Several lines of evidence suggest that combusted tobacco cigarettes have greater abuse potential than non-combusted, alternative nicotine delivery systems (ANDS) such as electronic cigarettes (ECs), smokeless tobacco, and nicotine replacement therapy (NRT). For example, combusted cigarettes are associated with higher rates of use among adults, greater reinforcement efficacy, and higher levels of dependence compared to ANDS (e.g., Etter and Eissenberg, 2015; Stein et al., 2017; Stiles et al., 2017; National Academies of Sciences, Engineering, and Medicine, 2018; Cornelius et al., 2020; Shiffman and Sembower, 2020). Understanding the product characteristics (e.g., constituents) that contribute to the differential abuse liability of cigarettes versus ANDS could inform FDA regulation of tobacco product design features and constituent levels. Such work could also provide insights into the basic behavioral and neurobiological mechanisms contributing to tobacco addiction and lead to the development of more effective treatments.

Nicotine is the primary addictive constituent in tobacco products, and the variance in nicotine content/yield between products could contribute to differences in abuse liability between cigarettes and ANDS. However, certain ANDS (e.g., newer-generation ECs) can produce levels of nicotine exposure similar to or even higher than combustible cigarettes (e.g., Lopez et al., 2016; Ramoa et al., 2016; Spindle et al., 2017; Hajek et al., 2020). Furthermore, several findings indicate that factors other than nicotine contribute to tobacco product abuse liability. For example, nicotine alone has limited abuse potential in humans and animals (Rose et al., 2000; Manzardo et al., 2002; Le Foll and Goldberg, 2009), and switching smokers to very low nicotine content cigarettes results in only a limited reduction in cigarettes smoked per day (Donny et al., 2007, 2015; Benowitz et al., 2012).

While there are numerous factors other than nicotine content/yield that could contribute to the greater abuse liability of cigarettes versus ANDs (e.g., sensory effects, marketing, etc.), cigarette smoke (CS) contains considerably higher levels of several behaviorally active non-nicotine constituents including monoamine oxidase (MAO) inhibitors (e.g., the β-carbolines harmane and norharmane, also called harman and norharman), minor tobacco alkaloids (e.g., nornicotine, anabasine), and volatile organic compounds (VOCs) (e.g., acetaldehyde, toluene) (e.g., Margham et al., 2016; National Academies of Sciences, Engineering, and Medicine, 2018; Belushkin et al., 2020). These compounds can mimic or enhance nicotine’s addiction-related effects in preclinical models, or may have abuse liability themselves (for review, see Hoffman and Evans, 2013; LeSage et al., 2018). The greater abuse liability of cigarettes versus ANDs may therefore reflect the higher levels of MAO inhibitors and other behaviorally active non-nicotine constituents in CS.

A useful preclinical approach for understanding determinants of tobacco abuse involves administration of extracts derived directly from tobacco products that contain nicotine and a wide range of non-nicotine constituents (e.g., Brennan et al., 2014, 2015; Costello et al., 2014). In contrast to traditional animal models of tobacco addiction that involve administration of nicotine alone or other isolated tobacco constituents, the use of extracts allows for evaluation of the aggregate effects of exposure to a range of tobacco constituents as occurs during actual product use. Although interactions can also be evaluated using inhalational models (e.g., Brennan et al., 2014; Chellian et al., 2021), parenteral (e.g., i.v., s.c.) administration of extracts allows the direct study of addiction-related central nervous system (CNS) effects of tobacco constituents largely independent of their peripheral (e.g., sensory) effects, avoidance of stressors associated with inhalational exposure, and more precise dosing (e.g., Brennan et al., 2014). Several studies have found greater addiction-related behavioral or neurobiological effects of CS extracts compared to nicotine alone (e.g., Brennan et al., 2014; Costello et al., 2014; Marusich et al., 2019), which may reflect the effects of behaviorally active non-nicotine constituents in the extracts.

While previous studies evaluating the addiction-related effects of CS extracts have been extremely valuable, they also have several limitations. First, nearly all of these studies have used extracts of only the particulate phase of CS, which excludes or limits levels of addiction-relevant constituents (e.g., VOCs such as acetaldehyde and toluene) that occur in the gas phase of CS. Second, several of these studies have used saline as a solvent for extract preparation (e.g., Costello et al., 2014; Gellner et al., 2016; Marusich et al., 2019), which limits or prevents extraction of constituents that have poor water solubility (e.g., β-carbolines). Third, chemical analyses of non-nicotine constituents in CS extracts have typically been limited to measuring 2–3 compounds (Brennan et al., 2013a,2015; Lee et al., 2016) or not conducted at all (Brennan et al., 2013b; Costello et al., 2014; Gellner et al., 2016; Cross et al., 2020; Levin et al., 2021), preventing identification of the non-nicotine constituents contributing to any unique effects of the extracts. Finally, comparisons of CS extracts and extracts of other classes of tobacco products (e.g., ECs) have rarely been conducted in the same study (Marusich et al., 2019), preventing characterization of their relative abuse liability. A goal of the present study was to address these limitations.

Abuse liability in this study was measured using intracranial self-stimulation (ICSS), which provides a sensitive index of the function of brain reinforcement systems. Low to moderate doses of nicotine reduce the lowest (threshold) stimulation intensity that maintains operant responding for ICSS, reflecting nicotine’s ability to enhance the reinforcing effects of other stimuli (Kornetsky et al., 1979; Huston-Lyons and Kornetsky, 1992; Rupprecht et al., 2015; Kenny et al., 2018). At high doses, nicotine attenuates the reinforcing effects of the brain stimulation and increases ICSS thresholds, providing a putative marker of nicotine’s acute aversive/anhedonic effects (Kenny et al., 2003; Spiller et al., 2009; Fowler et al., 2011). Nicotine’s reinforcement-enhancing and aversive effects may both influence tobacco abuse liability (Sellings et al., 2008; Fowler and Kenny, 2014; Rupprecht et al., 2015). For example, because nicotine’s aversive effects can limit its intake (e.g., Verendeev and Riley, 2013; Fowler and Kenny, 2014; Wills et al., 2022), reductions in nicotine aversion could increase tobacco use. The acute effects of a drug on ICSS are generally predictive of its abuse liability in other preclinical models (e.g., i.v. self-administration) and in humans (Wise, 1996; Negus and Miller, 2014). Supporting the utility and sensitivity of ICSS for tobacco constituent evaluation, we have used this methodology to evaluate the relative abuse liability of smokeless tobacco extracts (Harris et al., 2012, 2015b), EC liquids (LeSage et al., 2016; Harris et al., 2017, 2018b), and isolated non-nicotine constituents [i.e., β-carbolines, minor alkaloids, and the EC solvent propylene glycol (PG) (Harris et al., 2015a, 2018a, 2020)]. However, effects of CS or EC aerosol extracts on ICSS have not been evaluated.

The primary purpose of this study was to compare the acute effects of nicotine alone and CS extract or EC aerosol extract on ICSS. Extracts were prepared from either Marlboro Gold cigarettes or Vuse Solo Original (tobacco-flavor) ECs, two of the most popular brands of their respective classes. Vuse Solo Original is also of interest because it recently became the first EC approved for marketing by the FDA under a Premarket Tobacco Product Application (PMTA). Extracts were prepared using an approach that captures water-soluble and water-insoluble constituents from both the particulate and gas phase of CS and EC aerosol. To evaluate potential constituents that could account for any behavioral effects of extracts and to ensure consistency between batches of extracts, we evaluated levels of a range of behaviorally relevant non-nicotine constituents including MAO inhibitors (harmane and norharmane), minor alkaloids (e.g., nornicotine, anabasine), carbonyls (e.g., acetaldehyde, acetone), and glycols (e.g., PG). We also measured several constituents that are known toxicants but whose behavioral effects are unknown (e.g., crotonaldehyde, ethylene glycol). Effects of extracts and nicotine alone on brain MAO activity in vitro and in vivo were also studied to evaluate the potential role of MAO inhibition in our findings. MAO inhibition was studied because other CS extracts and β-carbolines present in cigarette smoke (e.g., harmane, norharmane) inhibit MAO activity in vitro (e.g., Herraiz and Chaparro, 2005; Lewis et al., 2012; Costello et al., 2014). This mechanism is also of interest because brain levels of the MAO-A and MAO-B isozymes are inhibited in smokers, an effect that may contribute to tobacco addiction (Fowler et al., 1996; Berlin and Anthenelli, 2001).

The current studies compared the relative abuse liability of Marlboro Gold CS extract or Vuse Solo Original EC aerosol extract to that of nicotine alone (NRT analog) using ICSS in rats. In summary, chemical analyses indicated that CS extract contained higher levels of several behaviorally active non-nicotine constituents [e.g., β-carbolines (MAO inhibitors), minor alkaloids] than EC extract. Nicotine alone reduced ICSS thresholds at a moderate nicotine dose, indicating a reinforcement-enhancing effect, and elevated ICSS thresholds at a high nicotine dose, suggesting an aversive/anhedonic effect. The significant main effect of formulation and formulation × dose interaction in Experiment 2a indicate that CS extract elevated ICSS thresholds compared to nicotine alone at high nicotine doses (1.0 and 1.25 mg/kg, see Figure 2A). Furthermore, a 1.0 mg/kg dose of CS extract significantly elevated ICSS thresholds while the same dose of nicotine alone did not, suggesting greater potency for CS extract in elevating ICSS thresholds. Effects of EC extract on ICSS did not differ from those of nicotine alone. CS extract, but not EC extract or nicotine alone, significantly inhibited total MAO activity measured in vitro. CS extract also exhibited greater potency at inhibiting MAO-B activity in vitro compared to MAO-A activity. None of the formulations inhibited MAO activity measured ex vivo.

The greater ICSS threshold-elevating effects of CS extract compared to nicotine alone suggests that it produces greater acute aversive/anhedonic effects and, hence, has lower abuse liability. To the extent that our findings are predictive of tobacco use in humans, they indicate that the greater abuse liability of cigarettes compared to ANDS reflects factors others than the centrally-mediated effects of MAO inhibitors and other non-nicotine constituents in CS (e.g., nicotine content/yield, sensory factors, marketing). However, confirmation of our findings using other approaches is needed. For example, while the current approach involving evaluation of acute drug exposure on ICSS is a reliable predictor of abuse potential (Wise, 1996; Negus and Miller, 2014), evaluating effects of chronic dosing of extracts and nicotine alone on ICSS would allow study of changes in tolerance to nicotine’s aversive effects and would better model the chronic nature of tobacco product use in humans. Comparison of the primary reinforcing effects of extracts and nicotine alone in an i.v. self-administration model is also needed to clarify the relevance of our findings to tobacco product consumption. Finally, given the considerable differences in constituent profiles between brands of cigarettes and ECs (e.g., Jain et al., 2019; Eshraghian and Al-Delaimy, 2021), evaluating the effects of extracts of other products is needed to better understand the generality of the current findings.

Further evaluation of the increased acute aversive/anhedonic effects of CS extract, including identification of the responsible constituent(s), could help inform FDA policy and product standards. For example, we have suggested that the ICSS threshold-elevating effects of high nicotine doses may be related to toxicity (LeSage et al., 2016; Harris et al., 2018b). As such, while the constituents accounting for the effects of CS extract would not be included on the FDA’s list of harmful or potentially harmful constituents (HPHCs) as addiction-related chemicals, they may still need to be considered for inclusion on the list as toxic compounds. Evaluating the role of neurobiological mechanisms implicated in nicotine aversion [e.g., activity of α5 nicotinic acetylcholine receptors in the habenula-interpeduncular pathway (Fowler et al., 2011; Fowler and Kenny, 2014; Wills et al., 2022)] in the effects of CS extract could also provide important insights into the basic mechanisms mediating tobacco addiction.

The current findings provide the most comprehensive chemical characterization of tobacco product extracts in a preclinical addiction study. The higher levels of most behaviorally active non-nicotine constituents in CS extract than in EC extract is consistent with prior analytical studies of CS and EC aerosol (e.g., Belushkin et al., 2020). The only exception was that PG levels were higher in EC extract, although this was expected given that PG is a primary ingredient in ECs, but not cigarettes (e.g., Margham et al., 2016). The rank-order prevalence of minor alkaloids, β-carbolines, and carbonyls in the current CS extract was generally similar to that reported for Marlboro Gold cigarette smoke in an analytical study conducted in the same laboratory (Jain et al., 2019). However, absolute levels of non-nicotine constituents (expressed as percent of nicotine) tended to be lower in our CS extract. This may reflect our use of a different solvent than those used for constituent extractions in the Jain et al. (2019) study, which are not safe for use in animals. These conclusions should be considered preliminary, however, until confirmed in a formal analytical study that contemporaneously compares the constituent profiles of extracts and cigarette smoke/EC aerosol itself using the same lot of tobacco products. Nonetheless, these findings support further use of extracts generated by the present methods to characterize determinants of the abuse liability of these or other tobacco products.

Several non-nicotine constituents found in CS extract could account for its greater aversive/anhedonic effects compared to nicotine alone. For example, we found that several β-carbolines (MAO inhibitors) (e.g., harmane, norharmane) and minor alkaloids (e.g., nornicotine, anabasine) elevated ICSS thresholds when administered alone (Harris et al., 2015a,2020). In addition, while their effects on ICSS thresholds have not been studied, the carbonyls acetaldehyde and formaldehyde can produce aversive effects in other assays (Weisinger et al., 1974; Brown et al., 1978). Although doses of these constituents that produced aversive effects in those studies were higher than those delivered in CS extract in this study, these and other constituents may have additive or synergistic effects that increase aversion when they are combined in CS extract. Future studies involving fractionation of extracts (e.g., Noya et al., 2013) or evaluation of cocktails of nicotine and one or more of these constituents are needed to isolate the constituent(s) accounting for our findings.

Effects of a drug on ICSS are often predictive of its abuse liability in an i.v. self-administration (SA) model (Negus and Miller, 2014). Our ICSS data therefore contrast with some studies reporting greater abuse liability for CS extract than nicotine alone on certain measures of i.v. SA (e.g., Costello et al., 2014; Brennan et al., 2015; Marusich et al., 2019). This discrepancy could reflect numerous differences between behavioral models including route of administration (s.c. versus i.v.), dosing regimen (acute versus chronic), and contingency of drug exposure (experimenter-administered versus self-administered). It should also be noted that the SA literature in this area is mixed, with some studies reporting lower levels of SA of CS extract than nicotine alone (Gellner et al., 2016; Levin et al., 2021). Factors that could contribute to these inconsistencies across SA studies include differences in tobacco products, extract preparation procedures (solvent, pH adjustment), training procedures (dose, prior food training), schedule of drug delivery, and form of response (nose-poke vs. lever press), among others. Our ICSS data also raise the possibility that findings from studies reporting lower levels of SA of CS extract than nicotine alone may have reflected greater aversive effects of CS extract. Direct comparison of i.v. SA of the current CS and EC extracts and nicotine alone is needed to assess the convergent validity of the present findings.

In contrast to our current findings with EC extract, we previously found that EC liquids for three other products had reduced ICSS threshold-elevating effects compared to nicotine alone (LeSage et al., 2016; Harris et al., 2018b), suggesting reduced aversive/anhedonic effects. PG itself also attenuated nicotine’s ICSS threshold-elevating effects, including at concentrations similar to those in the EC liquids (Harris et al., 2018a). A possible explanation for the current findings is that levels of PG in EC extract, while higher than in CS extract, were nonetheless considerably lower than in the EC liquids studied previously. For example, at a 1.0 mg/kg nicotine dose, EC extract contained ≈0.5% PG whereas the EC liquids contained ≈1.0–3.0% PG depending on the product. This difference in PG levels may reflect our use of an EC aerosol extract rather than an EC liquid and/or our use of a different product, as both of these factors could influence constituent levels (e.g., Eshraghian and Al-Delaimy, 2021). Regardless, the PG levels in EC extract may have been insufficient to attenuate the ICSS threshold-elevating effects of high nicotine doses. Other factors that could account for the discrepancy include differences in levels of constituents other than PG in Vuse EC extract versus the previously studied EC liquids, as well as differences in rat strains used across studies (Sprague Dawley vs. Holtzman).

Our finding that CS extract but not nicotine alone inhibited MAO activity in vitro is consistent with prior studies (e.g., Castagnoli et al., 2002; Lewis et al., 2012; Costello et al., 2014), and may be at least partially attributable to the effects of harmane and/or norharmane in CS extract (see Herraiz and Chaparro, 2005). These in vitro findings also complement reports of MAO-A and MAO-B inhibition in the brains of smokers (e.g., Fowler et al., 1996). The more potent inhibition of MAO-B than of MAO-A by CS extract contrasts with prior studies reporting more potent inhibition of MAO-A than MAO-B for other CS extracts (Costello et al., 2014; van der Toorn et al., 2019). One possible reason for this discrepancy is the use of different products across studies, as one study found that the relative inhibition of MAO-A versus MAO-B by CS extracts differed across brands of cigarettes (Lewis et al., 2012). Comparison of our findings to these prior studies is also complicated by differences in procedures for assaying MAO inhibition.

The lack of effects of Vuse Solo Original EC extract on MAO activity in vitro contrasts with a report that EC liquid of this same product inhibited MAO in vitro (Truman et al., 2019). However, the magnitude of that effect was among the weakest on the panel of EC liquids in that study. In addition, constituents accounting for the MAO inhibitory effects of Vuse EC liquid (potentially vanillin and ethyl vanillin, see Truman et al., 2019) may not have effectively transferred from EC liquid to EC aerosol during EC extract preparation. For example, ≈20% of vanillin can be lost during aerosolization of an EC liquid (Erythropel et al., 2019).

Our finding that CS extract inhibited MAO in vitro but not ex vivo is consistent with the findings of Costello et al. (2014). One explanation for this finding is that concentrations of CS extract that inhibited MAO in vitro in the current study (≥≈15 μM nicotine following dilution in the assay) were likely higher than peak brain nicotine levels produced following extract injection in the ICSS study [≤≈7.7 μM, based on Tuncok et al. (2001)]. However, such direct extrapolation of in vitro to in vivo data is often inappropriate (e.g., Yoon et al., 2012). It is also possible that CS extract produced only reversible MAO inhibition in vivo. In contrast to non-reversible MAO inhibition such as that produced by TCP, reversible MAO inhibition would likely be lost during brain homogenization for ex vivo analysis (see Costello et al., 2014; Smith et al., 2015). Administration of CS extract using a chronic dosing regimen that better simulates long-term smoking may also be required to inhibit MAO measured ex vivo. Use of other approaches (e.g., measurement of brain monoamine levels following CS extract exposure using in vivo microdialysis) is needed to clarify the potential involvement of MAO inhibition in our findings.

A potential concern is that our use of a saline/ETOH solvent for extract preparation may have influenced our findings due to the well-established abuse liability of ETOH and its potential effects on MAO activity (Truman et al., 2019), as well as the ability of ETOH to interact with nicotine on certain measures of abuse liability (e.g., Schaefer and Michael, 1992; Tizabi et al., 2007). We therefore studied whether the highest concentration of ETOH present in the extracts (32%) would itself affect ICSS or MAO activity when administered alone. In addition, to control for any interactions between nicotine and ETOH, the same ETOH concentrations present in the extract doses were added to the respective nicotine alone doses (e.g., 32% ETOH at 1.25 mg/kg nicotine, 24% at 1.0 mg/kg nicotine, etc.). The 32% concentration of ETOH had no effects on baseline ICSS when administered alone, and the current ICSS dose-effect function for nicotine alone delivered in a saline/ETOH vehicle was similar to that observed previously for nicotine alone delivered in only saline (e.g., LeSage et al., 2016; Harris et al., 2018b). ETOH also did not affect MAO activity when administered alone or in combination with nicotine alone or EC extract in Experiment 3. Given the small volumes used in the ICSS and MAO studies (≈0.3–0.4 ml and 20 μl, respectively) and the resulting ETOH doses or concentrations produced (≤≈250 mg/kg for ICSS and 1% following dilution in the MAO assay, respectively), the lack of effects of ETOH on ICSS or MAO activity when administered alone or in combination with nicotine is expected (Kornetsky et al., 1988; Schaefer and Michael, 1992; Truman et al., 2019) and suggest that our use of ETOH as a solvent was not a confound. Nonetheless, inclusion of a control condition of nicotine delivered in only saline in future studies would help clarify this issue further.

The MAO assay used in this study may represent a further limitation. We used this assay because it is commercially available and is widely used in the literature (e.g., Malin et al., 2013; Smith et al., 2015, 2016; Matthews et al., 2018), including for evaluating effects of tobacco constituents (e.g., harmane and norharmane) (Smith et al., 2015). Nonetheless, the selectivity and specificity of this assay can be compromised by the presence of interfering substances in the samples (e.g., phenolic compounds, see Herraiz et al., 2018). In addition, given that this assay involves detection of H₂O₂, any H₂O₂ present in the extracts could have artificially increased apparent MAO activity. This seems unlikely, however, given that CS extract reduced rather than increased MAO activity in vitro, while EC extract had no effect (see Figure 4B). Nonetheless, our findings should be confirmed using chromatographic assays that avoid limitations of the current MAO assay (see Herraiz et al., 2018).

Despite its limitations, our study suggests that the non-nicotine constituents in the present CS extract do not contribute to the greater abuse liability of cigarettes compared to ANDS apparent in humans, and may actually limit the abuse liability of cigarettes by enhancing their aversive effects at high nicotine doses. Studies using self-administration models are needed to examine how such aversive effects might influence the relative consumption of CS and ANDS extracts. Future studies of similarly-prepared extracts of these and other products could be useful to clarify further the role of MAO inhibition and other mechanisms in the addiction-related effects of CS extract and could help inform FDA regulation of tobacco products.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the Hennepin Healthcare Research Institute.

ML and AH supervised the conduct of the study and were responsible for the conception and design of the study. PM assisted with developing specific protocols, daily conduct of the experiment, and compiling data. IS and AA prepared the extracts and conducted the constituent analyses. AH wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or Food and Drug Administration.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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