Preclinical Evidence of the Effects of Sweet Flavors: Sweetness Increases Nicotine Intake and Seeking

Abstract

Introduction

The influence of early exposure to sweet and fruit flavors on subsequent nicotine use behaviors during adolescence remains poorly understood. To address this gap, we utilized a rodent model to simulate childhood flavor exposure and examine its effects on later nicotine use behaviors.

Aims and Methods

We employed a two-bottle choice paradigm in weanling rats to mimic human childhood flavor exposure. Subsequently, we investigated the impact of early exposure to sweet (saccharin) and cherry fruit (benzaldehyde) flavors on adolescent nicotine use behaviors. This included assessing subsequent nicotine acquisition, maintenance, and seeking behaviors using intraoral flavor and intravenous nicotine self-administration.

Results

Our findings reveal that rats exposed to saccharin during early development exhibited altered patterns of nicotine intake and seeking behaviors during adolescence. Specifically, rats demonstrated increased nicotine intake and seeking of saccharin flavored solutions when paired with nicotine, indicating a potential predisposition towards nicotine use following childhood flavor exposure. Intraoral benzaldehyde did not affect nicotine reinforcement in early sessions of self-administration, but significantly decreased nicotine reinforcement during later sessions. Both saccharin exposure alone or in combination with benzaldehyde promoted nicotine reinforcement. No significant sex differences in nicotine-related behaviors were observed.

Conclusions

This study highlights the significance of early flavor exposure in shaping adolescent nicotine use behaviors. The findings suggest that childhood exposure to sweet flavors may contribute to heightened susceptibility to nicotine use and addiction later in life. Understanding these early influences is crucial for developing targeted interventions to prevent the onset of nicotine addiction during adolescence.

Implications

Our study highlights the importance of how sweetness can contribute to and possibly even alter the effects of the flavor itself. The sweetness of a tobacco product does not solely stem from sweeteners, but it is also influenced by other components such as the propylene glycol (PG) and vegetable glycerin (VG) ratio (eg, higher VG) and the inclusion of flavors associated with sweetness (eg, vanillin and ethyl maltol). Therefore, sweet-associated constituents in tobacco products should be investigated further to inform regulatory policy. We believe that our findings can inform regulatory decisions on controlling sweet-associated constituents in tobacco products.

Introduction

In 2022, 3.08 million middle and high school students were current tobacco product users in the United States,¹ with 2.55 million current e-cigarette users, and 84.9% of them using flavored e-cigarettes.^1,2 The fruit flavors (69.1%) were the most popular, followed by candy, desserts, or other sweets (38.3%).² Despite the 2020 flavor ban (with exceptions for menthol and tobacco flavors; Food and Drug Administration [FDA], 2020), flavors are still available in disposable and personalized e-cigarettes, as well as in other tobacco products like dissolvable tobacco products and snus. Furthermore, flavors have been shown to increase the appeal of tobacco products in humans^3,4 including youth.⁵ In addition to characterizing flavors, sweeteners, such as sucralose, are commonly added to e-liquids and oral tobacco products.⁶ While flavors increase the appeal and use of tobacco products, the processes by which chemicals, such as primary chemical flavors or sweeteners, increase appeal are unclear. Therefore, dissecting the interaction between flavors, sweeteners, and nicotine reflects a critical gap in our understanding of tobacco product use behaviors, especially in adolescents. It is also important that we determine whether preconditioning to flavors and sweeteners during childhood influences the emergence of nicotine use behaviors and addiction.

Since candies and sweetened beverages are also part of the childhood diets,⁷ and given the surge in teen usage of fruit and sweet flavored tobacco products,² it is vital that we learn more about fruit and sweet flavor conditioning during childhood and its effects on development of adolescent flavored tobacco use. In this study, we first modeled human childhood fruit and sweet flavor exposure by using a two-bottle choice paradigm in weanling rats. We then investigated the impact of early fruit and sweet exposure on later nicotine use behaviors, including nicotine acquisition, maintenance, and seeking via intraoral flavor and intravenous nicotine self-administration during adolescence period. We used benzaldehyde as a chemical representing cherry fruit flavor and saccharin as an artificial sweetener representing sweet flavor in this study. Benzaldehyde is an aromatic compound found naturally in bitter almond oil and it primarily provides cherry, red berry, and almond flavors.⁸ Tobacco companies use benzaldehyde in various fruit and sweet-flavored products, including electronic nicotine delivery systems, smokeless, and dissolvable tobacco products.⁹ Moreover, saccharin is also listed among the ingredients in oral nicotine pouches, a rapidly growing tobacco product category.

Preventing the development of nicotine addiction in adolescence is crucial, as it can cause persistent tobacco product use, long-lasting negative consequences on physical health, mental well-being, and overall quality of life and economic burden. Our study provides preclinical evidence showing that preconditioning to sweet saccharin flavor increases use behavior for sweetened nicotine during adolescence, whereas preconditioning to the primary cherry fruit flavor, benzaldehyde had minimal effects on nicotine use behavior. In light of the emerging preclinical and clinical data discussed in this paper, we propose that controlling sweet associated constituents in the tobacco products can impact product use in adolescents.

Materials and Methods

Animals

Female and male adolescents (Post-natal day, PND, 21; weighing 60–80 g at the beginning of experiments) Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were housed individually in a temperature- and humidity-controlled husbandry room. Upon arrival, rats were individually housed and provided ad libitum food and water on a standard 12-hour light–dark cycle (7 am–7 pm). Rats received 24-hour habituation and the next day early exposure to flavorings started. The Yale University Institutional Animal Care and Use Committee (IACUC) approved all experiments in this study. Experiments were also conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Drugs and Chemicals

Nicotine tartrate salt (#211499) was purchased from MP Biomedicals, LLC (Solon, OH). Saccharin sodium salt hydrate (#S1002) and benzaldehyde (#B1334) were purchased from Sigma-Aldrich (St. Louis, MO). Oral delivery solutions were prepared in tap water with pH adjusted to 7.0, and solutions were used once on the test day. For intravenous (iv) delivery, nicotine was prepared in sterile saline. The pH was adjusted to 7.4 and filtered with a 0.22 µm filter. NaOH and HCl were used to stabilize the pH of the solutions. Nicotine (free base) was delivered at a rate of 30 µg/kg per infusion over a 6-second duration at a flow rate of 17.7 µL per second, resulting in a total volume of 106 µL. Flavored test solutions were delivered intraorally (i.o.) at a rate of 50 µL per second for a duration of 2 seconds, resulting in a total volume of 100 µL. The concentrations of test solutions used in this study were chosen based on our previous reports.^10,11

The benzaldehyde concentration used in our study (100 µg/mL) is within the range found in commercial products. Studies showed that flavored e-liquids, whether labeled as cherry or other flavors, can include benzaldehyde.⁹ Behar and colleagues tested 39 commercial e-liquids and found benzaldehyde at concentrations as high as 2.5 mg/mL. Kosmider et al. reported that benzaldehyde yields in non-cherry-flavored e-liquids ranged from 0.025 to 10.27 µg per 30 puffs.¹² In cherry-flavored e-liquids, concentrations were higher, ranging from 5.129 to 141.2 µg per 30 puffs. Kosmider et al. tested 145 e-cigarettes and detected benzaldehyde in 108 of them, with significantly higher yields in cherry-flavored products compared to non-cherry-flavored ones.

Two-Bottle Free-Choice Test

Rats arrived in the lab at PND 21. After rats were individually housed and habituated to their housing cages for 24 hours with plain water, the Two-Bottle Free-Choice (2BC) test was conducted in their housing cages (acrylic material, 30 cm × 28 cm × 17 cm dimensions). The rats were given two 300-mL plastic black colored bottles, each containing a metal spout. In a voluntary fashion, rats were given unrestricted access to drink from either bottle, with one bottle containing test solution, and the other containing plain water. The 2BC test was conducted over dark cycle period and took about 13 hours (started at 6 pm on the first rat and ended at 7:30 am on the last rat). After 13 hours, rats were given two bottles of plain tap water bottles. The following dark cycle, rats were given a test solution in one bottle and given water in the other bottle again. The 2BC exposure was continued from PND 22 to PND 28. To prevent the development of any side preferences, the side placement of the test solution was changed at each test session. After 13 hours of 2BC, fluid intake (mL) was measured. Preference for the test solution was determined by dividing the volume of test solution consumed by the total volume consumed, while the body weight of all the rats was measured at 72-hour intervals. The rats gained weight throughout the 2BC test; however, no significant differences were found between the groups. To accommodate for any spillage that might occur naturally, a cage devoid of animals was introduced every 13-hour session, equipped with two bottles—one containing the test solution and the other containing water.

Intraoral and Intravenous Catheter Implantation

On post-natal day (PND) 28 or 29, rats were anesthetized (intraperitoneal 80 mg/kg ketamine HCl and 10 mg/kg xylazine) and implanted with a handmade, oral catheter constructed with polyethylene-100 tubing and intravenous catheter, constructed with Silastic Laboratory tubing (#508002). IO and IV catheters were sterilized with ethylene oxide gas and steam, respectively. While the IO catheter was implanted laterally to the first maxillary molar,¹³ the IV catheter was implanted into the external jugular vein.¹⁰ Post-op carprofen analgesia (5 mg/kg, subcutaneously) was provided for three days, starting on surgery day. We administered gentamicin daily for seven days after IV catheter implantation to prevent bacterial infections. In cases of catheter blockage, we utilized heparinized IV saline to facilitate catheter clearing. Self-administration studies were started on PND 35 after the surgical recovery. Additionally, a group of rats were implanted with only IO catheters and another group of rats were implanted with only IV catheters.

Combined Model of Two-Bottle Free-Choice Test, Intraoral and Intravenous Self-administration

The two-bottle free-choice (2BC) test was first performed to introduce an early (PND 22-28) exposure to sweetened benzaldehyde and to mimic childhood beverage exposure in humans. During this time adolescent rats were given unlimited access to one bottle of water and one bottle test solution (water, benzaldehyde [100 µg/mL], saccharin [3.2 mg/mL], or benzaldehyde + saccharin) for 6 days. Daily fluid intake (mL) was measured, and the bottles were switched in position every 24 hours. At the end of the week, weekly average consumption was calculated, and rats were distributed to groups for intraoral and intravenous self-administration (IOIVSA) based on their test solution group. On PND 28 and 29, rats underwent IO and IV catheter implantation surgeries as described above. After surgical recovery, rats performed an operant task for intraoral self-administration of flavors and intravenously self-administer of nicotine.

For self-administration studies, rats were placed in individual operant chambers, containing an active and an inactive lever. At the beginning of every experiment, the IO catheter was flushed with water and the IV catheter was flushed with sterile saline (about 50–100 µL volume) while rats were in their home cages, to ensure catheter patency. During each session, active lever pressing led to IO delivery of test solution for 2 seconds and IV delivery of nicotine for 6 seconds with simultaneous cue light illumination (14 seconds), and termination of the house light for 14 seconds. At the end of 14 seconds timeout period (no drug available with lever pressing), the cue light and timeout period co-terminated, and the house light was turned back on. IOIVSA sessions were conducted on a daily basis, each lasting for 60 minutes, during which the sum of active and inactive lever presses was recorded. The rats followed a schedule consisting of 5 days of fixed ratio (FR)1, 3 days of FR2, 7 days of FR5, and 1 day of a progressive ratio task (PR). FR and PR schedules were used based on our established nicotine IVSA model in the laboratory.¹⁰ For PR, a geometric design was used [nj = 5e^j/5 − 5; where nj was the position in the following series of values: nj = 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, and 95]. We employed a modified criteria for self-administration behavior, adapted from previous reports,^14,15 which included the following conditions: (1) rats needed to self-administer infusions at least twice or more over a period of 3 days, (2) achieved a response rate of at least 65% on the active lever, and (3) received a minimum of >5 infusions per session on a fixed-ratio 5 (FR5) schedule. An increased FR schedule was utilized in our self-administration paradigms to accommodate the less potent reinforcing effects of nicotine compared to other substances. This adjusted criterion accounts for the unique reinforcing properties of nicotine, as noted in previous studies.^10,15 As a result, rats in our study successfully achieved the acquisition criteria for self-administration within a 15-day period. The breakpoint on PR was achieved when 20 minutes of inactivity on the active lever elapsed.

The criteria for exclusion in each experiment was established by evaluating catheter patency and the general health of the animals. Rats were removed from the study if their catheters became completely obstructed or if they exhibited any signs of illness. Thus, while we initially began with seven to nine rats in each group of the experiments, the sizes of the groups testing the solutions within each cohort fluctuated over time, contingent on whether the rats were not disqualified based on the exclusion criteria.

Study Design

Experiment 1

To study the effects of flavors on adolescent nicotine intake, we first established nicotine IVSA in adolescent rats at PND 35 (Figure 1; n = 7–9/sex/group). Rats received IV infusions of saline or nicotine.

Figure 1.

Intravenous nicotine self-administration in adolescent rats. (A) Active and inactive lever presses per hour over 15 days of intravenous self-administration of nicotine (30 µg/kg/infusion), in (B) female and male adolescent rats. (C) Average number of infusions per hour for day 15 of self-administration. (D) Breakpoint of self-administration session on day 16. Data were expressed as mean ± standard error of the mean (SEM) of n = 7–9/sex/group. Filled points indicate significantly different (p < .05) from the inactive lever press at a given time point. PND = post-natal day; IV = intravenous; FR = fixed ratio; PR = progressive ratio.

Experiment 2

To study the effects of flavors on adolescent nicotine intake, we then established flavor IOSA in adolescent rats starting at PND 35 (Figure 2; n = 8/group, n = 4/per sex). Rats received intraoral infusions of water, benzaldehyde, saccharin, or benzaldehyde plus saccharin.

Figure 2.

Intraoral flavor self-administration. (A) Active and (B) inactive lever presses per hour over 15 days of intraoral self-administration of water, benzaldehyde (100 µg/mL), saccharin (3.2 mg/ml), and combination of benzaldehyde and saccharin in female and male adolescent rats. (C) Average number of infusions per hour for day 15 of self-administration. (D) Breakpoint of self-administration session on day 16. Data were expressed as mean ± standard error of the mean (SEM) of n = 8/group (n = 4/per sex). Filled points indicate significant differences (p < .05) from the intraoral water group at a given time point. PND = post-natal day; IO = intraoral; FR = fixed ratio; PR = progressive ratio.

Experiment 3

The effects of early exposure to sweetened flavor on later nicotine intake were determined via a combined model of IO flavor and IV nicotine self-administration in adolescent rats (Figure 3; n = 13–14/group, n = 6–7/sex/group). Weanling rats were given a choice to consume water, benzaldehyde, saccharin, or benzaldehyde plus saccharin in 2BC between PND 22 and 28. Rats received IO infusions of water, benzaldehyde, saccharin, or benzaldehyde plus saccharin in the presence of IV nicotine by starting at PND 35.

Figure 3.

Impact of early exposure to sweet and fruit flavors on later nicotine intake and seeking behaviors. (A) Average preference to benzaldehyde (100 µg/mL), saccharin (3.2 mg/mL), and combination of benzaldehyde and saccharin in adolescent rats. (B) Active lever presses per hour over 15 days of intraoral self-administration of water, benzaldehyde, saccharin, and combination of benzaldehyde and saccharin in the presence of intravenous nicotine (30 µg/kg/infusion) self-administration. (C) Average number of infusions per hour for day 15 of self-administration. (D) Breakpoint of self-administration session on day 16. Data were expressed as mean ± standard error of the mean (SEM) of n = 13–14/group (n = 6–7/sex/group). *p < .05 vs. its water control. #p < .05 vs. its benzaldehyde control. Filled points indicate significant differences (p < .05) from the intraoral water + intravenous nicotine group at a given time point. PND = post-natal day; IO = intraoral; IV = intravenous; 2BC = two-bottle free-choice, FR = fixed ratio, PR = progressive ratio

These three experiments utilized different cohorts of rats.

Statistical Analyses

We used GraphPad Prism software (version 9; GraphPad Software, Inc., San Diego, CA) for the creation of graphs and the execution of statistical analyses. Prior to statistical analysis, we checked the data for adherence to the normal distribution. All other data passed these tests. However, in the nicotine self-administration studies, we applied log transformation because the data were skewed to the right. A two-way repeated measures analysis of variance (RM ANOVA with Greenhouse-Giesser correction) was used to evaluate the potential impact of the test solution on the taking behavior of each test over time. We tested the test solution by time interaction and if significant, we followed up with multiple comparisons among the solutions by time point, to infer when differences in taking behavior emerged. Multiple comparison tests were corrected using the false discovery rate (FDR) with the method of Benjamini and Hochberg.

Regarding the statistical analysis of infusions, breakpoints, and preferences, a t-test, one-way, or two-way ordinary ANOVA was performed, which was subsequently corrected for multiple comparisons using a Tukey test based on statistical hypothesis testing. Significance was determined at p < .05.

Results

Characterization of Nicotine Self-administration in Adolescent Rats

Rats acquired nicotine IVSA, as confirmed by significant main effects of nicotine (F_(1,30) = 53.230, p < .001), time (F_{(3.314, 99.41)} = 16.340, p < .001, Geisser-Greenhouse’s epsilon: 0.2367), and nicotine × time interaction (F_(14,420) = 11.023, p < .001; Figure 1A). Starting on day 3, rats showed significant active lever pressing until the end of the study, except on day 5 (Figure 1A). However, there were no sex differences on number of active lever responses (F_(1,14) = 1.134, p = .304; Figure 1B), number of nicotine infusions received (t = 1.059, df = 14, p = .307; Figure 1C), or nicotine seeking behavior as measured by breakpoint (t = 0.799, df = 14, p = .437; Figure 1D), between females and males.

Characterization of Flavor Self-administration in Adolescent Rats

Rats self-administered IO flavors which was confirmed by significant main effects of flavor (F_(3,27) = 37.270, p = .023), time (F_(14,378) = 3.803, p < .001), and flavor × time interaction (F_(42,378) = 1.449, p = .040; Figure 2A). Saccharin and benzaldehyde plus saccharin solutions elicited significant flavor intake by rats. Rats were able to distinguish active and inactive levers which was confirmed by no significant effects on inactive lever press of flavors (F_(3,27) = 0.192, p = .900; Figure 2B).

At a p-value of .05, rats showed no significant difference between flavor infusions (F_(3,27) = 2.943, p = .050; Figure 2C). However, there were significant differences in flavor seeking (F_(3,27) = 4.849, p = .008; Figure 2D). Rats were more motivated to receive benzaldehyde plus saccharin solution more than water and benzaldehyde.

Evaluation of Intraoral Flavor and Intravenous Nicotine Self-administration

Preference for Flavors During the Early Adolescence Period

In a voluntarily 2BC paradigm, rats orally self-administered flavors in the absence of nicotine during early adolescence (F_(3,50) = 44.96, p < .001; Figure 3A). Rats preferred saccharin and benzaldehyde plus saccharin solutions more than water (p < .05). In addition, the preference for combined solution was higher than benzaldehyde alone solution (p < .05).

Effects of Early Exposure to Flavors on Later Nicotine Intake

Rats self-administered IO flavors and IV nicotine which was confirmed by significant main effects of treatment (F_(3,50) = 17.200, p < .001), time (F_{(6.843,342.1)} = 146.600, p < .001, Geisser-Greenhouse’s epsilon: 0.488), and treatment × time interaction (F_(42,700) = 1.668, p = .005; Figure 3B). Saccharin and benzaldehyde plus saccharin solutions resulted in significantly higher nicotine self-administration compared to water group (s < .05). Although saccharin promoted the highest nicotine intake, it did not differ from the intake observed with benzaldehyde plus saccharin. Benzaldehyde significantly lowered nicotine self-administration on days 14 and 15 (p < .05).

Rats received a significant number of infusions of flavors and nicotine which was confirmed by significant effects of infusions (F_(3,50) = 4.837, p = .004; Figure 3C). Rats showed significantly higher number of deliveries in saccharin group compared to water group (p < .05). There were also significant differences on seeking behavior (F_(3,50) = 10.94, p < .001; Figure 3D). Rats were more motivated to receive IOIVSA, as reflected by a higher breakpoint in saccharin group compared to water group (p < .05). Moreover, benzaldehyde plus saccharin and saccharin alone induced higher breakpoints than benzaldehyde (p < .05).

Sex Differences in the Effects of Early Exposure to Flavors on Later Nicotine Taking and Seeking

To examine the impact of sex on IOIVSA of flavors and nicotine, we also compared preferences to flavors in 2BC were between two sexes (Figure 4). Significant main effects of flavor (F_(3,46) = 47.65, p < .001) were found, but there was no effect of sex (F_(1,46) = 0.192, p = .663; Figure 4A) or interaction (F_(3,46) = 2.181, p = .103). Although overall statistical analysis showed no sex differences, planned comparisons between flavors in each sex were also examined (Figure 4). In both sexes, rats preferred saccharin and benzaldehyde plus saccharin (p < .05). Planned comparisons also showed significant differences between females and males in preferences to benzaldehyde solution. Male rats preferred benzaldehyde significantly more than their female counterparts (p < .05).

Figure 4.

Intraoral flavor and intravenous nicotine taking and seeking behaviors in female and male adolescent rats. (A) Average preference to benzaldehyde (100 µg/mL), saccharin (3.2 mg/mL), and combination of benzaldehyde and saccharin in female and male rats. (B) Active lever presses per hour over 15 days of intraoral self-administration of water, benzaldehyde, saccharin, and combination of benzaldehyde and saccharin in the presence of intravenous nicotine (30 µg/kg/infusion) self-administration. (C) Breakpoint of self-administration session on day 16. Data were expressed as mean ± standard error of the mean (SEM) of n = 6–7/sex/group. *p < .05 vs. its water control. #p < .05 vs. its benzaldehyde control. + p < .05 vs. corresponding female control. Filled points indicate significant differences (p < .05) from the intraoral water + intravenous nicotine group at a given time point. PND = post-natal day; IO = intraoral; IV = intravenous; 2BC = two-bottle free-choice; FR = fixed ratio; PR = progressive ratio.

Figure 4B shows IOIVSA in females and males separately. Female rats acquired significant IOIVSA which was confirmed by significant main effects of treatment (F_(3,23) = 6.666, p = .002) and time (F_{(4.476,102.9)} = 67.920, p < .001, Geisser-Greenhouse’s epsilon: 0.319) without a significant effect of treatment × time interaction (F_(42,322) = 1.263, p = .137; Figure 4B). While saccharin significantly increased nicotine intake on days 6, 8, and 10, benzaldehyde plus saccharin pairing led to increased intake on days 8 and 10 (p < .05). Male rats also established significant IOIVSA which was confirmed by significant main effects of treatment (F_(3,23) = 11.53, p < .001) and time (F_{(6.445,148.2)} = 76.110, p < .001, Geisser-Greenhouse’s epsilon: 0.460) without a significant effect of treatment × time interaction (F_(42,322) = 1.183, p = .212; Figure 4B). Saccharin significantly increased nicotine self-administration on days 4, 5, 6, 9, and 10 compared to water group (p < .05). Benzaldehyde and saccharin also increased nicotine intake on days 5, 6, and 10 (p < .05). As seen in Figure 4B, males showed higher IOIVSA than females.

Furthermore, significant main effects of flavor (F_(3,46) = 10.26, p < .001), but not sex (F_(1,46) = 0.222, p = .639; Figure 4C) and interaction (F_(3,46) = 0.1219, p = .9467), were found in the flavor and nicotine IOIVSA experiments. Planned comparisons between flavors in each sex were shown in female and male panels. Saccharin induced the highest breakpoint level (p < .05).

Discussion

In this study, adolescent rats were provided with voluntary access to sweet or fruit-flavored solutions during adolescence, followed by the opportunity to self-administer nicotine in conjunction with these flavors in later periods. Our data revealed that animals with prior access to these flavors consumed significantly higher amounts of the sweetened solutions along with nicotine. Although no significant sex differences were observed in IOIVSA of flavors and nicotine, males showed >2-folds higher intake compared to females.

Adolescent rats successfully acquired nicotine IVSA, as evidenced by significant main effects of nicotine, time, and an interaction between nicotine and time (Figure 1). This suggests a robust and sustained motivation to self-administer nicotine in adolescent rats, which is consistent with the literature.^16,17 An intriguing aspect of the study is the investigation into potential sex differences in nicotine IVSA behavior in adolescent rats. Contrary to our hypothesis, the analysis revealed no significant differences between female and male rats in terms of the number of active lever responses, the number of nicotine infusions received, nor nicotine-seeking behavior as measured by breakpoint. This finding is also in line with a previous report in adolescent Sprague Dawley rats.¹⁸ However, it has been demonstrated that sex differences do exist in nicotine IVSA among adult Sprague-Dawley rats, and strain differences have also been observed between adult Sprague-Dawley and Long-Evans rats.¹⁹ Thus, research suggests that sex differences in nicotine IVSA are dependent on both age and strain. In summary, our data and previous results by Carreño et al.¹⁸ illustrate that male and female adolescent Sprague Dawley rats exhibit similar, robust nicotine self-administration.

Adolescent rats also effectively self-administered IO flavors in the absence of nicotine (Figure 2). Of particular interest is the differential response observed when self-administering saccharin and benzaldehyde solutions. Saccharin is an artificial sweetener. Artificial sweeteners are included in the ingredient list of oral nicotine pouches, a novel product category experiencing a rapid increase in market presence. Benzaldehyde is the primary chemical for characterizing cherry, red berries, and almond flavors in e-liquids and oral tobacco products. In this study, while saccharin elicited significant flavor intake by rats, the rats did not self-administer benzaldehyde. Other studies have also evaluated the motivational properties of benzaldehyde. One previous report showed that benzaldehyde is preferred and self-exposed by C. elegans.²⁰ Another study reported that male rats preferred commercial cherry e-liquid at 50% of total fluid intake in 2BC, indicating that cherry flavoring did not induce either reward or an aversion.²¹ Our previous study showed that adolescent male, but not female, rats preferred benzaldehyde over water in 2BC.¹¹ Therefore, our current findings contrast prior benzaldehyde self-administration studies in other species and prior benzaldehyde choice behaviors in rats. On the other hand, we found that, in the presence of saccharin, benzaldehyde induced self-administration. This suggests that the addition of saccharin may enhance the palatability or reinforcing properties of the benzaldehyde solution, resulting in a significant consumption across individuals.

Our study also provides preclinical insights into the sweetener (saccharin), cherry flavor (benzaldehyde), and tobacco (nicotine) interactions (Figures 3 and 4). Due to their appealing effects, characterizing flavors significantly impact youth experimentation and progression to regular tobacco use.⁴ This study represents the first investigation into the effects of oral saccharin and benzaldehyde on intravenous nicotine intake in adolescent rodents, which models the impact of sweet and fruit flavors on tobacco product use in adolescence.

Benzaldehyde alone did not alter nicotine reinforcement in early sessions of IOIVSA but decreased nicotine reinforcement during late sessions of IOIVSA. This suggests that benzaldehyde may be aversive at 100 µg/mL concentration during the later FR5 days. The aversive effect likely arises due to the increased number of infusions over time. In this operant paradigm, as the number of presses and infusions increases, the intraoral exposure to benzaldehyde might become more unpleasant, particularly in the later stages of the FR5 sessions. Thus, the decrease in nicotine intake observed in the later days could be attributed to the aversive taste of benzaldehyde after enhanced exposure and the aging of the rats. Indeed, we previously reported that intraoral benzaldehyde alone at 100 µg/mL is slightly aversive in adult rats,¹³ which may explain. Both saccharin alone and in combination with benzaldehyde promoted nicotine reinforcement. However, while saccharin alone robustly increased nicotine self-administration, combined benzaldehyde and saccharin resulted in self-administration behavior that was higher than nicotine alone group, but less than saccharin alone group. A previous report showed that artificial sweeteners, such as sucrose or saccharin can increase nicotine self-administration and phasic dopamine release in the nucleus accumbens in adult rats.¹⁰ Our findings in adolescent rats are consistent with the adults reported before.

In our previous study using the 2BC paradigm, we characterized the preference of benzaldehyde by running a dose-response analysis using the benzaldehyde concentrations of 20, 100, and 500 µg/mL.¹¹ Our dose-response analysis revealed a significant effect of benzaldehyde, with males showing increased preference for benzaldehyde at all tested concentrations compared to the water group without a concentration manner, while females did not exhibit a significant increase in preference at any concentrations. Therefore, we selected the 100 µg/mL concentration of benzaldehyde for the acute and chronic studies as a representative median dose in the previous study.¹¹ Furthermore, this previous dose−response study was conducted using the same rat strain (Sprague Dawley), and the animals used were of similar age (adolescent) and sex (both female and male) with this current study.¹¹ In terms of benzaldehyde concentration, rat strain and sex, our current study is also ensuring consistency with our previous study conditions. In previous study, we found that benzaldehyde did not alter nicotine’s orosensory properties, and did not alter acute and chronic nicotine use behaviors in adolescent female and male rats.¹¹ Indeed, the findings obtained with benzaldehyde in the current study is consistent with our previous published data in adolescence. Another previous study in adult rats revealed that benzaldehyde at 100 µg/mL concentration increased nicotine’s hedonic taste responses and decreased nicotine’s aversive taste responses.¹³ Age may be an important factor in the effects of benzaldehyde on nicotine use behaviors. In all these studies, we used one concentration of benzaldehyde, based on our dose response studies.^11,13 However, different concentrations of benzaldehyde may reveal differential interactions. In addition to age and concentration, the route of administration could be a possible contributor to the varying effects of benzaldehyde on nicotine use behaviors. For example, vapor inhalation of benzaldehyde and nicotine may result in differential interactions. Furthermore, our current study suggests that benzaldehyde, at this concentration, may not affect nicotine intake through taste-related mechanisms. However, benzaldehyde might act via other mechanisms, such as olfactory or direct central nervous system effects. Additionally, there is a possibility that propylene glycol and vegetable glycerin (PG and VG), either inert or when heated, could cause benzaldehyde to undergo secondary reactions, producing biologically active constituents.

Limited information is available regarding the influence of saccharin and benzaldehyde on the short-term and prolonged consequences of adolescent nicotine consumption behaviors. Cherry e-liquids containing benzaldehyde offer a sweet and fruity flavor profile.^22,23 When benzaldehyde was combined with saccharin, the results in both IOSA and IOIVSA experiments closely resembled those obtained with saccharin alone. Therefore, sweetness value may alter self-administration behaviors and modulate nicotine’s abuse liability. Indeed, the appeal of sweet flavors potentially play a role in the adoption of e-cigarettes in younger population.^24,25 Lastly, the investigation of sex and age in relation to the development of nicotine dependence in self-administration studies remains less explored.²⁶ Therefore, our study also contributes to the existing literature by examining the effects of sex and early exposure on continued usage patterns.

Conclusion

The findings from our preclinical study highlight how the importance of sweetness can strongly contribute to, or possibly even outweigh, the of effects of the flavor itself. Specially, we demonstrated that the sweetener saccharin heightened nicotine taking and seeking in a rodent self-administration model. Conversely, a commonly used fruit flavor, cherry (benzaldehyde), did not increase nicotine taking or seeking, but rather reduced nicotine self-administration on the last days of the test sessions. Furthermore, the effects of combination were driven by the saccharin when saccharin was combined with benzaldehyde, as benzaldehyde did not have an independent effect on increasing nicotine intake.

The sweetness of a tobacco product does not solely stem from sweeteners but is also influenced by other components such as the PG/VG ratio (eg, higher VG) and the inclusion of flavors associated with sweetness (eg, vanillin and ethyl maltol). Therefore, sweet-associated constituents in tobacco products, including sweeteners and flavor additives should also be investigated, to further inform regulatory policy.

Funding

This work was supported by grant number (U54DA036151) from the National Institute on Drug Abuse (NIDA) of the National Institutes of Health (NIH) and Food and Drug Administration (FDA) Center for Tobacco Products (CTP) and the State of Connecticut, Department of Mental Health and Addiction Services. The content is solely the responsibility of the authors and does not represent the official views of the NIH, the FDA, or the Department of Mental Health and Addiction Services or the State of Connecticut.

Declaration of Interests

[NAA] Royalties—Tyndale House Publishers. Speakers Bureau/ Consultation Fees—American Program Bureau.

Author Contributions

Deniz Bagdas (Conceptualization [equal], Data curation [lead], Formal analysis [lead], Investigation [lead], Methodology [equal], Project administration [equal], Writing—original draft [lead], Writing—review & editing [lead]) and Nii A. Addy (Conceptualization [equal], Funding acquisition [lead], Methodology [equal], Project administration [equal], Writing—original draft [supporting], Writing—review & editing [supporting])

Data Availability

The data underlying this study will be made available upon reasonable request to the corresponding author.