, heavy smoking is associated with shorter and more variable menstrual cycle lengths with the shorter menstrual cycle lengths occurring primarily in the follicular phase (Windham et al., 1999). While the biological mechanisms of these findings are not known, the literature suggests that the effects of smoking on hormone secretion and metabolism are mainly mediated by the pharmacological action of nicotine and its metabolite, cotinine (Marom-Haham and Shulman, 2016). Furthermore, a growing body of data, including epidemiological data, suggests that these effects may be non-linear [reviewed in Marom-Haham and Shulman (2016)]. More studies are needed to fully understand the complex pathobiology of cigarette smoking and its hormonal impact.

A preclinical study has shown that sex steroid hormones are involved in the acquisition of, motivation for, and reinstatement of drug taking. Female rats acquire drug self-administration in an operant conditioning chamber more rapidly than male rats (Lynch, 2006), which has been shown to be primarily due to circulating ovarian hormones in females; estradiol exposure enhances the rate of acquisition of drug taking (Lynch et al., 2001; Jackson et al., 2006; Hu and Becker, 2008). Female rats exhibit a higher breaking point to obtain drugs on a progressive ratio schedule than males, suggesting a greater drug motivation (Roberts et al., 1989; Cummings et al., 2011). This effect is enhanced by estradiol treatment in ovariectomized rats (Becker and Hu, 2008). With respect to reinstatement or relapse-like behavior, estradiol enhances while progesterone attenuates reinstatement in females (Fuchs et al., 2005; Feltenstein and See, 2007), through modulation of dopamine (Castner et al., 1993; Cummings et al., 2014). Because progesterone may attenuate some of the subjective effects of drugs of abuse and thereby decrease drug intake (Evans and Foltin, 2006; Jackson et al., 2006), sex steroid hormones may help direct treatments for addiction (Lynch et al., 2002).

The emerging body of literature on the interactions of dopamine and sex steroid hormones in addiction to nicotine has predominantly focused on ovarian hormones. For example, in castrated rats, estrogen differentially modulated nicotine-induced dopamine release in the striatum, enhancing dopamine release in female rats and reducing dopamine release in male rats (Dluzen and Anderson, 1997). In humans, cyclical changes in ovarian hormones over the course of the menstrual cycle alter sensitivity to the reinforcing effects of nicotine through interactions with the dopamine system (Carpenter et al., 2006). Taken together, this suggests that nicotine may interact with sex steroid hormones within the context of the dopamine system to impact the vulnerability of women to cigarette smoking. However, this topic is critically understudied, and this has not been directly tested.

Our group previously showed that dlPFC D₂R availability was significantly lower in people who smoke than healthy controls and that drug-induced dlPFC dopamine release (using amphetamine, a robust dopamine probe) is blunted in women who smoke compared to men who smoke and healthy control women (Zakiniaeiz et al., 2019). We also showed that lower dlPFC D₂R availability was associated with poorer cognitive function (Zakiniaeiz et al., 2022). However, we did not examine whether dlPFC D₂R availability before and after amphetamine administration was related to sex steroid hormones. The goal of this study was to relate dopamine metrics to sex steroid hormone levels in people who do and do not smoke. Based on dopamine-related findings from our group and prior literature, we hypothesized that in women, lower dlPFC D₂R availability before and after amphetamine administration would be related to lower estradiol and higher progesterone levels. Based on limited research, as an exploratory secondary analysis, we also hypothesized that lower dlPFC D₂R availability before and after amphetamine administration would be related to lower testosterone levels.

There were three main findings from this study: (1) women and men who smoke have different levels of sex steroid hormones compared to sex-matched counterparts, (2) for women only, lower levels of estradiol were significantly associated with lower dlPFC D₂R availability, and (3) for women only, trending lower levels of free testosterone were associated with lower dlPFC D₂R availability pre-amphetamine relative to post-amphetamine. In men, sex steroid levels were not significantly associated with dopamine metrics. A previously published study from our group showed that people who smoke tobacco had significantly lower dlPFC D₂R availability compared to healthy controls (Zakiniaeiz et al., 2019) and that lower dlPFC D₂R availability was associated with poorer cognitive function (Zakiniaeiz et al., 2022). This study extends these findings by relating dlPFC D₂R availability to sex steroid hormone levels in people who smoke tobacco, providing a better understanding of the influence of sex steroid hormones on the dopamine system (Zakiniaeiz and Cosgrove, 2020).

Lower estrogen levels have previously been observed in women who smoke (Westhoff et al., 1996), consistent with the trend observed in our study. Our findings are also consistent with previous human research reporting that men who smoke have higher concentrations of testosterone than their healthy control counterparts (English et al., 2001; Allen et al., 2002; Muller et al., 2003; Shiels et al., 2009; Liu et al., 2021). One study found that men who formerly smoked do not show this effect, suggesting that the association between testosterone and smoking is reversible (Shiels et al., 2009). Some studies have shown a dose-response relationship between the number of cigarettes smoked and testosterone levels (Tamimi et al., 2001; Allen et al., 2002) while others do not (Handa et al., 1997; Svartberg and Jorde, 2007). In contrast to our study, one study showed that male mice exposed to cigarette smoke have lower levels of testosterone relative to sex-matched controls (Yardimci et al., 1997). The relationship and mechanism between cigarette smoking and testosterone levels remains unclear. One possible mechanism is that cigarette smoke and nicotine may act as aromatase inhibitors, thus reducing the conversion of testosterone to estradiol. However, inconsistent with this theory, we also observed higher estradiol levels in men who smoke. Similar findings were previously reported (Svartberg and Jorde, 2007; Shiels et al., 2009), while other studies found no relationship between testosterone levels and smoking (Handa et al., 1997; English et al., 2001; Muller et al., 2003). Furthermore, our results show that women who smoke have lower levels of estradiol than their sex-matched counterparts while men who smoke have higher levels of estradiol than their sex-matched counterparts. This sexual dimorphism may be confounded by menstrual cycle phase which was not controlled in our study. Our findings and the current literature suggest that cigarette smoking alters sex steroid hormone levels, but more studies are needed to uncover the biological mechanism.

Among women, lower levels of estradiol were associated with lower dlPFC D₂R availability, consistent with our hypothesis. In line with this finding, a preclinical study showed that there is a sexually dimorphic effect of estradiol on the D₂R, where estradiol rapidly downregulates D₂ binding in females but not in males (Bazzett and Becker, 1994). Prior literature predominantly focused on the influence of ovarian hormone fluctuations on dopamine and the mesolimbic and nigrostriatal, but not on the mesocortical dopamine pathway. The relationship between menstrual phase-related fluctuations in estrogen and/or progesterone on dopamine has produced mixed results (Wong et al., 1988; Nordström et al., 1998; Munro et al., 2006). The current study serves as a snapshot of the relationship between dopamine receptor levels and sex steroid hormone levels rather than ovarian hormone fluctuations, in the mesocortical dopamine pathway. More studies are needed to determine whether there is a direct relationship between tobacco smoking, estradiol levels, and dopamine receptor levels, how this affects dopamine receptor functioning, and whether it is reversible with smoking cessation.

Among women, we also observed a trend such that lower levels of free testosterone were associated with lower dlPFC D₂R availability pre-amphetamine compared to post-amphetamine, consistent with our hypothesis. Literature on the relationship between dopamine and testosterone is limited as most observations have been reported in ovarian hormones. One study examining the effects of sex steroid neonatal exposure to sex steroid hormones showed that a single injection of testosterone in 1-day-old female rat pups showed an intermediate non-significant effect on morphine-induced dopamine release, whereas estradiol significantly enhanced dopamine release (Bonansco et al., 2018). Another study using the Study of Women Across the Nation (SWAN) dataset found that among perimenopausal women who engage in heavy alcohol use, those with higher levels of testosterone were less likely to reduce their drinking (Peltier et al., 2020). In our study, associations between sex steroid hormone levels and dopamine receptor levels were only observed in women suggesting that hormones may differently modulate dopamine in women vs. men. More studies are needed to untangle the complicated relationship between D_2/3Rs and sex steroid hormones, especially testosterone, which is critically understudied in women.

The strengths of this study include the relatively large PET dataset and plasma measures of sex steroids. This study also has some limitations that can be addressed in future studies. First, while the sample size allowed for systematic examinations between subgroups, missing plasma data in people who smoke reduced the sample size, which may have impacted our ability to detect the effect of smoking status on the relationship between dopamine and sex steroids. Second, data included in these analyses included variables (e.g., sex steroid hormone levels and dopamine levels) that were evaluated at only one-time point. Furthermore, because menstrual cycle phase was not controlled, we observed variability in sex steroid hormone levels which made it difficult to detect subgroup differences in sex steroid levels. However, literature has shown that the examination of sex steroid hormone levels vs. menstrual cycle phase more precisely quantifies the impact of dynamic changes in hormone levels throughout the cycle (Weinberger et al., 2015). This is a critical future direction (Allen et al., 2008; Mazure et al., 2010). Future studies should also focus on understanding the underlying biological mechanisms by which smoking may alter sex steroids and sex steroids may alter dopamine. Taken together, our findings suggest that cigarette smoking alters sex steroid levels and in women, sex steroids influence dopamine receptor levels which may impede abstinence from cigarette smoking.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Yale Human Investigation Committee and the Yale-New Haven Hospital Radiation Safety Committee. The patients/participants provided their written informed consent to participate in this study.

YZ: formal analysis, data curation, project administration, methodology, and writing—original draft. RG: formal analysis, methodology, and writing—reviewing and editing. MRP, TLV, and WR: methodology and writing—reviewing and editing. SAM: supervision, methodology, and writing—reviewing and editing. KPC: supervision, conceptualization, methodology, funding acquisition, and writing—reviewing and editing. All authors contributed to the article and approved the submitted version.