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. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: Drug Alcohol Depend. 2018 Feb 1;185:1–9. doi: 10.1016/j.drugalcdep.2017.11.032

Effects of nicotine-containing and “nicotine-free” e-cigarette refill liquids on intracranial self-stimulation in rats

Andrew C Harris 1,2,3, Peter Muelken 1, John R Smethells 1,4, Katrina Yershova 5, Irina Stepanov 5, Thao Tran Olson 6, Kenneth J Kellar 6, Mark G LeSage 1,2,3
PMCID: PMC5889751  NIHMSID: NIHMS942896  PMID: 29413432

Abstract

Background

Animal models are needed to inform FDA regulation of electronic cigarettes (ECs) because they avoid limitations associated with human studies. We previously reported that an EC refill liquid produced less aversive/anhedonic effects at a high nicotine dose than nicotine alone as measured by elevations in intracranial self-stimulation (ICSS) thresholds, which may reflect the presence of behaviorally active non-nicotine constituents (e.g., propylene glycol) in the EC liquids. The primary objective of this study was to assess the generality of our prior ICSS findings to two additional EC liquids. We also compared effects of “nicotine-free” varieties of these EC liquids on ICSS, as well as binding affinity and/or functional activity of nicotine alone, nicotine-containing EC liquids, and “nicotine-free” EC liquids at nicotinic acetylcholine receptors (nAChRs).

Methods and Results

Nicotine alone and nicotine dose-equivalent concentrations of both nicotine-containing EC liquids produced similar lowering of ICSS thresholds at low to moderate nicotine doses, indicating similar reinforcement-enhancing effects. At high nicotine doses, nicotine alone elevated ICSS thresholds (a measure of anhedonia-like behavior) while the EC liquids did not. Nicotine-containing EC liquids did not differ from nicotine alone in terms of binding affinity or functional activity at nAChRs. “Nicotine-free” EC liquids did not affect ICSS, but bound with low affinity at some (e.g., α4ß2) nAChRs.

Conclusions

These findings suggest that non-nicotine constituents in these EC liquids do not contribute to their reinforcement-enhancing effects. However, they may attenuate nicotine’s acute aversive/anhedonic and/or toxic effects, which may moderate the abuse liability and/or toxicity of ECs.

Keywords: Nicotine, intracranial self-stimulation, electronic cigarettes, Non-nicotine tobacco constituents, tobacco control policy

1. Introduction

Electronic cigarettes (ECs) are aerosol-producing devices designed to simulate the use of conventional tobacco cigarettes. Although ECs are often viewed as less addictive and safer than tobacco cigarettes, their abuse liability and other health consequences have not been well established (Brandon et al., 2015; Glasser et al., 2017; Walton et al., 2015). Nonetheless, ECs are becoming increasingly popular, especially among smokers and adolescents (e.g., Arrazola et al., 2015; Glasser et al., 2017). To address this growing health concern, the Food and Drug Administration Center for Tobacco Products (FDA CTP) recently extended their authority to regulate ECs in the same manner as cigarettes and other tobacco products (Food and Drug Administration, 2016). Development of appropriate methodology for evaluating abuse liability and other adverse effects of ECs is needed to inform FDA CTP regulation of these products.

Animal models are essential for tobacco product evaluation because they avoid limitations associated with human studies (e.g., inability to isolate the central nervous system (CNS) effects of nicotine and other tobacco constituents from peripheral sensory factors (e.g., taste, smell)) (Donny et al., 2012). An emerging approach for this purpose involves the use of extracts that are derived directly from tobacco or tobacco smoke and that contain a mixture of tobacco constituents (for review, see Brennan et al., 2014). In contrast to traditional preclinical models of tobacco addiction, which involve administration of nicotine alone or other isolated tobacco constituents, use of extracts provides insights into how the numerous constituents in a tobacco product act together to influence abuse liability. While such interactions can also be studied using exposure to actual cigarette smoke or EC aerosol (Bruijnzeel et al., 2011; Harris et al., 2010; Ponzoni et al., 2015; Small et al., 2010), these inhalational models do not allow dissociation of the direct CNS effects of smoke or EC aerosol from its sensory effects (e.g., taste, smell). Because extracts are administered systemically (i.p., s.c., or i.v.), they allow for the dissociation of these factors, as well as for more precise experimental control over dosing. Several studies have reported greater abuse liability for tobacco smoke extracts compared to nicotine alone (e.g., Brennan et al., 2015; Brennan et al., 2014; Costello et al., 2014), which may be due to the presence of certain constituents in the extracts (e.g., minor alkaloids, acetaldehyde) that can mimic or enhance nicotine’s addiction-related effects when studied in isolation (e.g., Arnold et al., 2014; Bardo et al., 1999; Belluzzi et al., 2005).

Preclinical evaluation of EC liquids, which often contain a combination of nicotine and other behaviorally relevant constituents (e.g., minor alkaloids, acetaldehyde, and propylene glycol (Etter et al., 2013; Goniewicz et al., 2014; Han et al., 2016)), provides similar advantages as study of tobacco extracts. We recently found that low to moderate doses of nicotine alone and nicotine dose-equivalent concentrations of an EC liquid (Aroma E-Juice Dark Honey) were similar in terms of their ability to lower intracranial self-stimulation (ICSS) thresholds (LeSage et al., 2016), a putative measure of nicotine’s ability to enhance the reinforcing effects of other stimuli (“reinforcement-enhancement”) (e.g., Caggiula et al., 2009; Harrison et al., 2002; Huston-Lyons and Kornetsky, 1992). At high nicotine doses, nicotine alone elevated ICSS thresholds while EC liquid did not, suggesting a reduction in nicotine’s acute aversive/anhedonic effects when delivered in EC liquid. Given that nicotine’s aversive/anhedonic effects can limit its intake (see Fowler and Kenny, 2012; Fowler and Kenny, 2014; Fowler et al., 2011), reduction of these effects would be expected to increase EC consumption. However, we found no differences in i.v. self-administration (SA) of nicotine alone versus EC liquid (see LeSage et al., 2016 and below for further discussion). Alternatively, the ICSS findings might reflect a reduction in nicotine’s toxic effects, which would be equally important given that product toxicity is a primary concern of the FDA CTP (Food and Drug Administration, 2016). Regardless of the interpretation of these findings, they suggest that at least this EC liquid contains behaviorally active levels of non-nicotine constituents as measured using ICSS. It is essential to evaluate the generality of these findings to additional EC liquids, which can differ substantially in levels of behaviorally relevant non-nicotine constituents including minor alkaloids (Etter et al., 2013; Goniewicz et al., 2014; Han et al., 2016).

The goal of this study was to further evaluate the acute effects of EC liquids on ICSS in order to understand the relative contribution of CNS effects of nicotine and non-nicotine constituents in EC abuse liability. Following an initial analysis of nicotine and minor alkaloid levels in 20 different EC liquids, we compared the ICSS threshold-altering effects of nicotine alone and nicotine dose-equivalent concentrations of EC liquids containing relatively high (Janty EC liquid) and low (NicVape EC liquid) levels of minor alkaloids relative to nicotine (Experiment 1). In contrast to the Aroma E-Juice EC liquid that we studied in LeSage et al. (2016), both Janty and NicVape EC liquids are available in a labeled nicotine concentration of 0 mg/ml. Therefore, Experiment 2 evaluated effects of these “nicotine-free” EC liquids on ICSS (Experiment 2). To complement the behavioral data, Experiment 3 compared binding affinity of nicotine alone, nicotine-containing Janty and NicVape EC liquids, and “nicotine-free” Janty and NicVape EC liquids at several nicotinic acetylcholine receptors (nAChRs), including the α4ß2 and α3ß4 nAChR subtypes, which are implicated in nicotine addiction (De Biasi and Salas, 2008; Fowler et al., 2008). Functional effects of nicotine alone and nicotine-containing EC liquids at α4ß2 and α3ß4 nAChRs were also compared in a rubidium efflux assay.

2. Methods

2.1. Animals

Male adult Holtzman rats (Harlan/Envigo, Indianapolis, IN) weighing 300-350 grams at arrival were individually housed in a temperature- and humidity-controlled colony room with unlimited access to water under a reversed 12-h light/dark cycle. Rats were food restricted to 18 g/day to facilitate operant performance and avoid detrimental health effects of long-term ad libitum feeding (Keenan et al., 1997; Keenan et al., 1999). Protocols were approved by the Institutional Animal Care and Use Committee of the Minneapolis Medical Research Foundation in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council 2011).

2.2. Analysis of Nicotine and Minor Alkaloids in EC Liquids

2.2.1. Initial EC Liquid Alkaloid Analysis

Concentrations of nicotine and the minor alkaloids nornicotine, anabasine, and anatabine were analyzed in 20 EC liquids using liquid chromatography-tandem mass spectrometry (LC-MS/MS) by modification of a previously described method (Rangiah et al., 2011). Briefly, each EC liquid was mixed with stable isotope-labeled nicotine and nornicotine, anatabine, and anabasine (internal standards), diluted with 10 mM ammonium acetate containing 5% methanol, and analyzed by LC-MS/MS on a Hypercarb column (Thermo Scientific), using 10 mM ammonium acetate (with 0.001% formic acid) and methanol as mobile phase. EC liquids were chosen based on their local popularity (e.g., TC Vape), their previous alkaloid characterization in Etter et al. (2013) (e.g., Johnson Creek), or because they were advertised as containing higher levels of minor alkaloids than other ECs (Aroma E-Juice). EC liquids were purchased in the Minneapolis area (TC Vape) or ordered online through their manufacturer (all other EC liquids). Following completion of the initial multi-product alkaloid comparison and selection of two EC liquids for the behavioral studies that contained relatively high and low levels of minor alkaloids relative to nicotine (i.e., Janty and NicVape, see below), nicotine and minor alkaloid levels in the “nicotine-free” variety of these products were analyzed in the same manner.

2.2.2. Routine Nicotine Assay

Nicotine concentrations in solutions of nicotine alone and Janty and NicVape EC liquid used in Experiments 2 and 3 were measured by gas chromatography (GC) with nitrogen phosphorus detection, according to standard protocol in our laboratory (Hieda et al., 1999),. The measured nicotine concentrations for Janty and NicVape EC liquid vials used for dose preparation in Experiment 1 (labeled nicotine content = 24.0 mg/ml) were 22.10 mg/ml and 22.52 mg/ml, respectively. The average measured nicotine concentrations ± SEM for Janty and NicVape vials used for Experiments 2 and 3a (labeled nicotine content = 0 mg/ml) were 0.0058 ± 0.0017 mg/ml (range 0.0042 to 0.0075 mg/ml) and 0.0008 ± 0.0006 mg/ml (range 0.0001 to 0.0014 mg/ml), respectively.

2.3. Drugs

Nicotine bitartrate was obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in sterile saline. Janty EC refill liquid (DK Port flavor) and NicVape EC refill liquid (Fruit Stripe Gum / Fruit Twist flavor) were obtained from Janty USA (http://www.usa.janty.com, Blasdell, NY) and NicVape (http://www.nicvape.com, Spartanburg, SC), respectively. According to the manufacturer, the Janty refill liquid contained 66.1% propylene glycol (PG), 15.0% vanillin tincture, 1.0% peach aldehyde, and 2.0% 2,5-dimethylpyrazine. The remaining 15.9% of the product’s ingredients were unaccounted for. NicVape refill liquid was advertised as containing 50% PG and 50% vegetable glycerine. Labeled nicotine concentrations for both Janty and NicVape EC liquids was 24 mg/ml or 0 mg/ml. Actual nicotine concentration for each vial of Janty or NicVape EC liquid was determined (see above), and the EC liquids were diluted in saline to the concentrations required for the current studies and filter-sterilized. The pH for all solutions was adjusted to 7.4 with dilute NaOH or HCL. Nicotine doses are expressed as the base.

2.4. Intracranial Self-Stimulation

Surgery, apparatus, and training procedures used here are described in detail elsewhere (Harris et al., 2010; Harris et al., 2011). Briefly, animals were anesthetized with i.m. ketamine (75 mg/kg)/dexmedetomidine (0.025 mg) and implanted with a bipolar stainless steel electrode in the medial forebrain bundle. Rats were later trained to respond for electrical brain stimulation using a modified version of the Kornetsky and Esposito (1979) discrete-trial current-threshold procedure (Harris et al., 2010; Harris et al., 2011; Markou and Koob, 1992). Each trial was initiated with presentation of a non-contingent stimulus (0.1 ms cathodal squarewave pulses at a frequency of 100 Hz for 500 ms) followed by a 7.5-sec window during which a positive response on the wheel manipulandum produced a second, contingent stimulation identical to the first. Lack of responding in the 7.5-second time window was considered a negative response. Each positive or negative response was followed by a variable inter-trial interval averaging 10 sec (range = 7.5 to 12.5 sec), during which time additional responses delayed onset of the subsequent trial by 12.5 sec. Stimulus intensities were presented in four alternating descending and ascending series (step size = 5 uA), with five trials presented at each current intensity step. The current threshold for each series was defined as the midpoint between two consecutive current intensity steps that yielded three or more positive responses and two consecutive current intensity steps that yielded three or more negative responses. The overall threshold for the approximately 45 min session was defined as the mean of the current thresholds from the four alternating series. To assess performance effects (e.g., motor disruption), response latencies (time between onset of the non-contingent stimulus and a positive response) were averaged across all trials in which a positive response was made (Markou and Koob, 1992).

2.5. Experiment 1: Effects of Nicotine Alone and Nicotine-Containing EC Liquids on ICSS

Rats (N = 13) were tested in daily ICSS sessions conducted Mon-Fri until thresholds were stable (i.e., less than 10% coefficient of variation over a 5-day period and no apparent trend). To habituate animals to the injection procedure, saline was administered s.c. 10 minutes prior to ICSS at least once and until thresholds were unaltered by the injection. Effects of 10-minute pretreatment with s.c. nicotine alone, Janty EC liquid, or NicVape EC liquid were subsequently determined at nicotine doses of 0, 0.06, 0.125, 0.25, 0.50, 0.75, 1.0, or 1.25 mg/kg. These doses reduce or increase ICSS thresholds when administered acutely (Harris et al., 2012; Harrison et al., 2002; Spiller et al., 2009). Nicotine and EC liquid injections typically occurred on Tuesdays and Fridays, provided that thresholds were within baseline range on intervening days, and doses of each formulation were administered in a counterbalanced order. Effects of all 3 formulations were tested in each rat, and order of the 3 formulations was counterbalanced across animals. Animals were tested under drug-free conditions for at least 2 weeks and until ICSS thresholds were stable between testing each formulation.

2.6. Experiment 2: Effects of “Nicotine-Free” EC Liquids on ICSS

A separate group of rats (N = 8) was trained for ICSS and tested using the same general procedure described for Experiment 1, except that animals were administered “nicotine-free” Janty or NicVape EC liquid s.c. at concentrations of 0 (i.e., saline alone), 5, 12.5, 25, 50, 75, or 100%.

2.7. Experiment 3: Binding Affinities and Receptor Activation of Nicotine Alone and EC Liquids at nAChRs

Radioligand binding (Experiments 3a and 3b) and rubidium efflux (Experiment 3c) assays were conducted using solutions of nicotine alone, nicotine-containing Janty and NicVape EC liquids, and/or “nicotine-free” Janty EC and NicVape EC liquids as described in the Supplementary Methods1.

2.8. Statistical Analyses

2.8.1. Experiments 1 and 2

Only seven of the 13 rats in Experiment 1 were tested with all three formulations, owing to attrition due to ICSS headcap removal, loss of stability of ICSS thresholds, or other problems. Our primary interest was the comparison of each EC liquid to nicotine alone rather than between the two EC liquids. Therefore, data were analyzed separately for the subset of animals completing dose-response testing for nicotine alone and Janty EC liquid (n = 9) and the subset receiving nicotine alone and NicVape EC liquid (n = 8). Within each of these subsets, baseline ICSS thresholds (a measure of the function of brain reinforcement pathways, in μA) and response latencies (a measure of non-specific (e.g., motor) effects, in sec) were compared between formulations using separate paired-samples t-tests. ICSS threshold and latency values during test sessions, expressed as a percent of baseline (i.e., mean during last 5 sessions prior to each dose-response determination), were subsequently compared using separate two-factor ANOVAs with formulation and dose as within-subject factors. Paired sample t-tests with a Bonferroni correction were used for between-formulation comparisons at each dose. Data within each formulation were also analyzed using a one-factor ANOVA with dose as a within-subject factor, followed by Dunnett’s post hoc tests comparing each dose to saline. Degrees of freedom for all ANOVAs were adjusted using the Greenhouse-Geisser correction to account for possible violations of sphericity. Pearson’s correlation analysis was used to compare the effects of nicotine alone on ICSS thresholds and response latencies under some conditions (see below). Data for Experiment 2 were analyzed in the same manner. In both experiments, p-values < 0.05 were considered statistically significant.

2.8.2. Experiment 3

Data for Experiment 3a (binding affinity) and 3c (functional activity) were analyzed by nonlinear least-squares regression to obtain Ki or EC50 values, respectively (see http://pdsp.med.unc.edu. for further details). Statistically significant differences between formulations were defined as non-overlapping 95% confidence intervals for Ki or EC50.

3. Results

3.1. Initial EC Liquid Constituent Analysis

Actual (measured) nicotine levels were 91.3 ± 3.5% (range = 57.4 to 125.1%) of labeled nicotine levels in the 20 EC liquids analyzed using LC-MS/MS (Table 1). There was considerable variability in the relative levels of the minor alkaloids nornicotine, anatabine, and anabasine (expressed as % of nicotine) between products (Table 1).

Table 1.

Levels of nicotine (mg/mL) and minor alkaloids (μg/mL) in EC liquids.

Brand / Flavor Nicotine, mg/mL Minor alkaloids, μg/mL (% of nicotine)
Labeled Actual Nornicotine Anabasine Anatabine Total Minor
Ecig express / USA Mix 6 5.6 7.7 (0.14%) 9.7 (0.17%) 119.8 (2.12%) 137.20 (2.43%)
Janty / DK Port 24 19.3 4.9 (0.03%) 31.1 (0.16%) 402.7 (2.09%) 438.7 (2.28%)
Vapor4Life / 555 Nobacco Juice 24 21.9 12.0 (0.05%) 41.3 (0.19%) 301.4 (1.38%) 354.7 (1.62%)
TC Vape / Organic Normal Tobacco 24 22.5 14.0 (0.06%) 36.5 (0.16%) 286.0 (1.27%) 336.5 (1.50%)
Vapor4life / Nobacco Juice Gunslinger 6 4.53 2.3 (0.05%) 24.7 (0.54%) 38.3 (0.85%) 65.3 (1.44%)
TC Vape / Normal Tobacco 12 9.54 3.8 (0.03%) 17.3 (0.18%) 100.3 (1.05%) 121.4 (1.27%)
Aroma E-Juice / Menthol WTA 18 14.7 10.1 (0.07%) 25.4 (0.17%) 139.9 (0.95%) 175.4 (1.20%)
Aroma E-Juice / Dark Honey WTA 24 20.7 7.1 (0.03%) 37.1 (0.18%) 192.8 (0.93%) 237.0 (1.15%)
Janty / Gold Sahara 24 30.0 4.4 (0.01%) 42.3 (0.14%) 243.4 (0.81%) 290.1 (0.97%)
TC Vape / Menthol Tobacco 24 17.5 4.4 (0.025%) 32.4 (0.19%) 108.2 (0.62%) 145.0 (0.83%)
Janty / Elixir, Koreanesque 24 23.2 4.8 (0.02%) 42.1 (0.18%) 144.1 (0.62%) 191.0 (0.82%)
Aroma E-Juice / 555 WTA 24 26.8 9.5 (0.04%) 50.2 (0.19%) 160.3 (0.60%) 220.0 (0.82%)
Aroma E-Juice / Nuport WTA 24 21.3 7.6 (0.04%) 45.7 (0.21%) 91.8 (0.43%) 145.1 (0.68%)
Aroma E-Juice / Flue-Cured WTA 24 26.2 6.8 (0.03%) 42.9 (0.16%) 79.2 (0.30%) 129.0 (0.49%)
NicVape / American Menthol 24 20.4 9.5 (0.05%) 35.4 (0.17%) 34.5 (0.17%) 79.4 (0.39%)
Totally Wicked E-Liquid / Tobacco 18 20.4 6.0 (0.03%) 11.4 (0.06%) 48.1 (0.23%) 65.5 (0.32%)
NicVape / Fruit Stripe Gum 24 13.8 2.9 (0.02%) 9.2 (0.07%) 3.3 (0.02%) 15.4 (0.11%)
Patriot Range / Tobacco 18 19.0 7.8 (0.04%) 7.1 (0.04%) 5.1 (0.03%) 20.0 (0.11%)
NicVape / Tobacco 16 13.7 6.1 (0.04%) 3.1 (0.02%) 3.7 (0.03%) 12.9 (0.09%)
Johnson Creek – JC Original 24 23.6 4.8 (0.02%) 8.1 (0.03%) 5.7 (0.02%) 18.6 (0.08%)
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Both labeled and actual (measured) levels of nicotine are shown. Data in parentheses indicate relative levels of each minor alkaloid (expressed as % of nicotine) in that solution. Total Minor = Nornicotine + Anabasine + Anatabine. Alkaloid data for Aroma E-Juice Dark Honey WTA were previously reported in LeSage et al. (2016)

Nicotine levels in undiluted, “nicotine-free” Janty and NicVape EC liquids were 0.008 mg/mL and below the limit of detection, respectively. Nornicotine, anatabine, and anabasine were not detected in either “nicotine-free” EC liquid.

3.2. Experiment 1: Effects of Nicotine Alone and Nicotine-Containing EC Liquids on ICSS

3.2.1. Nicotine Alone Versus Janty EC Liquid

There were no differences in baseline ICSS thresholds (97.2 ± 7.7 μA versus 104.3 ± 11.9 μA) or response latencies (2.44 ± 0.11 seconds versus 2.37 ± 0.08 seconds) in the subset of animals receiving nicotine alone and Janty EC liquid (n = 9).

Analysis of ICSS threshold data for these animals indicated a significant main effect of dose (F(2.54,20.35)=24.9, p <0.0001), no effect of formulation (F(1,8)=4.98, p = 0.056), and a significant dose × formulation interaction (F(2.43,19.46)=3.3, p < 0.05) (Fig 1A). Thresholds did not differ significantly (i.e., p < 0.05) between formulations at any dose. Subsequent one-factor ANOVAs on data for each formulation indicated a significant effect of dose for nicotine alone (F(2.8, 22.7)=31.0, p <0.0001), with thresholds reduced compared to saline at 0.06, 0.125, and 0.25 mg/kg (Dunnett q = 3.58-4.50, p < 0.05 or 0.01), and elevated compared to saline at 1.0 and 1.25 mg/kg (q = 6.86 and 5.18, respectively, p < 0.01) (Fig 1A). There was also a significant effect of dose for Janty EC liquid (F(2.7, 21.6)=8.1, p <0.01), with ICSS thresholds reduced compared to saline at 0.06 and 0.125 mg/kg (q = 4.29 and 4.41, respectively, p < 0.05). No dose of Janty EC liquid produced a significant increase in ICSS thresholds.

Figure 1.

Figure 1

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ICSS thresholds (A) and response latencies (B) (expressed as percent of baseline, mean ± SEM) following injection of nicotine alone or Janty EC liquid (0 - 1.25 mg/kg) in Experiment 1. ICSS threshold and latency data for nicotine alone and NicVape EC liquid are shown in (C) and (D). *,** Significantly different from saline (0 mg/kg) for that formulation, p < 0.05 or 0.01, respectively. # Significantly different from nicotine alone at that dose, p < 0.05.

There was a significant main effect of dose on ICSS response latencies (F(3.8,30.58)=7.0, p<0.0001), but no effect of formulation or dose × formulation interaction (Fig 1B). Latencies were significantly increased compared to saline at the 1.0 mg/kg dose of nicotine alone (q = 4.67, p < 0.01), but did not differ from saline at any dose of Janty EC liquid (Fig 1B). Elevations in ICSS thresholds and response latencies at the 1.0 mg/kg dose of nicotine alone were not correlated (r = -0.22, p = 0.56), suggesting that effects of nicotine on these measures of ICSS were independent.

3.2.2. Nicotine Alone Versus NicVape EC Liquid

Baseline ICSS thresholds (94.2 ± 7.5 μA versus 97.9 ± 9.2 μA) and response latencies (2.46 ± 0.15 seconds versus 2.39 ± 0.09 seconds) were similar for the nicotine alone and NicVape EC liquid dose-response determinations in the 8 animals that received both of these formulations.

There were significant main effects of dose (F(2.65,18.54)=22.2, p <0.0001) and formulation (F(1,7)=8.6, p < 0.05) on ICSS thresholds, as well as a significant dose × formulation interaction (F(3.02, 21.17)=6.26, p < 0.0001) (Fig 1C). Thresholds were lower for NicVape EC liquid compared to nicotine alone at 1.0 mg/kg (t(7) = 4.8, p = 0.015), but did not differ between formulations at any other dose. There was a significant effect of dose for the nicotine alone condition (F(2.7, 19.04)=25.16, p <0.0001), with ICSS thresholds significantly reduced compared to saline at 0.25 mg/kg (q = 3.94, p < 0.05), and elevated compared to saline at 1.0 and 1.25 mg/kg (q = 7.24 and 4.90, respectively, p < 0.01) (Fig 1C). There was also a significant effect of dose for NicVape EC liquid (F(2.42, 16.93)=9.3, p <0.01). ICSS thresholds were significantly reduced compared to saline at 0.25 mg/kg (q = 5.6, p < 0.05), but did not differ from saline at any other dose.

Analysis of ICSS response latency data indicated a significant main effect of dose (F(2.86,20.0)=2.86, p <0.001), but no significant effect of formulation or dose × formulation interaction (Fig 1D). Latencies did not significantly differ from saline at any dose of nicotine alone or NicVape EC liquid (Fig 1D).

3.3. Experiment 2: Effects of “Nicotine-Free” EC Liquids on ICSS

Baseline ICSS thresholds (105.0 ± 15.2 μA versus 108.1 ± 15.7 μA) and response latencies (3.04 ± 0.46 seconds versus 2.68 ± 0.15 seconds) did not differ between the “nicotine-free” Janty and NicVape EC liquid concentration-response determinations.

Neither “nicotine-free” EC liquid affected ICSS. There was no effect of concentration, formulation, or concentration × formulation interaction for either ICSS thresholds (Fig 2A) or latencies (Fig 2B).

Figure 2.

Figure 2

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ICSS thresholds (A) and response latencies (B) (expressed as percent of baseline, mean ± SEM) following injection of “nicotine-free” Janty and NicVape EC liquids (0 – 100%).

3.4. Experiment 3: nAChR Binding Affinity and Activation Profiles of Nicotine Alone and EC Liquids

3.4.1. Experiment 3a: nAChR binding affinity

Nicotine alone, Janty EC liquid, and NicVape EC liquid (nicotine concentration = 0.6 mg/ml for all formulations, 3.7 mM) produced similar, marked (>90%) inhibition of labeled epibatidine binding to all nAChRs in primary binding assays (data not shown). “Nicotine-free” Janty EC liquid produced significant (>50%) inhibition of α4ß2, α2ß2, and α3ß2 nAChRs expressed in HEK cells and α4ß2 and α7 nAChRs expressed in rat brain (i.e., α4ß2* and α7*) in primary assays, but not at α3ß4, α2ß4, α4ß4, or α7 nAChRs expressed in HEK cells. “Nicotine-free” NicVape EC liquid only produced >50% inhibition at α4ß2 nAChRs expressed in HEK cells.

Results of secondary binding analyses indicated that nicotine alone and both nicotine-containing EC liquids had similar nAChR binding affinities at all nAChR subtypes studied (Table 2, see Fig. 3A and 3B for competition binding curves for α4ß2* and α7* nAChRs). “Nicotine-free” Janty EC liquid bound to α4ß2, α2ß2, α3ß2, α4ß2* and α7* nAChRs with considerably lower affinity (higher Ki values) than the nicotine-containing solutions (Table 2). Ki values for NicVape EC liquid were even higher than those for Janty at these nAChRs.

Table 2.

Binding affinities for nicotine alone and EC liquids at nAChRs expressed in HEK cells or rat brain (denoted by *) and labeled by [3H]-epibatidine.

Nicotine Alone Janty EC Liquid NicVape EC Liquid “Nicotine-Free” Janty EC Liquid “Nicotine-Free” NicVape EC Liquid
α4ß2 5.6 (4.2-7.5) 4.9 (3.7-6.6) 4.3 (3.2-5.7) 167.6 (125.3- 224.2)# 886.4 (663.7- 1184.0)#+
α4ß2* 9.3 (7.0-12.5) 9.0 (6.7-12.0) 7.4 (5.5-9.9) 267.1 (199.2- 358.0)# 1890.0 (1413.0- 2529.7)#+
α7 208.7 (72.6- 599.7) 277.6 (97.0- 794.4) 352.6 (124.0- 1002.0) ND ND
α7* 9.2 (6.0-14.0) 6.4 (4.2-9.7) 6.6 (4.4-10.1) 178.0 (117.2- 270.2)# 1008.0 (665.7- 1526.0)#+
α3ß4 303.4 (237.4- 387.7) 288.4 (225.5- 368.8) 307.1 (240.3- 392.4) ND ND
α2ß2 4.0 (3.1-5.1) 3.8 (2.9-4.9) 4.8 (3.7-6.2) 111.9 (86.4- 144.9)# 681.9 (527.3- 881.9)#+
α2ß4 73.3 (46.0- 116.7) 52.8 (33.0- 84.2) 88.7 (55.8- 140.8) ND ND
α3ß2 7.6 (4.6- 124.8) 6.9 (4.2-11.3) 6.1 (3.7-10.0) 258.5 (157.2- 425.0)# 1603.0 (972.0- 2644.0)#+
α4ß4 22.6 (15.9- 32.2) 23.2 (16.3- 33.0) 23.8 (16.7- 33.9) ND ND
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Values represent Ki (nM) and 95% CI (in parentheses) derived from a single competition binding curve at each nAChR subtype. ND = Not determined.

#

Different from nicotine-containing solutions, p < 0.05.

+

Different from “nicotine-free” Janty EC liquid, p < 0.05.

Figure 3.

Figure 3

Open in a new tab

Competition by nicotine alone and nicotine-containing EC liquids for α4ß2 (A) or α7 (B) nAChR binding sites expressed in rat brain and labeled by [3H]-epibatidine in Experiment 3a. Ki values for formulations at these and other nAChRs are shown in Table 2. Fig (C) shows % inhibition of [3H]-epibatidine binding by nicotine alone and “nicotine-free” EC liquids at α4ß2 nAChRs expressed in HEK cells (Experiment 3b). Fig (D) and (E) show 86Rb+ efflux stimulated by nicotine alone and EC liquids in α4ß2 or α3ß4 nAChRs expressed in HEK cells (Experiment 3).

3.4.2. Experiment 3b: Additional nAChR Binding Assay

Consistent with the results of Experiment 3a, “nicotine-free” Janty and NicVape EC liquids after dilution of the commercial stock samples (1:20 to 1:2000) produced a concentration-dependent inhibition of [3H]-epibatidine binding at α4ß2 nAChRs expressed in HEK cells (Fig 3C). The magnitude of inhibition was greater for “nicotine-free” Janty EC liquid than for “nicotine-free” NicVape EC liquid. The stock solution of nicotine alone (positive control) fully inhibited [3H]-epibatidine binding at all concentrations studied (Fig 3C).

3.4.3. Experiment 3c: nAChR Activation Profile

Nicotine alone and nicotine-containing Janty and NicVape EC liquid produced similar, robust α4ß2 or α3ß4 nAChR activation in HEK cells in both primary (data not shown) and secondary (Fig 3D and 3E) functional assays. EC50 values for formulations at α4ß2 or α3ß4 nAChRs did not differ significantly (data not shown). There was no evidence of antagonist activity for any of the formulations at either α4ß2 or α3ß4 nAChRs (data not shown).

4. Discussion

Nicotine-containing Janty and NicVape EC liquids reduced ICSS thresholds to a similar degree as nicotine alone at low to moderate nicotine doses, but were less effective than nicotine alone in increasing ICSS thresholds at high nicotine doses. These EC liquids did not differ from nicotine alone in terms of binding affinity or functional activity at a range of nAChR subtypes. “Nicotine-free” Janty and NicVape EC liquids did not affect ICSS, but bound with low affinity at some (e.g., α4ß2) nAChRs. Novel findings of this study include: 1) extension of the generality of our previous findings using another EC liquid (Aroma E-Juice) that contained differing levels of behaviorally relevant non-nicotine constituents (minor alkaloids, PG) than the current EC liquids, 2) the first characterization of the effects of “nicotine-free” EC liquids on ICSS and nAChR binding / activation, and 3) one of the most extensive evaluations of nicotine and minor alkaloid levels in EC liquids conducted to date (see also Etter et al., 2013; Han et al., 2016; Lisko et al., 2015), including evaluation of several previously unstudied EC liquids (e.g., NicVape EC liquid).

The ability of high nicotine doses to elicit elevations in ICSS thresholds has been proposed to reflect nicotine’s acute aversive effects that limit its intake (Fowler and Kenny, 2012, 2014; Fowler et al., 2011; Fowler et al., 2013). Our ICSS findings may therefore reflect an attenuation of nicotine’s aversive effects by non-nicotine constituents in EC liquid, an effect that could increase EC consumption. However, we previously found no differences in i.v. SA of nicotine alone and Aroma E-Juice EC liquid, despite the fact that the ICSS threshold-elevating effects of this EC liquid were also attenuated compared to nicotine alone (LeSage et al., 2016). This lack of translation across behavioral models may reflect differences in route of administration, nicotine dose, or contingency, (see LeSage et al., 2016). In addition, tolerance develops rapidly to nicotine’s aversive/anhedonic effects, but not to its reinforcement-enhancing effects, upon repeated administration (Bauco and Wise, 1994; Bozarth et al., 1998; Freitas et al., 2016). This may limit the impact of an attenuation of nicotine’s aversive/anhedonic effects in drug SA procedures, which necessarily involve repeated administration. Accordingly, differences between EC liquid and nicotine alone may be evident during the early stages of SA acquisition, prior to the development of tolerance to nicotine’s aversive/anhedonic effects. However, no such differences were observed in LeSage et al. (2016). Our ICSS findings may also reflect an attenuation of nicotine toxicity, which represents a key adverse consequence of EC use, particularly among children, and a primary concern of the FDA CTP (Kong and Krishnan-Sarin, 2017; LoVecchio and Zoph, 2015). Comparison of EC liquid to nicotine alone using well-established measures of toxicity (e.g., seizures, LD50s) is needed to evaluate this issue.

We recently reported that magnitude of ICSS threshold elevations during nAChR antagonist-precipitated withdrawal did not differ between rats receiving a chronic infusion of nicotine alone versus Aroma E-Juice, Janty, or NicVape EC liquids (Harris et al., 2017). These findings contrast with the attenuated ICSS threshold-elevating effects of acute injection these same EC liquids, and suggest a differential role for non-nicotine constituents in elevations in ICSS threshold elevations induced by chronic versus acute nicotine exposure.

It is unclear which non-nicotine constituent(s) in EC liquid mediate the attenuation of nicotine’s ICSS threshold-elevating effects. An involvement of minor alkaloids is unlikely for several reasons. First, Janty and NicVape EC liquid differed considerably in terms of their relative levels of minor alkaloids (see Table 1), yet produced a similar attenuation of nicotine’s ICSS threshold-elevating effects (compare Fig 1A and 1C). Second, while minor alkaloids act as agonists at nAChRs (Copeland et al., 1991; Hoffman and Evans, 2013; Kem et al., 1997), the similar nAChR binding affinity and functional activity of EC liquids and nicotine alone indicate that levels of minor alkaloids in EC liquids were too low to produce such effects. Third, nornicotine and anabasine can produce nicotine-like effects on ICSS thresholds when administered in isolation, but only at concentrations much higher than those in the current EC liquids (Harris et al., 2015a). Finally, minor alkaloids typically enhance nicotine’s effects when administered in combination with nicotine (Clemens et al., 2009; Desai et al., 2016), which is inconsistent with the attenuation of the effects of high-dose nicotine in EC liquids observed in this study.

The alcohol PG represents a more likely moderator of the differential effects of EC liquids and nicotine alone on ICSS. PG is a major ingredient in all three of the EC liquids that we have studied and can have reinforcing or other behavioral effects in animals (Lin et al., 1998; Singh et al., 1982). Furthermore, ethanol can attenuate nicotine aversion under some conditions (Kunin et al., 1999). The role of PG in our ICSS findings could be addressed by evaluating effects of EC liquids that do not contain PG. Studying cocktails containing high-dose nicotine combined with concentrations of PG that are similar to those in EC liquids would also be informative.

It is unlikely that differences in nicotine pharmacokinetics between nicotine alone and EC liquids contributed to their different effects on ICSS, as we previously found that nicotine alone and Aroma E-Juice EC liquid (0.1 mg/kg, i.v.) produced similar nicotine serum and brain levels (LeSage et al., 2016). Nonetheless, future studies should compare nicotine pharmacokinetics between formulations using the current dosing conditions, which involved s.c. administration and higher nicotine doses than were studied in LeSage et al. (2016). Furthermore, the current doses of EC liquid contain higher doses of non-nicotine constituents including PG, which can affect drug absorption (Yu and Kent, 1982).

The lack of differences in nAChR binding/activation between nicotine alone and the current EC liquids is consistent with our previous study using Aroma E-Juice EC liquid (LeSage et al., 2016), as well as other studies indicating that non-nicotine constituents do not affect nicotine binding or function at α4ß2 and other nAChRs (Costello et al., 2014; Harris et al., 2015b; Ponzoni et al., 2015). Nonetheless, future work should compare binding affinity/functional activity of nicotine alone and EC liquid at nAChRs containing the α5 subunit, which can affect nicotine’s acute aversive effects including elevations in ICSS thresholds (Fowler and Kenny, 2014; Fowler et al., 2011). Evaluation of the ability of these formulations to elicit other neurobiological effects implicated in nicotine aversion (see Fowler and Kenny, 2014; Sellings et al., 2008) is also an important area for future work.

The similar ICSS threshold-lowering effects of EC liquids and nicotine alone in this and our earlier study (LeSage et al., 2016) are consistent with our previous findings using smokeless tobacco extracts (Harris et al., 2015b). Together, these data indicate a primary role for nicotine in the reinforcement-enhancing effects of ECs and smokeless tobacco, at least as far as these products are concerned. However, as mentioned above, the minor alkaloids nornicotine and anabasine can lower ICSS thresholds and produce other addiction-related behavioral effects when administered alone at much higher doses than are in EC liquids or smokeless tobacco extracts (i.e., by at least 10-fold) (Bardo et al., 1999; Caine et al., 2014; Harris et al., 2015a). Levels of these and other minor alkaloids in ECs and smokeless tobacco should continue to be closely monitored by the FDA CTP to ensure that their levels are not significantly increased.

Despite their lack of effects on ICSS, both “nicotine-free” EC liquids bound to several nAChR subtypes including α4ß2 nAChRs, albeit with markedly lower affinity than nicotine-containing EC liquids. Using LC-MS/MS or GC, we detected nicotine in “nicotine-free” Janty EC liquid, which is consistent with analyses of other “nicotine-free” EC liquids; it presumably reflects contamination occurring during manufacturing (Goniewicz et al., 2015; Goniewicz et al., 2013; Han et al., 2016), and nicotine levels in this EC liquid (0.006-0.008 mg/ml, 37-49 nM) were sufficiently high to bind to and possibly even desensitize nAChRs. However, nicotine levels in NicVape were either very low (0.0008 mg/ml) or undetectable depending on the assay (GC versus LC-MS/MS), and neither EC liquid contained detectable levels of the minor alkaloids nornicotine, anabasine, or anatabine. Regardless, the lack of effects of both “nicotine-free” EC liquids on ICSS suggests that this effect is not behaviorally relevant, at least in this assay. Further work with these “nicotine-free” EC liquids, including evaluating whether addition of high doses of nicotine to these EC liquids attenuates nicotine’s aversive/anhedonic effects, is of interest.

A limitation of this study is that ICSS provided the sole measure of abuse potential. Future studies should extend our findings to other preclinical models of addiction vulnerability, particularly i.v. SA. Nonetheless, the validity, sensitivity, and utility of ICSS in the evaluation of abuse liability is widely recognized, and findings from ICSS studies are generally concordant with i.v. SA studies (Negus and Miller, 2014; Wise, 1996, 2002). Therefore, our evaluation of the effects of the current EC liquids on ICSS represents an important advance in understanding the abuse potential of ECs.

A potential concern is that non-specific (e.g., motoric) effects of high doses of nicotine may have compromised our findings. Indeed, the 1.0 mg/kg dose of nicotine alone produced a small but significant increase in response latencies in the subset of animals tested using nicotine alone and Janty EC liquid (see Fig 1B), suggesting motoric disruption (see Markou and Koob, 1992). However, ICSS thresholds in the present discrete-trial current-threshold procedure are relatively independent of response rates (Harris et al., 2010; Harrison and Markou, 2001; Markou and Koob, 1992), and elevations in ICSS thresholds and response latencies at the 1.0 mg/kg dose of nicotine alone were not correlated. Finally, ICSS latencies were not significantly increased under any other condition (see Fig 1B and 1D). It is therefore unlikely that our findings were influenced by general motoric effects of nicotine alone or EC liquids.

Together, our findings suggest that non-nicotine constituents in these EC liquids do not contribute to their reinforcement-enhancing effects, but may attenuate nicotine’s aversive/anhedonic and/or toxic effects. Comparison of EC liquids to nicotine alone in more established models of aversion (e.g., conditioned place or taste aversion), as well as evaluation of i.v. SA of these formulations under conditions in which nicotine intake is likely limited by its aversive effects (e.g., at high nicotine unit doses), is needed to better understand the relevance of our findings to nicotine aversion and consumption. Finally, evaluating the behavioral effects of inhalation of EC aerosol could also provide insights into the role of sensory factors (e.g., taste, smell) in EC abuse liability.

Supplementary Material

supplement

Highlights.

  • We tested effects of e-cigarette (EC) liquids on intracranial self-stimulation.

  • Nicotine alone and EC liquids produced similar reinforcement-enhancing effects.

  • EC liquids had attenuated aversive/anhedonic effects compared to nicotine alone.

  • “Nicotine-free” EC liquids did not affect intracranial self-stimulation.

  • Non-nicotine constituents in EC liquid may attenuate nicotine aversion.

Acknowledgments

The authors thank Danielle Burroughs, Laura Tally, Theresa Harmon, Andrew Banal, Christine Egan, Nettie Enshayan, Danielle Motz, Annika Skansberg, Mary Krueger, Zachary Haave, and Vipin Jain for their excellent technical assistance in conducting the experiments. Ki determinations and agonist/antagonist functional data for Experiments 3a and 3c were generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract # HHSN- 271-2008-00025-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA.

Footnotes

1

Supplementary material can be found by accessing the online version of this paper at http://dx.doi.org and by entering doi:…

Author Disclosures

Role of Funding Source

Funding for this study was provided by NIH/NCI grant U19- CA157345 (Hatsukami DH and Shields P, MPI; LeSage MG, PL), NIH/ NIDA grant RO3 DA042009 (Harris, AC, PI), NIDA training grant T32 DA007097 (Smethells, JR; Molitor T, PI), the Minneapolis Medical Research Foundation (MMRF) Career Development Award (LeSage, MG, Harris, AC) and the MMRF Translational Addiction Research Program (Harris, AC). These funding institutions had no role in the study design, data collection, data analysis, interpretation of the data, manuscript preparation, or decision to submit the manuscript for publication. 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.

Contributors

Mark LeSage and Andrew Harris supervised the conduct of the study and were responsible for the conception and design of the study. Peter Muelken and Jack Smethells assisted with developing specific protocols, daily conduct of the experiment, and compiling data. Irina Stepanov and Katrina Yershova conducted the alkaloid analysis. Thao Tran Olson and Kenneth Kellar conducted the additional nAChR binding assay (Experiment 3b). Andrew Harris wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.

Conflict of Interest

All authors declare that they have no conflict of interest.

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