Nicotine & Tobacco Research, Volume 15, Number 3 (March 2013) 622–632
Review
Abuse Potential of Non-Nicotine Tobacco Smoke
Components: Acetaldehyde, Nornicotine, Cotinine,
and Anabasine
Allison C. Hoffman PhD, Sarah E. Evans PhD
U.S. Food and Drug Administration, Rockville, MD
Corresponding Author: Allison C. Hoffman, PhD, U.S. Food and Drug Administration, Rockville, MD 20850, USA. Telephone:
301-796-9203; Fax: 240-276-3655; E-mail: [email protected]
Received January 3, 2012; accepted July 8, 2012
Abstract
Introduction: This review identified published animal studies evaluating the possible abuse potential of acetaldehyde, nornicotine, cotinine, and anabasine based on five commonly used paradigms. These include their effects on midbrain dopamine (DA)
levels, drug discrimination and substitution for known drugs of abuse, place conditioning, self-administration behavior, and
somatic withdrawal symptoms.
Results: Acetaldehyde had mixed effects on midbrain DA levels and drug discrimination; however, it consistently produced a
conditioned place preference and supported self-administration. The single available study on withdrawal found that cessation of
acetaldehyde administration resulted in a somatic withdrawal syndrome. Nornicotine increased DA in the midbrain, especially in
the nucleus accumbens. Although there are no data on place conditioning, it substituted for nicotine in drug discrimination testing, partially substituted for cocaine and amphetamine, and, though only a single study, supported self-administration. Anabasine
increased midbrain DA levels and that it partially substituted for nicotine in drug discrimination testing. Cotinine increased
midbrain DA levels and substituted for nicotine.
Conclusions: The existing literature suggests that acetaldehyde and nornicotine likely possess abuse potential, with anabasine
having possible abuse potential. Although some cotinine data were available, it was insufficient to draw conclusions about p­ ossible
abuse potential. Further research is needed to determine the role of minor alkaloids on tobacco dependence.
Introduction
Since the 1964 publication of the U.S. Surgeon General’s
Report (SGR) first established the link between smoking and
various disease outcomes, it has been clear that continued
use of tobacco is harmful to health. Despite known negative
health outcomes, people continue to smoke. The SGR established that “habitual use … [is] perpetuated by the pharmacological action of nicotine on the central nervous system”
(U.S. Public Health Service, 1964, p. 34). Over the last four
decades, it has been well established that nicotine is the primary drug that promotes and supports tobacco addiction
(Benowitz, 2010; Markou, 2008; Watkins, Koob, & Markou,
2000). Recent peer-reviewed studies suggest that chemical
constituents other than nicotine present in tobacco smoke
may have abuse potential. Tobacco constituents such as acetaldehyde (a product of the combustion of (poly)saccharides)
and nicotine alkaloids may possess abuse potential independent of nicotine. That is, although these chemicals may alter
nicotine’s effects, this review focuses on the abuse potential
of these chemicals in and of themselves.
Tobacco smoke contains several aldehyde compounds
(Houlgate, Dhingra, Nash, & Evans, 1989; Xie, Yin, Sun, Xie,
Zhang, & Guo, 2009), however, the form most commonly referenced in addiction literature is acetaldehyde. Acetaldehyde
can be present in tobacco smoke in concentrations as high
as half of the nicotine content itself (i.e., 400–1400 µg/cigarette for acetaldehyde and 100–3000 µg/cigarette for nicotine;
Hoffmann, 2001). The effective average amount of acetaldehyde per cigarette is 427 µg (Smith & Hansch, 2000), so a
person who smokes a pack a day is exposed to 8.5 mg of acetaldehyde. Much of the research on the abuse potential associated
with acetaldehyde comes from the ethanol literature, as acetaldehyde is also a metabolite of ethanol (Amit & Smith, 1985;
Correa et al., 2012; Deng & Deitrich, 2008). The acetaldehyde/
ethanol literature is particularly useful in studies of tobacco
abuse potential (Amit & Smith, 1985; Correa et al., 2012;
Deng & Deitrich, 2008), as well-designed studies comparing
the effects of acetaldehyde and ethanol (discussed below in the
Self-Administration Acetaldehyde section) provide information both for acetaldehyde alone as well as in comparison to a
drug of abuse (ethanol).
doi:10.1093/ntr/nts192
Advance Access publication September 18, 2012
Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco 2012.
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Nicotine & Tobacco Research, Volume 15, Number 3 (March 2013)
Tobacco smoke also contains nicotine alkaloids that are less
potent than nicotine, but are still pharmacologically active (Wu,
Ashley, & Watson, 2002). These minor nicotine alkaloids may
also play a role in tobacco abuse potential. There are five minor
nicotine alkaloids: nornicotine, cotinine, anabasine, anatabine,
and myosmine, all of which are structurally similar to nicotine and are present in tobacco or tobacco smoke (Rodgman &
Perfetti, 2009). Typical quantities of the minor alkaloids in the
smoke of one cigarette are nornicotine (27–88 pg), cotinine (9–50
pg), anabasine (3–12 pg), anatabine (4–14 pg), and myosmine (9
pg) (U.S. Public Health Service, 1988, p. 27). In some varieties
of nicotine, nornicotine concentrations exceed those of nicotine
(Schmeltz & Hoffmann, 1976). Cotinine is also a metabolite of
nicotine and a specific indicator of nicotine exposure.
A single study that administered cocktails of nicotine with
one or more of these five alkaloids (anabasine, nornicotine, anatabine, cotinine, and myosmine) administered to rats was identified (Clemens, Caille, Stinus, & Cador, 2009); however, the
individual contributions of each chemical were not evaluated.
No other research was found on anatabine or myosmine; however, published data were found for the nornicotine, cotinine, and
anabasine. Therefore, the current assessment includes nornicotine, cotinine, and anabasine, but not anatabine or myosmine.
Abuse potential is studied utilizing a variety of instruments
and models. Most commonly reported are effects on midbrain
dopamine (DA) levels, drug discrimination and substitution for
known drugs of abuse, place conditioning, self-administration
behavior, and somatic withdrawal symptoms. Each paradigm
is valuable; however, few investigators have the resources to
utilize all five in a single study. The purpose of this review is to
evaluate the literature on abuse potential of acetaldehyde, nornicotine, cotinine, and anabasine using outcomes from five of
the most common abuse potential paradigms. Given the limited
data on anatabine and myosmine, information presented will
include only the minor alkaloids nornicotine, cotinine, and anabasine. By bringing together the literature on abuse potential of
these chemical constituents of tobacco and tobacco smoke, we
hope to identify research needs in the field of tobacco addiction.
Methods
Literature Search and Inclusion Criteria
This review is based on a search of publicly available published literature (English language only) performed using the
PubMed database during September 2011. Because the stated
goal of this search is abuse potential, drug effects on the brain
were included; however, effects on peripheral tissues were not
included. Studies were limited to nonhuman studies. Both in
vitro and in vivo animal studies were included; cell culture studies were excluded. The following search terms were entered
into the PubMed database: aldehyde, acetaldehyde, nornicotine,
cotinine, nicotine alkaloid, minor nicotine alkaloid, anabasine,
anatabine, myosmine, DA, nicotine, reward, reinforcement,
self-administration, drug discrimination, and addiction.
Results
Midbrain DA
The techniques used to measure midbrain DA (e.g.,
microdialysis, electrophysiology) and activity of dopaminergic
neurons (e.g., voltammetry) have been described in detail in
previous studies (Diggory & Buckett, 1984; Glowinsky &
Iversen, 1966; Melis et al., 2006; Pidoplichko et al., 2004). The
primary outcome measurement is of DA release or activity of
dopaminergic neurons following drug administration.
Rewarding or reinforcing stimuli (e.g., psychostimulants
such as amphetamine, cocaine, nicotine) produce changes in
dopaminergic activity (as measured by release or neuronal activity/firing) in the midbrain of animals and humans (Carlezon &
Thomas, 2009; Di Chiara et al., 2004; Howell & Wilcox, 2002;
Volkow, Fowler, & Wang, 2002). The nucleus accumbens is a
focal point in the midbrain and thought to be one of the primary
regions involved in reward and reinforcement (Alcaro, Huber,
& Panksepp, 2007; Deadwyler, 2010; Markou, 2008). Nicotine
has long been known to stimulate DA release in a variety of
midbrain regions (Balfour, Wright, Benwell, & Birrell, 2000;
Benowitz, 2010; Livingstone & Wonnacott, 2009). In particular, it is well documented that nicotine increases dopaminergic neuronal firing in the ventral tegmental area (VTA), which
results in DA release from the nucleus accumbens (Grenhoff,
Aston-Jones, & Svensson, 1986; Li, Doyon, & Dani, 2011). It
also directly increases DA release in the nucleus accumbens and
the striatum (caudate/putamen) (Bassareo, Tanda, Petromilli,
Giua, & Di Chiara, 1996; Benwell, Balfour, & Khadra, 1994;
Di Chiara & Imperato, 1988; Janhunen & Ahtee, 2004; Mifsud,
Hernandez, & Hoebel, 1989). Functional effects of addictive
drugs on midbrain DA correlate with self-reported measures
of drug liking in humans (Di Chiara et al., 2004; Volkow et al.,
2002), suggesting that a significant increase in midbrain DA
may be a marker for drugs that are abused in people. Based on
these reported findings, drug effects on midbrain DA level and/
or dopaminergic neuronal activity was chosen as a first-level
indicator of possible abuse potential for the current analysis.
Acetaldehyde
Acetaldehyde stimulates dopaminergic neuronal activity in the
VTA of the brain. When administered intravenously (5–20 mg/
kg) to male Sprague-Dawley rats, acetaldehyde dose-dependently increased the firing rates, spike/burst, and burst firing of
DA-containing VTA neurons with both the 10 and 20 mg/kg
doses producing significant increases as compared baseline
responding (Foddai, Dosia, Spiga, & Diana, 2004). Similar
findings by Melis and colleagues (2007) report that male
Wistar rats treated with intragastric acetaldehyde (20 mg/kg)
had a spike in DA release in the nucleus accumbens (Melis,
Enrico, Peana, & Diana, 2007). In contrast, a single dose
(200 mg/kg, intraperitoneal [i.p.]) of acetaldehyde was reported
to decrease striatal DA content when administered systemically
to adult male Wistar rats (Padilla-de la Torre et al., 2008). Two
studies administered systemic acetaldehyde at doses of 20 or
100 mg/kg, i.p. (Ward, Colantuoni, Dahchour, Quertemont, &
De Witte, 1997) or 11, 22, or 44 mg/kg, i.p. (Wang et al., 2007)
to adult male Wistar rats. DA was reduced in both the nucleus
accumbens (Padilla-de la Torre et al., 2008). The differences
in findings may be attributable to the doses and routes used.
If acetaldehyde’s effects are similar to ethanol (for which it is
a metabolite), then like ethanol, it may have a complex biphasic effect across time and dose (Enrico et al., 2009). At low
doses, acetaldehyde has been shown to possess an important
psychomotor stimulant effects (Correa, Arizzi, Betz, Mingote,
& Salamone, 2003; Tambour, Didone, Tirelli, & Quertemont,
2006). Acetaldehyde also acts mainly as a depressive drug
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Abuse potential of non-nicotine tobacco smoke components
when it is systemically administered; however, it can act as a
stimulant when administered directly in the brain (Quertemont,
Tambour, Bernaerts, Zimatkin, & Tirelli, 2004; Tambour et al.,
2006). Thus, it is difficult to directly compare the results of
these studies, as they cross a range of doses, routes of administration, and outcome measurements.
Nornicotine
Two studies have investigated the effects of nornicotine on
midbrain DA. When brain slices of male Sprague-Dawley rat
striatum were superfused with various concentrations of nornicotine, investigators report a dose-dependent increase in
electrically evoked DA release. Although less than 1 µM concentrations of nornicotine were relatively ineffective, there was
a steep increase in release between 10 and 100 µM concentrations of nornicotine (Dwoskin, Buxton, Jewell, & Crooks,
1993; Dwoskin, Teng, Buxton, Ravard, Deo, & Crooks, 1995).
Cotinine
Cotinine has been found to stimulate DA release from midbrain neurons. When male Sprague-Dawley rat striatal brain
slices were bathed in cotinine (1–3000 µM solution), there was
a significant dose-dependent release of DA (Dwoskin, Teng,
Buxton, & Crooks, 1999). However, in an in vivo study, Sziraki
and colleagues (1999) failed to find a significant effect when
cotinine (100 or 500 µg/kg, intravenously administered) was
administered to male Sprague-Dawley rats, with no significant
change in extracellular DA levels in the nucleus accumbens as
measured using microdialysis (Sziraki et al., 1999). According
to the study authors, the dose that reached the brain was either
60 or 300 µg/kg, both of which are higher than the dose of
12.5 µg/kg that an 80-kg person would take in over a 3–6 min
of smoking a cigarette. The lack of support by Sziraki et al.
(1999) for the positive finding of Dwoskin et al. (1999) may
be explained by significant differences in methodology (e.g.,
brain slices vs. microdialysis) and a lack of comparable doses
(bath solution vs. systemic administration), which prevent
direct comparisons of outcomes.
Anabasine
A single study investigated the effect of anabasine on midbrain
DA levels. Dwoskin and colleagues (1995) reported increased
DA release in male Sprague-Dawley rats exposed to anabasine
in vitro. Rat striatal slices were preloaded with [3H] DA, allowing for quantification of evoked DA release. Though limited,
this study found that anabasine stimulated DA release. When
anabasine was added to the superfusion buffer, there was a
concentration-dependent increase in DA release between 1 and
100 µM anabasine (Dwoskin et al., 1995).
Drug Discrimination
Drug discrimination is effective in evaluating shared central
mechanisms of action, with a test drug being compared with
the training drug (Balster, 1991; Thompson & Pickens, 1971).
In this model, an animal learns to press one lever (salinepaired) when pretreated with vehicle and another lever when
pretreated with drug (drug-paired). Responding on the active
lever (the lever paired with one of the two cues) will result
the delivery of a food pellet or other nondrug reinforcer.
Responding on the inactive lever has no reinforcement
624
consequence; however, it may result in a “reset” of the active
lever such that the reinforcement schedule (e.g., fixed ratio)
returns to the original setting. The pretreatment provides an
interoceptive cue as to which lever is active. Animals will
learn to reliably discriminate between the interoceptive cues
produced by the different pretreatments and will press the
appropriate lever. When a test drug is administered before
the behavioral session, both levers are active and result in
reinforcer delivery. If the test drug shares interoceptive cues
with the training drug, that drug will “substitute for” or
“generalize to” the training drug (or training stimulus); the
animal will press the lever that has been previously paired
with that drug, so-called “drug appropriate” responding,
whereas pressing the lever associated with saline would be
“saline appropriate.” Challenge with a drug results in a %
responding on the drug-paired lever, which is compared with
the % responding produced by the training drug (Colpaert &
Rosecrans, 1978).
Test drugs that are in the same drug class (e.g., stimulant
drugs) partially or fully substitute for one another. Nicotine
produces reliable drug discrimination in a variety of animal
models, including mice, rats, and nonhuman primates (Desai,
Barber, & Terry, 2003; Goldberg, Risner, Stolerman, Reavill,
& Garcha, 1989; Jackson et al., 2010). Other stimulant drugs,
such as caffeine, cocaine, and amphetamine, partially substitute (typically between 20% and 80% responding on the drug
appropriate lever) or fully substitute (at least 80% responding on the drug appropriate lever) for nicotine and vice versa
(Justinova et al., 2009; Gatch, Flores, & Forster, 2008; Bardo,
Bevins, Klebaur, Crooks, & Dwoskin, 1997; Desai, Barber,
& Terry, 1999).
Acetaldehyde
Like nicotine and other drugs of abuse, acetaldehyde has been
demonstrated to have discriminative cue properties. Male rats
can be trained to reliably discriminate between acetaldehyde
(0.2 or 0.3 g/kg, i.p.) and saline (Redila, Aliatas, Smith, &
Amit, 2002), indicating that acetaldehyde produces interoceptive cues. Despite similar ethanol training doses (1.0 or
2.0 g/kg) and acetaldehyde challenge doses (100–300 mg/kg),
there have been mixed results. Although some studies have
failed to find any shared discriminative stimulus properties
with ethanol across a range of acetaldehyde doses (0–300 mg/
kg, i.p.; Quertemont & Grant, 2002), other studies report limited substitution for the training stimulus (Jarbe, Hiltunen, &
Swedberg, 1982). This may be due to differences in species,
as Quertemont & Grant (2002) used male Long-Evans rats,
whereas Jarbe et al. (1982) used male and female mongolian
gerbils. It may also have been impacted by differences in methodologies, as Quertemont & Grant (2002) used a cumulative
dosing procedure and tested 10 min after injection, whereas
Jarbe et al. (1982) gave discrete doses and tested 5 min after
injection (with the exception of a separate group given 300 mg/
kg acetaldehyde and tested 10-min postinjection).
Interestingly, a novel paradigm of drug discrimination has
been combined with a conditioned taste procedure, as acetaldehyde administration has been shown to attenuate several
ethanol-induced behaviors, such as a conditioned taste aversion
(Aragon, Abitbol, & Amit, 1986). That is, experience with acetaldehyde, administered systemically 3 days before the start of a
conditioned taste aversion procedure, augments the outcomes
Nicotine & Tobacco Research, Volume 15, Number 3 (March 2013)
of that procedure such that the conditioned taste aversion produced by acetaldehyde is even more robust. It was found that
ethanol produced significant substitution when administered to
animals trained using an acetaldehyde as the training drug; the
reverse association was also reported despite a range of acetaldehyde doses (0.05–0.3 g/kg) (Redila et al., 2002; Redila,
Smith, & Amit, 2000). Acetaldehyde appears to share interoceptive properties with ethanol. Because acetaldehyde is a
metabolite of alcohol, this is not surprising. Comparisons to
other drugs of abuse have not been made.
Nornicotine
Nornicotine has been found be a partial substitute for several drugs of abuse (Rosecrans, Kallman, & Glennon, 1978).
Squirrel monkeys trained to discriminate cumulative doses of
l-nicotine. (0.003–0.36 mg/kg) or d-nicotine (0.003–3.2 mg/kg)
from saline also displayed dose-related substitution (increases
in the percentage of drug-appropriate responses emitted, from
predominately saline-appropriate responses after low doses, to
predominately drug-appropriate responses at the highest doses)
when given l-nornicotine camsylate (0.03–1.94 mg/kg) indicating that the nornicotine interoceptive cue is similar to that of
nicotine (Takada, Swedberg, Goldberg, & Katz, 1989).
In male Lister hooded rats trained to discriminate between
systemically administered nicotine (0.1 mg/kg) and saline,
challenge with nornicotine (0.1–3.2 mg/kg) resulted in partial
substitution for nicotine, with animals responding on the nicotine-appropriate lever in a dose-related manner (fixed-interval
response rates first increased and then decreased, whereas
fixed-ratio response rates only decreased, with increasing
doses of nicotine). This difference in responding is typical with
interval versus ratio schedules of reinforcement (Goldberg
et al., 1989). In a similar study, male Sprague-Dawley rats were
trained to discriminate between systemically administered
nicotine (0.1 mg/kg) and saline. Challenge with nornicotine
(3 mg/kg) resulted in partial substitution for nicotine (maximum = 76% nicotine lever responding), with animals responding on the nicotine-appropriate lever in a dose-related manner
(Desai et al., 1999). In another study by the same group, adult
male Sprague-Dawley rats were trained to discriminate systemic cocaine (8.9 mg/kg) from saline. When challenged with
nornicotine (1–5.6 mg/kg), responding indicated partial generalization to the cocaine cue in a dose-related manner, with a
maximum of 44% responding on the cocaine-appropriate lever
produced by a nornicotine (Desai et al., 2003). A similar study
in adult male Sprague-Dawley rats, challenge with nornicotine (0.1–1.0 mg/kg/infusion) produced partial substitution for
amphetamine (0.0625–2.0 mg/kg) with maximal responding
of 50% (Bardo et al., 1997). Thus, like nicotine, nornicotine
appears to share interoceptive cues with a variety of stimulant
drugs of abuse.
Cotinine
Among male Lister hooded rats trained to discriminate
nicotine (0.1 mg/kg) from saline, systemic cotinine (3.2–
100 mg/kg) produced dose-dependent substitution, with the
highest dose substituting fully for nicotine (Goldberg et al.,
1989). The authors note that, in this particular study, this
may be due to a significant amount of nicotine present as an
impurity and should be interpreted with caution. That is, the
authors suggest that the dose-dependent generalization may,
in fact, be due to the presence of increasing amounts of the
training drug (nicotine), rather than cotinine per se. Although
such an impurity may have contributed to the rate-increasing
effects of cotinine, the authors discuss several reasons why
impurities alone couldn’t explain their results. Similar work
with squirrel monkeys trained to discriminate cumulative
doses of L-nicotine (0.02–2.2 μmol/kg) and D-nicotine
(0.02–19.7 μmol/kg) from saline reported some dose-related
generalized responses when given cotinine (10–100 mg/kg),
indicating partial substitution (Takada et al., 1989). It is not
surprising that cotinine shares interoceptive cues with nicotine
because they are very similar molecules.
Anabasine
In male Wistar rats trained to discriminate nicotine (0.3 mg/
kg, i.p.) from saline, treatment with anabasine (0.31 and 1 mg/
kg) resulted in almost full substitution at the highest dose,
with rats pressing the nicotine-associated lever almost 80%
of the time (Brioni, Kim, O’Neill, Williams, & Decker, 1994).
Others have found similar results, with anabasine substituting
for nicotine (the training stimulus) across a different route of
administration (subcutaneous [s.c.]), training dose of nicotine
(0.4 mg/kg, s.c.), challenge doses of anabasine (1–4 mg/kg),
and species of rat (male Sprague-Dawley; male Lister hooded)
(Pratt, Stolerman, Garcha, Giardini, & Feyerabend, 1983;
Stolerman, Garcha, Pratt, & Kumar, 1984; Romano, Goldstein,
& Jewell, 1981). Additional work with male Sprague-Dawley
rats trained to discriminate d-methamphetamine (0.3 mg/kg,
i.p.) and saline reported that treatment with anabasine (0.3–
10 mg/kg, i.p.) using a cumulative dosing procedure resulted
in a dose-related substitution. Increased responding on the
d-methamphetamine-associated lever reached a plateau at the
intermediate dose (Desai and Bergman, 2010). Like nornicotine and nicotine, anabasine shares interoceptive cues with a
multiple stimulant drugs of abuse.
Place Conditioning
Place conditioning is a paradigm that evaluates the rewarding
(“place preference”) or aversive (“place avoidance”) effects of
drugs (Tzschentke, 1998). It is used as an assessment of “liking,” which is a component of abuse potential. In this paradigm, animals are pretreated with a drug and placed in a unique
environment. On alternate days, they are pretreated with a control such as saline and subsequently placed in a second unique
environment. After a series of pairings, the untreated animal
is given a choice of environments. If the animal spends significantly more time in the environment paired with the drug,
the drug is thought to be rewarding. If the animal spends significantly less time in the environment paired with the drug,
the drug is thought to be aversive (Prus, James, & Rosecrans,
2009). Place conditioning with drugs of abuse such as nicotine,
cocaine, amphetamine, morphine, and ethanol results in preference, often following an inverted U-shaped dose-response
curve, with moderate doses producing peak preferences and
higher doses resulting in a return to baseline or even a place
aversion (Becker, Schmitz, & Grecksch, 2006; Font, Aragon,
& Miquel, 2006; Fudala, Teoh, & Iwamoto, 1985; Orsini,
Buchini, Piazza, Puglisi-Allegra, & Cabib, 2004). In this paradigm, an inverted U-shaped dose-response curve indicates that,
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Abuse potential of non-nicotine tobacco smoke components
at higher doses, the effects are aversive rather than rewarding.
Nicotine produces reliable place preference in a variety of
adult and adolescent rats (Le Foll & Goldberg, 2006; Le Foll &
Goldberg, 2009).
Acetaldehyde
Quertemont and de Witte (2001) paired acetaldehyde and
saline with two distinct olfactory cues in a place conditioning test. Five groups of male Wistar rats (0, 10, 20, 100, and
150 mg/kg, i.p.) were given eight conditioning sessions (four
saline and four acetaldehyde) before testing. The control
group was not observed to have significant changes in the
amount of time spend in the nonpreferred side after introduction of the olfactory cue, whereas the experimental groups
showed a significant place preference (p < .001). There was
an inverted-U shaped dose-response curve for the active
acetaldehyde conditions; with peak preference seen with the
20 mg/kg group and a return to baseline at the highest dose
(Quertemont & De Witte, 2001). There was no evidence of significant preference or aversion for the olfactory stimulus. Low
and moderate doses (10, 20, and 40 mg/kg) of systemically
administered acetaldehyde in male Wistar rats resulted in a
significant (p < .005) dose-dependent preference (Peana et al.,
2008). Intragastrically administered acetaldehyde (20 mg/kg)
is also reported to produce a conditioned place preference
in male Sprague-Dawley rats (Spina et al., 2010). However,
not all studies found significant place conditioning. In male
Wistar rats, doses of systemically administered acetaldehyde
(5, 20, and 200 mg/kg, i.p.) failed to produce any significant
changes in place preference, with some reported movement
toward place aversion at the highest dose (Suzuki, Shiozaki,
Moriizumi, & Misawa, 1992). Interestingly, there may be an
effect of genetics. A small study of ethanol-naïve male rats of
each line (UChA and UChB), bred for low alcohol drinking,
but not those bred for high alcohol drinking, develop a significant (p < .001) place preference following conditioning with
50 mg/kg (i.p.) acetaldehyde (Quintanilla & Tampier, 2003).
However, because the same rat strain and similar doses and
route of administration were used in both the Quertemont and
de Witte (2001) and Suzuki et al. (1992) studies, these do not
explain Suzuki’s negative results.
Nornicotine, Cotinine, and Anabasine
No published articles were found that examined the effect of
nornicotine, cotinine, or anabasine on place conditioning.
Self-Administration
Self-administration procedures allow an animal to perform
a behavior in order to receive a dose of drug (Panlilio
and Goldberg, 2007). Typically, the drug is administered
systemically via an intravenous catheter, but drugs can also be
administered through oral solutions. The role of specific brain
areas in a drug’s reinforcing properties can be investigated by
using in-dwelling cannulae, which allow local administration
of the drug to discrete brain areas. A drug that supports the
development and maintenance of self-administration behavior
is thought to have strong abuse potential. Most known drugs
that are abused by people are self-administered by animals
626
(Panlilio & Goldberg, 2007; Thomsen & Caine, 2007). Thus,
the ability of a drug to develop and maintain self-administration
behavior in animals is not only an indicator of abuse potential,
but it is considered to be the gold standard of abuse potential
indicators. Like place conditioning, it is common for drugs
of abuse, such as nicotine, to have an inverted U-shaped dose
response curve, with increased responding for low and moderate
doses and decreased levels of responding at higher doses
(Corrigall & Coen, 1989, 1991; Donny, Caggiula, Knopf, &
Brown, 1995; Goldberg, Spealman, & Goldberg, 1981; Risner
& Goldberg, 1983; Shoaib, 1997). Unlike in place conditioning,
in this paradigm, the descending limb of the dose-response
curve may indicate that the animal is titrating its blood levels
so that at higher doses, fewer administrations are needed, rather
than being due to aversive effects (Corrigall & Coen, 1989).
Nicotine supports self-administration, with stable responding
for intravenous nicotine administration occurring with 0.03 and
0.06 mg/kg/infusion dose. However, rates differ depending on a
variety of factors, including dose (as mentioned previously) and
schedule of reinforcement (e.g., fixed ratio vs. progressive ratio).
Usually, stable and robust behavior is typically obtained on fixedratio schedules up to fixed-ratio five levels of responding, with
the animal receiving nicotine every x number of lever presses (up
to the fixed-ratio five level). For progressive ratio responding, the
breakpoint (the maximum point at which the animal will work
to receive nicotine) depends on the steepness of the progression
and session length. In addition, the age, sex, strain of animals,
and secondary reinforcers can also impact performance on selfadministration (Caille, Clemens, Stinus, & Cador, 2012; Fattore,
Fadda, & Fratta, 2009; O’Dell & Khroyan, 2009).
Acetaldehyde
Several studies have found that acetaldehyde supports selfadministration behavior when administered systemically.
Male Fisher rats allowed to self-administer one of three doses
(0.088, 0.026, or 0.79 mg/kg/infusion; 3–5 rats/group) of intravenous acetaldehyde for 24 hr over 22–28 days earned double
the number of infusions as those self-administering ethanol
(Takayama & Uyeno, 1985). Although not all animals initiated
self-administration (33%–40%), the numbers were comparable
to, or better than, those who initiated ethanol self-administration (Takayama & Uyeno, 1985). Across a wide range of doses
(0.835–13.33 mg/kg/infusion) and conditions (e.g., free-feeding
vs. food restricted, different environmental conditions), intravenous acetaldehyde has supported the development and maintenance of self-administration in male Long-Evans rats (Myers,
Ng, & Singer, 1982; Myers, Ng, & Singer, 1984; Myers, Ng,
Marzuki, Myers, & Singer, 1984; Myers, Ng, Singer, Smythe,
& Duncan, 1985). In a study with a wide dose range (0.835,
1.67, 2.89, 6.66, or 13.33 mg/kg/infusion), the dose-response
curve had an inverted U-shape with male Long-Evans rats
responding produced by the 2.89 mg/kg infusion, but not lower
doses (Myers et al., 1982). Thus, it is not surprising that a very
low dose of intravenous acetaldehyde (0.016 µg/kg/inj) failed
to support self-administration in adolescent and adult male
Sprague-Dawley rats (Sershen et al., 2009), as it is well below
the behaviorally active dose identified by Myers et al. (1982).
Acetaldehyde also supports the development and maintenance of self-administration when given orally (Peana,
Muggironi, & Diana, 2010). Using a nose-poke operant
Nicotine & Tobacco Research, Volume 15, Number 3 (March 2013)
procedure with both active and inactive nose-poke holes, male
Wistar rats responded for 0.1%–3.2% acetaldehyde solution.
Responding on the active nose-poke hole followed an invertedU shaped dose-response curve, with the three moderate doses
producing significant dose-related increases in responding.
Acetaldehyde did not alter the number of inactive nose-pokes,
suggesting this is a specific behavioral effect (Peana et al.,
2010). A similar second study by the same group confirmed
that 2% acetaldehyde solution supported oral self-administration in male Wistar rats (Peana et al., 2011).
In addition to self-administering acetaldehyde via systemic
administration, animals will administer acetaldehyde directly
into the brain (Amit, Brown, & Rockman, 1977; Arizzi, Correa,
Betz, Wisniecki, & Salamone, 2003; Myers et al., 1985; Smith
& Amit, 1985). In two early studies, male Wistar rats were
placed in operant chambers equipped with levers which, when
pressed, would infuse acetaldehyde into the lateral ventricles.
At the end of 11 days in the operant chamber, and without any
experimenter-directed training, rats receiving 1%, 2%, or 5%
pressed the lever at rates above those of control animals (Amit
et al., 1977; Brown, Amit, & Rockman, 1979). Using the same
experimental procedure, a second study found that male Wistar
rats also self-administered 2% v/v acetaldehyde solution into
the cerebral ventricles at a rate significantly higher than control
animals (Brown, Amit, & Smith, 1980). Additional work supported these findings in larger groups of animals, over different
dose ranges of acetaldehyde, and varying schedules of reinforcement (Arizzi et al., 2003; Smith & Amit, 1985). Together,
these data suggest that acetaldehyde supports the development
and maintenance of self-administration behavior through central mechanisms.
Further investigation into particular brain areas has found
that the VTA may be involved in acetaldehyde self-administration (Rodd-Henricks et al., 2002). Female adult alcohol naïve
rats pressed an active lever to self-administer acetaldehyde
solution (0.13–15.6 mg/kg) directly into the VTA, with no significant change in activity on an inactive lever. Like Peana et al.
(2010), there was an inverted-U shaped dose-response curve.
The lowest concentration of 0.13 mg/kg did not significantly
affect lever-pressing behavior, whereas concentrations of
0.26–3.9 mg/kg significantly increased pressing of active lever.
Peak responding occurred at the 1.0 mg/kg concentration. The
two highest concentrations of acetaldehyde solution (7.8 and
15.6 mg/kg) did not produce any significant lever press behavior (Rodd-Henricks et al., 2002).
Nornicotine
In a study by Bardo and colleagues (1999), male SpragueDawley rats were allowed to self-administer intravenous
nornicotine. Because available behavioral data suggest a 10-fold
shift in relative potencies between nicotine and nornicotine
(Risner, Cone, Benowitz, & Jacob, 1988), a training dose of
nornicotine (0.3 mg/kg/infusion) was chosen, which is 10-fold
greater than that used to establish nicotine self-administration.
Nornicotine produced significant self-administration. The
dose-response curve followed an inverted U-shaped pattern,
with the lowest dose (0.075 mg/kg/infusion) failing to support
self-administration and the middle doses (0.15 and 0.3 mg/kg/
infusion) supporting the most self-administration behavior. The
highest dose (0.6 mg/kg/infusion) still produced significant
self-administration, but the mean number of responses was
slightly lower than those seen with 0.3 mg/kg/infusion. When
total drug intake was measured, there was a dose-dependent
increase (Bardo, Green, Crooks, & Dwoskin, 1999).
Cotinine and Anabasine
No published articles were found that examined the effect of
cotinine or anabasine on self-administration behavior.
Withdrawal
Withdrawal is experienced upon discontinuation of use, typically
undesirable, and is thought to be a manifestation of dependence. In humans, both psychological (e.g., craving, anxiety,
stress, anhedonia) and physical withdrawal (e.g., reduced concentration, increased appetite) of nicotine can be assessed (De
Biasi & Dani, 2011; Benowitz, 2010). In animals, studies are
limited to behavioral or physical manifestations of withdrawal
from nicotine and include anxiety-like behavior, teeth-chattering, chewing, gasps, palpebral ptosis, tremors, shakes, yawns,
scratching, and/or jumping (Benowitz, 2010; De Biasi & Dani,
2011; Malin & Goyarzu, 2009). Although other methods used
to evaluate withdrawal, such as intracranial self-stimulation
(Bauzo & Bruijnzeel, 2012; Bruijnzeel, Zislis, Wilson, & Gold,
2007; Johnson, Hollander, & Kenny, 2008; Stoker, Semenova, &
Markou, 2008), to date these have not been used to assess withdrawal from acetaldehyde, nornicotine, cotinine, or anabasine.
Acetaldehyde
A study by Ortiz and colleagues (1974) found that male mice
(T/O strain) exposed to acetaldehyde vapor for up to 10 days
display withdrawal upon cessation of exposure. Behavioral
and physiological symptoms included ataxia, tremor, piloerection, tail lift, and convulsions on handling. These symptoms
typically subsided after 2 hr. Caution should be taken when
interpreting these results, as the doses of inhaled acetaldehyde
were, according to the authors, extremely high (750 µg liter−1
to 4 mg liter−1) over the course of 10 days, and exposure was
not well quantified. Concentrations were only given approximately because they were very close to toxic levels and often
had to be adjusted to prevent large numbers of deaths in some
groups. About 5%–10% of treated mice died when exposure
extended beyond 5 days, and 20% died when exposure lasted
10 days (Ortiz, Griffiths, & Littleton, 1974).
Nornicotine, Cotinine, and Anabasine
No published articles were found that examined the effect of
nornicotine, cotinine, or anabasine on withdrawal.
Conclusions
Tobacco dependence is a complicated phenomenon. Broadening
our thinking beyond nicotine may aid in our understanding and
allow for minimization of dependence following initiation.
Based on this review, both acetaldehyde and nornicotine likely
have independent abuse potential, with some suggestion of anabasine having abuse potential. Too little is known about cotinine
to infer possible abuse potential.
627
Abuse potential of non-nicotine tobacco smoke components
Acetaldehyde has abuse potential because it induces conditioned place preference (Quertemont & De Witte, 2001),
is self-administered within the VTA (Rodd-Henricks et al.,
2002), and enhances the activity of VTA DA neurons (Foddai
et al., 2004). Although acetaldehyde effects on midbrain DA
levels are inconsistent, the majority of work to date suggests
DA levels are increased in response to acetaldehyde exposure.
Nornicotine has abuse potential because it increases DA in
the midbrain, especially in the nucleus accumbens. (Dwoskin
et al., 1993; Dwoskin et al., 1995), substitutes for nicotine in
drug discrimination testing (Desai et al., 1999; Goldberg et al.,
1989; Takada et al., 1989) and partially substitutes for cocaine
and amphetamine (Bardo et al., 1997). Furthermore, though
limited to a single study (Bardo et al., 1999), self-administration is the strongest indicator of abuse potential.
There is some evidence that anabasine has abuse potential.
The limited evidence presented suggests that it increases midbrain DA levels (Dwoskin et al., 1999) and that it fully substitutes for nicotine (Brioni et al., 1994) and partially substitutes
for d-methamphetamine (Desai & Bergman, 2010) in drug discrimination testing.
Of the chemicals reviewed, we identified only two studies of
abuse potential and cotinine (Dwoskin et al.,1999; Sziraki et al.,
1999). Although there are many studies investigating the abuse
potential of nicotine (Benowitz, 2010; Markou, 2008; Watkins,
Koob, & Markou, 2000), only few studies evaluate its downstream metabolite cotinine as a separate chemical of interest.
This is an identified area of future tobacco dependence research.
In addition to independent abuse potential, there is limited
evidence that acetaldehyde and the five nicotine alkaloids (anabasine, nornicotine, anatabine, cotinine, and myosmine) may
augment nicotine’s abuse potential. Animals self-administered
a mixture of nicotine plus anabasine, nornicotine, anatabine,
cotinine, and myosmine at significantly higher rates as compared with rats receiving nicotine alone (Clemens et al., 2009).
Although not tested separately, with no assessment of the individual contributions of components, this finding that the minor
nicotine alkaloids plus nicotine were more reinforcing than
nicotine alone suggests an augmentation of nicotine’s abuse
potential. Acetaldehyde plus nicotine has also been shown to
support self-administration behavior at doses that, by themselves, do not (Belluzzi, Wang, & Leslie, 2005).
An important limitation to this review is the absence of
data from human studies. Although animal studies can give
indications of abuse potential, human laboratory studies are
needed to truly ascertain the effect of each of these chemicals
on tobacco addiction. Alone, animal studies merely serve as a
model of human abuse potential. Additional research is needed
to investigate how these chemicals affect progress from initiation to dependence, something that can be investigated using
both animal and human studies.
Another limitation is that this review does not include
assessment of the relationships between the doses that tobacco
users might be exposed to and dependence liability. Rather, the
focus is on the individual chemicals themselves. In some cases,
the doses administered to laboratory animals far exceed that
which pack-a-day smokers are likely exposed. In others, there
appears to be a fair amount of overlap (e.g., acetaldehyde).
Although the route of administration is different for smokers,
the fast-onset of intravenous administrations has been used
as an alternative to pulmonary delivery, with peak delivery
628
to the brain within 30 s (Rose, Behm, Westman, & Coleman,
1999). In addition to differences with individual dosing (doses
and routes) in humans as compared with animal studies, it is
unknown how the short-term doses used in animal experiments
relate to very long-term exposure to these chemicals, such as
that experienced by smokers (numerous exposures over several
years or more). Dose-appropriate human laboratory studies
would contribute to understanding the abuse potential of acetaldehyde, nornicotine, anabasine, and cotinine in a real-world
setting.
In conclusion, current data suggest that acetaldehyde and
nornicotine likely possess abuse potential independent from
nicotine. Work done till date on anabasine is suggestive only;
more evidence is needed. Evidence supporting any claim that
cotinine has abuse potential is weak due to the very limited data
available. However, this review finds support for the hypothesis
that there are non-nicotine chemicals in tobacco and tobacco
smoke, such as acetaldehyde and nornicotine that may have
abuse potential. The role of acetaldehyde and the minor alkaloids on tobacco dependence needs to be studied further, and
future studies should include analysis of the additional constituents when the study design allows.
Declaration of Interests
This article reflects the views of the authors and should not be
construed to represent FDA’s views or policies. There were no
external funding sources.
Acknowledgments
The authors thank Dana van Bemmel for editorial help and
insight.
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