other than nicotine that rnay contribute to behavioral effects of
cigarette smoking.

The toxicity of nicotine is discussed in detail in Appendix B.

Nicotine and Other Alkaloids in Various Tobacco Products

Nicotine is a tertiary amine composed of a pyridine and a
pgrrolidine ring (Figure 1). Nicotine may exist in two different three-
dimensionally structured shapes,  called stereoisomers. Tobacco
contains only (S)-nicotine (also called l-nicotine), which is the most
pharmacoloqicaliy active form. Tobacco smoke also contains the less
potent (Rj-nicotine (also called d-nicotine) in quantities up to 10
percent of the total nicotine present (Pool, Godin, Crooks 1985).
Presumably some racemization occurs during the combustion pro-
cess.

The nicotine yield of cigarettes, as determined by standardized
smoking machine tests, is available for most brands. However, the
amount of nicotine in cigarettes or other tobacco products is not
specified by manufacturers. Because tobacco is a plant product, there
are differences in the amount of nicotine among and within different
types and strains of tobacco, including variations in different parts of
the plant, as well as differences related to growing conditions. Table
1 shows concentrations of nicotine and other alkaloids in several
different tobacco leaves used in making commercial tobacco prod-
ucts. Witliin a tobacco plant, leaves harvested from higher stalk
positions have higher concentrations of nicotine than from lower
stalk positions; ribs and stems of the leaves have the least (Rath-
kamp, Tso, Hoffmann 19731. Combining different varieties of tobacco
and different parts of the plant is a way to change the nicotine
concentration of commercial tobacco.

In a study of amounts of nicotine in the tobacco of 15 American
cigarette brands of differing machine-determined yields (Benowitz,
Hall et al. 19831, tobacco contained on average 1.5 percent nicotine
by weight. Nicotine yield of the cigarettes, as defined by Federal
Trade Commission smoking machine tests, was correlated inversely
with nicotine concentrations in the tobacco. Thus. tobacco of lower-
yield cigarettes tended to have higher concentrations of nicotine
than did tobacco of higher-yield cigarettes. However, lower-yield
cigarettes also contained less tobacco per cigarette, so the total
amount of nicotine contained per cigarette, averaging 8.4 mg, was
similar in different brands. Thus, low-yield cigarettes are low yield
not because of lower concentrations of nicotine in the tobacco, but
because they contain less tobacco and have characteristics which
remove tar and nicotine by filtration or dilution of smoke with air.
Concentrations of nicotine in commercial tobacco products are
summarized in Table 2.

26


NICOTINE

NORNICOTINE

N'-NITROSONORNICOTINE

NICOTINE N-OXIDE
(OXYNlCOTINEl

ANATAGINE        ANABASINE

N'-METHYLANATABINE   N`-METHYLANABASINE

NIMTYRINE       NORNICOTYRINE

COTININE       6'.OXOANABASINE

ANABASEINE

2.3'.DIPYPIDYL

METANICOTINE

PSEUDCOXYNICOTINE

FIGURE L-Chemical structures of nicotine and minor
        tobacco alkaloids
sOL'K(`E IRIP IY*:i

Although the major alkaloid in t.obacco is nicotine, there are other
alkaloids in tobacco which may be of pharmacologic importance.
These include nornicotine, anabasine, myosmine, nicotyrine, and
anatabine (Figure 1). These substances make up 8 to 12 percent of
the total alkaloid content of tobacco products (Table 1) (Piade and
Hoffmann 1980). In some varieties of tobacco, nornicotine concentra-
tions exceed those of nicotine (Schmeltz and Hoffmann 1977).

Typical quantities of the minor alkaloids in the smoke of one
cigarette are: nornicotine (27 to 88 pg), cotinine (9 to 50 pg),
anabasine (3 to 12 pgl, anatabine (4 to 14 pg). myosmine (9 pg), and
2,3' dipyridyl (7 to 27 pg). N'-methylanabasine, nicotyrine, nornicoty-
rine, and nicotine-N'-oxide have also been identified in cigarette
smoke (Schmeltz and Hoffmann 1977). Puffing characteristics,
especially puff frequency, influence the delivery of the component
alkaloids (Bush, Griinwald, Davis 1972).

27


TABLE l.-Alkaloid content of various tobaccos (mg/kg,
      dry basis)

Dar-k commercial tobacco

Anstabme              360       3x0       570       600

Anabasine

140       150        99       150

cotlnlne                195       140        90        40

M\oam,ne               45        50        60        30

2.3 -Dlpwld~l             1w       110        30        10

TABLE 2.-Nicotine content of various tobacco products

Nornicotine and anabasine have pharmacologic activity qualita-
tively similar to that of nicotine, with potencies of 20 to 75 percent
compared with that of nicotine, depending on the test system and the
animal (Clark, Rand, Vanov 1965). In addition to direct activity,
some of the minor alkaloids may influence the effects of nicotine. For
example, nicotyrine inhibits the metabolism of nicotine in animals
(Stalhandske and Slanina 1982).

The pharmacology of the minor tobacco alkaloids is discussed in
more detail in the last section of this Chapter.

28


Pharmacokinetics and Metabolism of Nicotine

Absorption of Nicotine

Nicotine is distilled from burning tobacco and is carried proximal-
ly on tar droplets (mass median diameter 0.3 to 0.5 urn) and probably
also in the vapor phase (Eudy et al. 19851, which are inhaled.
Absorption of nicotine across biological membranes depends on pH
(Armitage and Turner 1970; Schievelbein et al. 1973). Nicotine is a
weak base with a pKa (index of ionic dissociation) of 8.0 (aqueous
solution, 25oC). This means that at pH 8.0, 50 percent of nicotine is
ionized and 50 percent is nonionized. In its ionized state, such as in
acidic environments, nicotine does not rapidly cross membranes.

The pH of tobacco smoke is important in determining absorption
of nicotine from different sites within the body. The pH of individual
puffs of cigarettes made of flue-cured tobacco, the predominant
tobacco in most American cigarettes, is acidic and decreases progres-
sively with sequential puffs from pH 6.0 to 5.5 (Brunnemann and
Hoffmann 1974). At these pHs, the nicotine is almost completely
ionized. As a consequence, there is little buccal absorption of nicotine
from cigarette smoke, even when it is held in the mouth (Gori,
Benowitz, Lynch 1986). The smoke from air-cured tobaccos, the
predominant tobacco in pipes, cigars, and in a few European
cigarettes, is alkaline with progressive puffs increasing its pH from
6.5 to 7.5 or higher (Brunneman and Hoffmann 1974). At alkaline
pH, nicotine is largely nonionized and readily crosses membranes.
Nicotine from products delivering smoke of alkaline pH is well
absorbed through the mouth (Armitage et al. 1978; Russell, Raw,
Jarvis 1980).

When tobacco smoke reaches the small airways and alveoli of the
lung, the nicotine is rapidly absorbed. The rapid absorption of
nicotine from cigarette smoke through the lung occurs because of the
huge surface area of the alveoli and small airways and because of
dissolution of nicotine at physiological pH (approximately 7.4), which
facilitates transfer across cell membranes. Concentrations of nic-
otine in blood rise quickly during cigarette smoking and peak at its
completion (Figure 2). Armitage and coworkers (19751, measuring
exhalation of radiolabeled nicotine, found that four cigarette smok-
ers absorbed 82 to 92 percent of the nicotine in mainstream smoke,
another smoker presumed to be a noninhaler absorbed 29 percent,
and three nonsmokers (who were instructed to smoke as deeply as
possible) absorbed 30 to 66 percent.

Chewing tobacco, snuff, and nicotine polacrilex gum are of
alkaline pH as a result of tobacco selection and/or buffering with
additives by the manufacturer. The alkaline pH facilitates absorp-
tion of nicotine through mucous membranes. The rate of nicotine
absorption from smokeless tobacco depends on the product and the

29


M Cigarettes
A-----A Oral snuff
O--Q Chewing tobacco
F--O Nicotine gum
       N=lo

-10  0       30      60      90      120

            Minutes

FIGURE 2.-Blood nicotine concentrations during and after
      smoking cigarettes (1 l/3 cigarettes), using oral
      snuff (2.5 g), using chewing tobacco (average,
       7.9 g), and chewing nicotine gum (two 2-mg
       pieces)
`0, `I<(`?: Ht.ni.x!:,  ,`<?,rii,.r i'l :i, 1""

route of administration. With fine-ground nasal snuff, blood levels of
nicotine rise almost as fast as those observed after cigarette smoking

30


(Russell et al. 1981). The rate of nicotine absorption with the use of
oral snuff and chewing tobacco is more gradual. PVicotine is poorly
absorbed from the stomach due to the acidity of gastric fluid (Travel1
1960), but is well absorbed in the small intestine (Jenner, Gorrod,
Beckett 19731, which has a more alkaline pH and a large surface
area. Bioavailability of nicotine from the gastrointestinal tract (that
is, swallowed nicotine) is incomplete because of presystemic (first
pass) metabolism. whereby? after absorption into the portal venous
circulation, nicotine is metabolized by the liver before it reaches the
systemic venous circulation. This is in contrast to nicotine absorbed
through the lungs or oral,`nasal mucosa, which reaches the systemic
circulation without first passing through the liver. Nicotine base can
be absorbed through the skin, and there have been cases of poisoning
after skin contact with pesticides containing nicotine ~Faulkner
1933; Benowitz, Lake et al. 1987; Saxena and Scheman 1985).
Likewise, there is evidence of cutaneous absorption of and toxicity
from nicotine in tobacco field workers (Gehlbach et al. 19751.

Because of the complexity of cigarette smoking processes and use
of smokeless tobacco products, the dose of nicotine cannot be
predicted from the nicotine content of the tobacco or its absorption
characteristics. To determine the dose, one needs to measure blood
levels and know how fast the individual eliminates nicotine. This
topic, estimation of systemic doses of nicotine consumed from various
tobacco products, will be considered in a later section after discussion
of relevant pharmacokinetic issues.

Distribution of Nicotine in Body Tissues

After absorption into the blood, which is at pH 7.4, about 69
percent of the nicotine is ionized and 31 percent nonionized. Binding
to plasma proteins is less than 5 percent (Benowitz, Jacob et al.
1982). The drug is distributed extensively to body tissues with a
steady state volume of distribution averaging 180 liters (2.6 times
body weight (in kilograms)) (Table 3). This means that when nicotine
concentrations have fully equilibrated, the amount of nicotine in the
body tissues is 2.6 times the amount predicted by the product of
blood concentration and body weight. The pattern of tissue uptake
cannot be studied in humans, but it has been examined in tissues of
rabbits by measuring concentrations of nicotine in various tissues
after infusion of nicotine to steady state (Table 41. Spleen, liver,
lungs, and brain have high affinity for nicotine, whereas the affinity
of adipose tissue is relatively low.

After rapid intravenous (i.v.1 injection, concentrations of nicotine
decline rapidly because of tissue uptake of the drug. Shortly after i.v.
injection, concentrations in arterial blood. lung, and brain are high,
while concentrations in tissues such as muscle and adipose (major
storage tissues at steady state) are low. The consequence of this

31


TABLE 3.-Human pharmacokinetics of nicotine and
        cotinine

Nicotine           Cotinine

\`olumr of dknbutmn          180 L             88 L

1.300 mL/min


200 mL!min
(acid urinel

7`2 mL/min


12 mL/min

.sonrerx~l Clt31.3nCC           1,100 mlimin         60 mL/min

TABLE 4.-Steady state distribution of nicotine

Tissue to blood ratio

Blood

10

distribution pattern is that uptake into the brain is rapid, occurring
within 1 or 2 min, and blood levels fall because of peripheral tissue
uptake for 20 or 30 min after administration. Thereafter, blood
concentrations decline more slowly, as determined by rates of
elimination and rates of distribution out of storage tissues.

Rapid nicotine uptake into the brain has been demonstrated in
animal studies. Oldendorf (1974) showed a high degree of nicotine
uptake from blood in the first pass through the brains of rats.
Schmiterlow and colleagues (1967) showed by autoradiographic
techniques that high levels of nicotine were present in the brain 5
min after i.v. injections in mice and that most nicotine had been


cleared from the brain by 30 min. Stalhandske (1970) showed that
intravenously injected "C-nicotine is immediately taken up in the
brains of mice, reaching a maximum concentration within 1 min
after injection. Similar findings based on positron emission tomogra-
phy of the brain were seen after injection of "C-nicotine in monkeys
(Ma&re et al. 1976).

Nicotine inhaled in tobacco smoke enters the blood almost as
rapidly as after rapid i.v. injection except that the entry point into
the circulation is pulmonary rather than systemic venous. Because
of delivery into the lung, peak nicotine levels may be higher and lag
time between smoking and entry into the brain shorter than after
i.v. injection. After smoking, the action of nicotine on the brain is
expected to occur quickly. Rapid onset of effects after a puff is
believed to provide optimal reinforcement for the development of
drug dependence. The effect of nicotine deciines as it is distributed to
other tissues. The distribution half-life, which describes the move-
ment of nicotine from the blood and other rapidly perfused tissues,
such as the brain, to other body tissues, is about 9 min (Feyerabend
et al. 1985). Distribution kinetics, rather than elimination kinetics
(half-life, about 2 hr), determine the time course of central nervous
system (CNS) actions of nicotine after smoking a single cigarette.

Nicotine is secreted into saliva (Russell and Feyerabend 1978).
Passage of saliva containing nicotine into the stomach, combined
with the trapping of nicotine in the acidic gastric fluid and
reabsorption from the small bowel, provides a potential route for
enteric nicotine recirculation. This recirculation may account for
some of the oscillations in the terminal decline phase of nicotine
blood levels after iv. nicotine infusion or cessation of smoking
(Russell 1976).

Nicotine freely crosses the placenta and has been found in
amniotic fluid and the umbilical cord blood of neonates (Hibberd,
O'Connor, Gorrod 1978; Luck et al. 1982; Van Vunakis, Langone,
Milunsky 1974). Nicotine is found in breast milk and the breast fluid
of nonlactating women (Petrakis et al. 1978; Hill and Wynder 1979)
and in cervical mucous secretions (Sasson et al. 1985). Nicotine is
also found in the freshly shampooed hair of smokers and of
nonsmokers environmentally exposed to tobacco smoke (Haley and
Hoffmann 1985).

Elimination of Nicotine

Nicot.ine is extensively metabolized, primarily in the liver, but also
to a small extent in the lung (Turner et al. 1975). Renal excretion of
unchanged nicotine depends on urinary pH and urine flow, and may
range from 2 to 35 percent, but typically accounts for 5 to 10 percent
of total elimination (Benowitz, Kuyt et al. 1983; Rosenberg et al.
1980).

33


fi --p- Q%j
  NICOTINE     L 5'4iYDROXYNICOTINE       NICOTINE IMINIUM
1 \,                       ION

FLAVOPROTEIN

ALDEHYDE
OXIDASE

URINARY
EXCRETION

FIGURE 3.-Major pathways of nicotine metabolism

Pathlcays of ,Vicotine Metabolism

The primary metabolites of nicotine are cotinine and nicotine-N'-
oxide (Figure 3). Cotinine is formed in the liver in a two-step process,
the first of which involves oxidation of position 5 of the pyrrolidine
ring in a cytochrome P-450-mediated process to nicotine-A' l5 I-imini-
urn ion (Peterson, Trevor, Castagnoli 19871. In the second step the
iminium ion is metabolized by a cytoplasmic aldehyde oxidase to
cotinine (Hibberd and Gorrod 1983).

Cotinine itself is also extensively metabolized, with only about 17
percent excreted unchanged in the urine (Benowitz. Kuyt et al.
1983). Several metabolites of cotinine have been reported, including
trans-3'-hydroxycotinine (McKennis, Turnbull et al. 19631, 5'-hydrox-
ycotinine (Bowman and McKennis 19621, cotinine-N-oxide (Shulgin
et al. 19871, and cotinine methonium ion (McKennis, Turnbull,
Bowman 1963) (see Figure 4). Little is known about the quantitative
importance of these metabolites. Trans-3'-hydroxycotinine appears
to be a major metabolite (Jacob, Benowitz, Shulgin 1988; Neurath et
al. 1987), with urinary concentrations exceeding cotinine concentra-
tions by twofold to threefold. Cotinine N-oxide is a minor metabolite
in humans, accounting for approximately 3 percent of ingested
nicotine (Shulgin et al. 1987). Subsequent oxidative degradation of
the pyrrolidine ring gives rise to 3-pyridylacetic acid. This compound
has been identified in human urine (McKennis, Schwartz, Bowman
1964), but no quantitative data are available.

34


NICOTINE

COTNNE

TRANS - J' HYDROXYCOTININE       COTININE-N-OXIDE

NICOTINE-N-OXIDE

NORNICOTINE

5'. HYDROXYCOTININE      r. 13. PYR,DYL, -`d-0X0-to-METHYLBUTYRAMIDE

NICOTINE ISOMETHONIUM ION

CCTININE METHONIUM ION

FIGURE 4.-Structures of nicotine and its major
       metabolites

SOURCE P Jacob 111 tw,th perm,simnl

35


Nicotine-l'-N-oxide is quantitatively a minor metabolite of nic-
otine. Oxidation of the nitrogen atom of the pyrrolidine ring depends
on a microsomal flavoprotein syst.em and produces a mixture of the
two diasterisomers, l'-(R)-2'-(S)-&- and l'-(S)-2'-(S)-trurts-nicotine-l'-
N/-oxide (Booth and Boyland 1970). After i.v. injection, 100 percent of
nicotine-N'-oxide is excreted unchanged in the urine, indicating no
further metabolism (Beckett, Gorrod, Jenner 1971a). However, after
oral administration only 30 percent is recovered in the urine as
nicotine-N'-oxide; the remainder is recovered as nicotine and its
metabolites. To evaluate the possibility of reduction of nicotine-N'-
oxide in the gastrointestinal tract, rectal administration of nicotine-
N'-oxide was performed for experimental purposes. Less than 10
percent was recovered in the urine as nicotine-N'-oxide (Beckett,
Gorrod, Jenner 1970). These findings indicate reduction of nicotine-
N'-oxide back to nicotine within the human gastrointestinal tract,
believed to be a consequence of bacterial action.

Experiments in rats indicate that significant amounts of nicotine-
N'-oxide are converted to nicotine both in vitro and in vivo (Dajani,
Gorrod, Beckett 1975a,b). Nicotine and cotinine have been measured
in the blood of rats administered nicotine-N,N'-dioxide and nicotine-
N'-oxide in drinking water (Sepkovic et al. 1984, 1986). Thus, while
reduction of nicotine-N'-oxide to nicotine appears to be bacterial in
humans, it may be mediated by endogenous enzymes in other
species.

Quantitative aspects of the conversion of nicotine to its metabo-
lites have not been well defined. Studies of cotinine excretion in
urine collected for 24 hr after i.v. nicotine injection indicate less than
10 percent of nicotine is excreted as cotinine in nonsmokers
compared with an average of 25 percent in smokers (Beckett, Gorrod,
Jenner 1971b). Another study, comparing 24-hr urinary excretion of
cotinine with nicotine content of cigarette butts after smoking,
indicated 46 percent recovery as cotinine (Schievelbein 1982).
However, both of these studies underestimate the conversion of
nicotine to cotinine because the urine collection period was too short.
In cigarette smokers, cotinine has a half-life averaging 18 to 20 hr
(Benowitz, Kuyt et al. 1983), so that in 24 hr only a little more than
half of cotinine is recovered. Urine collection for at least 72 hr is
necessary to recover more than 90 percent of cotinine in most
subjects. In addition, since only 17 percent of cotinine is excreted
unchanged (Benowitz, Kuyt et al. 1983), urinary recovery analysis
underestimates the cotinine generation rate.

At steady stat.e, the rate of metabolite excretion reflects the rate at
which the metabolites are generated. After i.v. dosing, 100 percent of
nicotine-N'-oxide but only 17 percent of cotinine are excreted
unchanged in the urine. Based on a ratio of urinary cotinine to
nicotine-N'-oxide of 2.9 and based on excretion of that 17 percent of

36


cotinine and 100 percent of nicotine-N'-oxide unchanged in the
urine, the relative generation rate of cotinine compared with that of
nicotine-N'-oxide is calculated to be 17 to 1 (Benowitz 1986b).
Because 4 percent of nicotine is excreted as nicotine-N'-oxide (Jacob
et al. 1986; Beckett, Gorrod, Jenner 1971a), about 70 percent of
nicotine appears to be converted to cotinine. Quantitative data on
other metabolites that may have pharmacologic activity, such as
nicotine isomethonium ion and nornicotine, are not available.

Rate of Nicotine Metabolism

The rate of nicotine metabolism can be determined by measuring
blood levels after administration of a known nicotine dose. In one
study, cigarette smokers were given iv. infusions of nicotine for 30 to
60 min, and total and renal clearances were computed (Benowitz,
Jacob et al. 1982). Total clearance ia term which describes the
capacity to eliminate a drug) averaged 1,300 ml/mix-i. Nonrenal
clearance averaged 1,100 mL/min (Table 31, which represents about
70 percent of liver blood flow. Because nicotine is metabolized
mainly by the liver (data in animals indicate only a small degree of
metabolism by the lung) (Turner, Sillett, McNicol 19771, this means
that about 70 percent of the drug is extracted from the blood in each
pass through the liver. On the average, 85 or 90 percent of nicotine is
metabolized by the liver.

Renal Excretion

Nicotine is excreted by glomerular filtration and tubular secretion
within the kidney. Depending on urinary pH and urine flow rate,
variable amounts of nicotine are reabsorbed by the kidney tubules.
In acidic urine, where nicotine is mostly ionized and tubular
reabsorption is minimized, renal clearance of nicotine may be as
high as 600 mL/min (urinary pH 4.4) (Benowitz, Kuyt et al. 1983;
Rosenberg et al. 1980). In alkaline urine, a larger fraction of nicotine
is not ionized. Tubular reabsorption of nonionized nicotine results in
lower rate of excretion and reduced renal clearances as low as 17
mL/min (urine pH 7.0). When urine pH is uncontrolled, averaging
5.8, renal clearance averages about 100 mL/min, accounting for the
elimination of 10 to 15 percent of the daily nicotine intake.

Nicotine and Cotinine Blood Levels During Tobacco Use
Nicotine Levels

Plasma nicotine concentrations (or concentrations in blood, which
are similar) sampled in the afternoon in smokers generally range
from 10 to 50 ng/mL. The increment in blood nicotine concentration
after smoking a single cigarette ranges from 5 to 30 ng/mL,
depending on how the cigarette is smoked (Armitage et al. 1975;

37


Herning et al. 1983; Isaac and Rand 1972). Peak blood levels of
nicotine are snnilar, although the rate of nicotine increase is slower
for cigar smokers and snuff and chewing tobacco users compared
with that for cigarette smokers (Armitage et al. 1978; Turner, Sillett,
McNicol 1977: Gritz et al. 1981; Russell, Raw, Jarvis 1980; Russell et
al. 1981) (Figure 2). Pipe smokers, particularly those who have
previously smoked cigarettes and who inhale, may have blood and
urine levels of nicotine as high as those of cigarette smokers
(McCusker, McNabb, Bone 1982; Turner, Sillett, McNicol 1977; Wald
et al. 1984).

The earliest published studies of nicotine elimination kinetics
reported half-lives of 20 to 40 min (Armitage et al. 1975; Isaac and
Rand 1972). In those studies, drug blood levels were followed only for
30 to 60 min, which is not long enough to determine the elimination
half-life. Thus, half-lives were based on blood levels which included
the distribution phase. When blood levels are followed for several
hours after the end of nicotine infusion, a log-linear decline of blood
levels with a half-life of about 2 hr is observed (Benowitz, Jacob et al.
1982; Feyerabend, Ings, Russell 1985).

The half-life of a drug is useful in predicting its accumulation rate
in the body with repetitive doses and the time course of its decline
after cessation of dosing. Assuming a half-life of 2 hr, one would
predict nicotine to accumulate over 6 to 8 hr (3 to 4 half-lives) of
regular smoking and persist at significant nicotine levels for 6 to 8 hr
after cessation of smoking. If a smoker smokes until bedtime,
significant nicotine levels should persist all night. Studies of blood
levels in regular cigarette smokers confirm these predictions (Figure
5) (Russell and Feyerabend 1978; Benowitz, Kuyt, Jacob 1982). Peaks
and troughs follow the use of each cigarette, but as the day
progresses, trough levels rise and the influence of peak levels
becomes less important. Thus, nicotine is not a drug to which people
are exposed intermittently and that is eliminated rapidly from the
body. To the contrary, smoking represents a multiple dosing
situation with considerable accumulation during smoking and with
persistent levels for 24 hr of each day.

Cotinine Leljels

Cotinine levels are of particular interest as qualitative markers of
tobacco use and quantitative indicators of nicotine intake. Cotinine
is present in the blood of smokers in much higher concentrations
than nicotine. Cotinine blood levels average about 250 to 300 ng/mL
in groups of cigarette smokers (Benowitz, Hall et al. 1983; Haley,
Axelrad, Tilton 1983; Langone, Van Vunakis, Hill 1975; Zeidenberg
et al. 1977). After stopping smoking, levels decline with a half-life
averaging 18 to 20 hr (range 11 to 37 hr). But because of the long
half-life, there is much less fluctuation in cotinine concentrations

38


  7         --o-- Usual brand
        T
-      T          -A-  Low nicotine
           High nicotine

N = 10

l2 r

N=7

2-l      I      I      I      I            I
0800 ,200 16.X 2016 24'" 0~20 oa3C

             Clock time

FIGURE S-Blood nicotine and carboxyhemoglobin
      concentrations in subjects smoking high
      nicotine (`2.5 mg) and low nicotine (0.4 mg)
      Kentucky reference cigarettes and their usual
      brand (average nicotine yield, 1.2 mg) of
       cigarettes
VOTE Sub,rcts .n,flhrd rin LI i,\id si.hddr n, , ~,c..w,,e I)\, ,> /h`,,' !,.`A' /PI, 1 . :11,1 1, 10 I, SW ,I 11, ,111 :! 1$11,,1
o, 13, c,~z,w,~P, da>. blood ..1,11~1~?, nrrr <1?llrrlK ,.1-l !W11111~ !I ? 11?\1 .l.!>l~l:ilA x IL,i'i'!`.'
SOL'K(`E Rrnm\,t/ ir;~i( .l,ic ,h l'lhl

39


throughout the day than in nicotine concentrations. As expected,
there is a gradual increase in cotinine levels during the day, peaking
at the end of smoking and persisting in high concentrations
overnight.

Intake of Nicotine

Cigarette Smoking

Nicotine intake from single cigarettes has been measured by
spiking cigarettes with "C-labeled nicotine (Armitage et al. 1975).
That study of eight subjects, each smoking a single filter-tipped
cigarette, indicated an intake range of 0.36 to 2.62 mg. Intake was
higher in smokers than in nonsmokers. Intake of nicotine from
smoking a single cigarette or with daily cigarette smoking has been
estimated by methods similar to those used in drug bioavailability
studies (Benowitz and Jacob 1984; Feyerabend, Ings, Russell 1985).
Metabolic clearance of nicotine was determined after i.v. injection.
Metabolic clearance data were then used in conjunction with blood
and urinary concentrations of nicotine measured during a period of
smoking to determine the intake of nicotine. In five subjects, average
intake of nicotine per cigarette was 1.06 mg (range, 0.58 to 1.49 mg)
(Feyerabend, Ings, Russell 19851. In 22 cigarette smokers, 13 men
and 9 women who smoked an average of 36 cigarettes/day (range 20
to 621, the average daily intake was 37.6 mg, with a range from 10.5
to 78.6 mg (Benowitz and Jacob 1984). Nicotine intake per cigarette
averaged 1.0 mg (range 0.37 to 1.56 mg). Intake per cigarette did not
correlate with yields obtained by smoking machine using standard
Federal Trade Commission methods. This is because smoking
machines smoke cigarettes in a uniform way, using a fixed puff
volume (35 mL1, flow rate (over 2 secl, and interval (every minute).
Smokers smoke cigarettes differently, changing their puffing behav-
ior to obtain the desired amount of tobacco smoke and nicotine.

Elimination Rate as a Determinant of Nicotine Intake by
Cigarette Smoking

There is considerable evidence that smokers adjust their smoking
behavior to try to regulate or maintain a particular level of nicotine
in the body (Gritz 1980; Russell 1976). For example, when the
availability of cigarettes is restricted, habitual smokers can increase
intake of nicotine per cigarette 300 percent compared with the
intake of unrestrict,ed smoking (Benowitz, Jacob, Koslowski et al.
1986).
Techniques for measuring daily intake of nicotine (Benowitz and
Jacob 1984) have been applied to study the influence of elimination
on nicotine intake. The rate of renal elimination of nicotine was
manipulated by administration of ammonium chloride or sodium

40


bicarbonate to acidify or alkalinize the urine: respectively (Benowitz
and Jacob 1985). Compared with daily excretion during placebo
treatment (3.9 mg nicotine/day), acid loading increased (to 12
mglday) and alkaline loading decreased (to 0.9 mglday) daily
excretion of nicotine. The total intake of nicotine averaged 38
mg/day. Average blood nicotine concentrations were similar in
placebo and bicarbonate treatment conditions but were 15 percent
lower during ammonium chloride treatment. Daily intake of nicotine
was 18 percent higher during acid loading, indicating compensation
for increased urinary loss. The compensatory increase in nicotine
consumption was only partial, replacing about half of the excess
urinary nicotine loss. Bicarbonate treatment had no effect on
nicotine consumption, consistent with the small magnitude of effect
on excretions of nicotine in comparison to total daily intake.

These results seem compatible with the suggestion of Schachter
(1978) that emotional stress, which results in more acidic urine,
might accelerate nicotine elimination from the body and thereby
increase cigarette smoking. But caution must be exercised in
applying these findings to usual smoking situations. These studies
were performed under conditions of extreme urinary acidification or
alkalinization, so that the changes in renal clearance would be
maximized, Even with extreme differences in urinary pH, differ-
ences in overall nicotine elimination rate and smoking behavior
were modest. This is because renal excretion is a minor pathway for
elimination of nicotine; most is metabolized. Smaller changes in
urinary pH, such as occur spontaneously throughout the day or that
might be related to stressful events, would not be expected to
substantially influence nicotine elimination or smoking behavior.

Biochemical Markers of Nicotine Intake

Absorption of nicotine from tobacco smoke provides a means of
verification and quantitation of tobacco consumption. The general
strategy is to measure concentrations of nicotine, its metabolites
(such as cotinine), or other chemicals associated with tobacco smoke
in biological fluids such as blood, urine, or saliva. Different measures
vary in sensitivity, specificity, and difficulty of analysis. Different
investigators have used blood or urinary nicotine concentrations,
blood or salivary or urinary cotinine concentrations, expired carbon
monoxide or carboxyhemoglobin concentrations, or plasma or sali-
vary thiocyanate (a metabolite of hydrogen cyanide, a vapor phase
constituent) concentrations as measures of tobacco smoke consump-
tion.

Relationships among daily intake of nicotine, daily exposure to
nicotine (that is, blood concentrations of nicotine integrated over 24
hr), various parameters of cigarette consumption, and different
measures of nicotine intake have been examined experimentally

41


during ad libitum cigarette smoking on a research ward (Benowitz
and Jacob 1984). The best biochemical correlate to nicotine intake
and exposure in this study was a random blood nicotine concentra-
tion measured at 4 p.m. This level did not depend on when the last
cigarette was smoked. This finding is consistent with the observation
that nicotine levels accumulate throughout the day and plateau in
the early afternoon (see Figure 5). At steady state, with regular
smoking throughout the day, there should be a reasonably good
correlation between nicotine concentrations and daily intake. Car-
boxyhemoglobin (COHb) concentrations in the afternoon were the
next best markers of nicotine intake. Also, morning (8 a.m.) levels of
nicotine and COHb correlated with intake, presumably reflecting
persistence of nicotine and COHb in the blood from exposure on the
previous day.

Although cotinine is a highly specific marker for nicotine expo-
sure, blood levels of cotinine across subjects in this study did not
correlate as closely with nicotine intake as did blood levels of
nicotine or COHb (Benowitz and Jacob 1984). This is probably due to
individual variability in fractional conversion of nicotine to cotinine
and in the elimination rate of cotinine itself.

Because of its relatively long half-life, cotinine levels are less
sensitive than nicotine levels to smoking pattern, that is, when the
last cigarette was smoked. For longitudinal within-subject studies,
the cotinine level would be expected to be a good marker of changes
in nicotine intake. Cotinine measurements have become the most
widely accepted method for assessing the intake of nicotine in long-
term studies of tobacco use (see also Chapter V).

As expected by the known variation in renal clearance due to
effects of urinary flow and pH, urinary concentrations of nicotine did
not correlate well with nicotine intake (Benowitz and Jacob 1984). In
contrast, urinary cotinine, which is less influenced by urinary flow
or pH, was as good a marker as blood cotinine concentration.
Salivary and urinary cotinine concentrations correlate well (r = 0.8
to 0.9) with blood cotinine concentrations (Haley, Axelrad, Tilton
1983; Jarvis et al. 1984). Therefore, salivary or urine cotinine
concentrations should be almost as useful as blood levels in
indicating nicotine intake.

Analytical Methods for Measuring Nicotine and Cotinine in
Biological Fluids

Determination of nicotine concentrations in biological fluids
requires a sensitive and specific method, because concentrations of
nicotine in smokers' blood are generally in the low nanogram per
milliliter range and a number of metabolites are also present.
Cotinine concentrations in blood are generally about tenfold greater
than nicotine concentrations, and as a result, less sensitive analyti-

42


cal methodology may be acceptable. Methods with adequate sensitiv-
ity for determination of nicotine and cotinine in smokers' blood
include gas chromatography (GC) (Curvall, Kazemi-Vala, Enzell
1982; Davis 1986; Feyerabend, Levitt, Russell 1975; Hengen and
Hengen 1978; Jacob, Wilson, Benowitz 1981; Vereby, DePace, Mule
1982), radioimmunoassay (RIA) (Langone, Gjika, Van Vunakis 1973;
Castro et al. 1979; Knight et al. 1985), enzyme-linked immunosorbent
assay (ELISA) (Bjercke et al. 1986), high performance liquid chroma-
tography (HPLC! (Machacek and Jiang 1986; Chien, Diana, Crooks,
in press), and combined gas chromatograph-mass spectrometry (GC-
MS) (Dow and Hall 1978; Gruenke et al. 1979; Jones et al. 1982;
Daenens et al. 1985). For reasons of sensitivity, specificity, and
economy, GC and RIA are the most frequently used methods. GC-MS
is a highly sensitive and specific technique, but the expense has
discouraged its routine use. HPLC is less sensitive than GC for
nicotine and cotinine determination. Although recently reported
methods (Machacek and Jiang 1986; Chien, Diana, Crooks, in press)
appear to have adequate sensitivity for determining concentrations
in plasma, relatively large sample volumes are required. Concentra-
tions of nicotine and cotinine in urine are tenfold to hundredfold
greater than concentrations in plasma or saliva (Jarvis et al. 1984),
and a variety of chromatographic and immunoassay techniques meet
sensitivity requirements.

The choice of a particular method depends on the biological fluid
to be assayed; the need for sensitivity, precision, and accuracy; and
economic considerations. Chromatographic methods, particularly
those utilizing high-resolution capillary columns and specific detec-
tors such as nitrogen-phosphorus detectors or a mass spectrometer,
provide the greatest specificity. On the other hand, immunoassay
techniques are operationally simpler, generally require smaller
samples, and may be less expensive than chromatographic methods.
A drawback to immunoassay methods is the potential for cross-
reactivity of the antibody with metabolites or endogenous sub-
stances. There is generally a good correlation between results
obtained by GC and RIA for plasma cotinine concentrations (r = 0.94)
(Gritz et al. 1981; Biber et al. 1987). In an interlaboratory comparison
study (Biber et al. 19871, cotinine concentrations in smokers' urine
measured by RIA were generally higher than concentrations deter-
mined by GC, whereas in nonsmokers' urine spiked with cotinine
RIA and GC values were similar. These results suggest that nicotine
metabolites cross-react with the antibody against cotinine, at least in
some of the RIA methods.

Pharmacodynamics of Nicotine
General Considerations

This Section will focus on the relationship between nicotine levels
in the body and their effects on behavior and physiological function

43


(pharmacodynamics). These data show how pharmacodynamic fac-
tors determine some of the consequences of cigarette smoking, Two
issues are particularly relevant in understanding the pharmacody-
namics of nicotine: a complex dose-response relationship and the
level of tolerance that is either preexisting or is produced by
administration of nicotine.

Dose-Response

The relationship between the dose of nicotine and the resulting
response (dose-response relationship) is complex and varies with the
specific response that is measured. In pharmacology textbooks,
nicotine is commonly mentioned as an example of a drug which in
low doses causes ganglionic stimulation and in high doses causes
ganglionic blockade following brief stimulation (Comroe 1960). This
type of effect pattern is referred to as "biphasic." Dose-response
characteristics in functioning organisms (in vivo) are often biphasic
as well, although the mechanisms are far more complex. For
example, at very low doses, similar to those seen during cigarette
smoking, cardiovascular effects appear to be mediated by the CNS,
either through activation of chemoreceptor afferent pathways or by
direct effects on the brain stem (Comroe 1960; Su 1982). The net
result is sympathetic neural discharge with an increase in blood
pressure and heart rate. At higher doses, nicotine may act directly
on the peripheral nervous system, producing ganglionic stimulation
and the release of adrenal catecholamines. With high doses or rapid
administration, nicotine produces hypotension and slowing of heart
rate, mediated either by peripheral vagal activation or by direct
central depressor effects (Ingenito, Barrett, Procita 1972; Porsius
and Van Zwieten 1978; Henningfield, Miyasato, Jasinski 1985).

Tolerance

A second pharmacologic issue of importance is development of
tolerance; that is, after repeated doses, a given dose of a drug
produces less effect or increasing doses are required to achieve a
specified intensity of response. Functional or pharmacodynamic
tolerance can be further defined as where a particular drug
concentration at a receptor site (in humans approximated by the
concentration in blood) produces less effect than it did after a prior
exposure. Dispositional or pharmacokinetic tolerance refers to
accelerated drug elimination as a mechanism for diminished effect
after repeated doses of a drug. Behavioral tolerance refers to
compensatory behaviors that reduce the impact of a drug to
adversely affect performance. Such tolerance can occur following
intermittent exposures to a drug such that there is minimal
development of functional or dispositional tolerance.

44


Most studies of drug tolerance have focused on tolerance which
develops as a drug is chronically administered. If the tolerance
develops within one or two doses, it is referred to as acute tolerance
or tachyphylaxis. If tolerance develops after more prolonged use, the
tolerance is referred to as acquired or chronic tolerance. Individual
differences in sensitivity to the first dose of a drug also frequently
exist. Those individuals who exhibit a reduced response to a specified
drug dose or require a greater dose to elicit a specified level of
response are said to be tolerant to the drug. This form of tolerance is
referred to as first-dose tolerance, drug sensitivity, or innate drug
responsiveness. For sake of clarity, this Report will reserve the term
tolerance to describe reduction in the response to nicotine during the
course of or following a previous exposure and will use acute drug
sensitivity to describe responsiveness to an initial dose.

Studies of tolerance to nicotine began in the late 19th century. In a
series of studies of fundamental importance to the understanding of
the nervous system, as well as to understanding the pharmacology of
nicotine, Langley (1905) and Dixon and Lee (1912) studied the effects
of repeated nicotine administration on a variety of animal species
and on in vitro tissue preparations. Several findings emerged which
have been widely verified and extended to other species and
responses. These include: (1) With repeated dosing, responses dimin-
ished to nearly negligible levels; (2) After tolerance occurred,
responsiveness could be restored by increasing the size of the dose; (3)
After a few hours without nicotine, responsiveness was partially or
fully restored.

After smoking a cigarette, people who have not smoked before
("naive smokers") usually experience a number of effects that
become generally uncommon among experienced smokers. For
example, retrospective reports by smokers indicate that initial
exposure to tobacco smoke produced dizziness, nausea, vomiting,
headaches, and dysphoria, effects that disappear with continued
smoking and are rarely reported by chronic smokers (Russell 1976;
Gritz 1980). Tolerance may also develop to toxic effects, such as
nausea, vomiting, and pallor, during the course of nicotine poisoning,
despite persistence of nicotine in the blood in extremely high
concentrations (200 to 300 ng/mL! (Benowitz, Lake et al. 1987).

A systematic analysis of the various forms of tobacco smoke
tolerance has not been carried out. There are a few studies
comparing the effects elicited by an acute exposure to tobacco in
nonsmokers and smokers. Clark and Rand (1968) studied the effect of
smoking cigarettes of varying nicotine content on the knee-jerk
reflex and reported that high-nicotine cigarettes suppressed this
reflex to a greater degree than did low-nicotine cigarettes. This effect
was more pronounced at each nicotine dose in nonsmokers and light
smokers compared to heavy smokers. These findings suggested that

45


tolerance is due to altered sensitivity to nicotine. Tolerance to
nicotine is not complete because even the heaviest smokers experi-
ence symptoms such as dizziness, nausea, and dysphoria when they
suddenly increase their smoking rates (Danaher 1977). Evidence
indicates that the majority of the psychological actions of tobacco
smoke result from nicotine (Russell 1976; Chapter VII). Thus, most of
the tolerance to effects of tobacco smoke that occurs following
chronic tobacco use is due to the development of tolerance to
nicotine.

Acute Sensitivity
Human Studies

Studies which have indicated that individuals differ in response to
tobacco smoke or nicotine have used smokers as the experimental
subjects. Consequently, whether individual differences are due to
differences in acute sensitivity to nicotine that have persisted during
chronic tobacco use or are due to differences in the development of
tolerance is unknown.

Nesbitt (1973) and Jones (1986) noted that individual smokers
differ with respect to the effects of smoking a standard cigarette on
heart rate, but it is not clear from these studies whether these
differences in responsiveness are due to differences in sensitivity to
nicotine or to differences in the dose and kinetics of nicotine.
Benowitz and colleagues (1982) observed individual differences in the
effects of iv. injections of nicotine on heart rate, blood pressure, and
fingertip skin temperature. Differences were not explained by
differences in blood levels, indicating differential sensitivity to
nicotine.

Animal Studies

Studies using laboratory animals indicate that differences in acute
sensitivity to nicotine exist. Inbred rat and mouse strains differ in
sensitivity to the effects of nicotine on locomotor activity (Garg 1969;
Battig et al. 1976; Schlatter and Battig 1979; Hatchell and Collins
1980; Marks, Burch, Collins 1983b). Mouse strains also differ in the
direction of the effect (increased or decreased activity). The mouse
strains that differ in sensitivity to the effects of injected nicotine on
locomotor activity also differ in the magnitude of response to a
standard dose of tobacco smoke (Baer, McClearn, Wilson 1980).
Inbred mouse strains also differ in sensitivity to the effects of
nicotine on body temperature, heart rate, and acoustic startle
response (Marks, Burch, Collins 1983a; Marks et al. 1985, 1986), as
well as in sensitivity to nicotine-induced seizures (Tepper, Wilson,
Schlesinger 1979; Miner, Marks, Collins 1984, 1986). These findings
indicate that genetic factors may influence the sensitivity of rats and

46


mice to the first dose of nicotine. The importance of genetically
determined differences in human sensitivity to the effects of nicotine
administered in tobacco smoke remains to be determined.

Mechanisms of Differences in Acute Sensiticit?

Differences between inbred mouse and rat strains in sensitivity to
the effects elicited by a single injected dose of nicotine do not appear
to result from differences in rate of nicotine metabolism (Petersen,
Norris, Thompson 1984) or from differences in brain nicotine
concentration following intraperitoneal injection !Hatchell and
Collins 1980; Rosecrans 1972; Rosecrans and Schechter 1972). Thus,
rat and mouse strains differ in tissue sensitivity to the effects of
nicotine. Differences among mouse strains in sensitivity to nicotine
do not appear to be due to differences in the number or affinity of
brain nicotine receptors that are measured via the binding of 3H-
nicotine (Marks, Burch, Collins, 1983b). Mouse stocks that are more
sensitive to nicotine-induced seizures do have greater numbers of
hippocampal nicotine receptors that bind "`1-bungarotoxin (BTX)
(Miner, Marks, Collins 1984, 1986). Some of the differences in
sensitivity to nicotine between genetically defined stocks of animals
may be related to differences in the number of nicotine receptors in
specific regions of the brain.

Tachyphylaxis (Acute Tolerance)
Human Studies

Systematic studies of tachyphylaxis or acute tolerance to effects of
tobacco in nonsmokers have not been reported. There is evidence
that tachyphylaxis does develop to effects of tobacco and nicotine in
humans. Smokers frequently report that the first cigarette of the day
is the best and that subsequent cigarettes are "tasteless" (Russell
1976; Henning-field 1984). Smoking a single standard cigarette after
24 hr of abstinence increases heart rate, whereas smoking an
identical cigarette during the course of a normal day fails to change
heart rate (West and Russell 19871. Fewer standard puffs were
required to produce nausea at the beginning of the day (following 8
to 10 hr of tobacco abstinence) or from high-nicotine cigarettes than
at the end of the day or from low-nicotine cigarettes (Henningfield
1984). Complete tolerance to nausea and vomiting developed over 8
hr in a woman in the course of an accidental nicotine poisoning,
despite persistently toxic blood levels of nicotine (Benowitz, Lake et
al. 1987). These findings suggest that tolerance which is lost and
regained during short periods of abstinence from tobacco is tolerance
to nicotine.

Tolerance develops very rapidly to several effects of nicotine.
Rosenberg and colleagues (1980) studied the effects of i.v. nicotine

47


injections on arousal level, heart rate, and blood pressure. In these
experiments, six healthy smokers, 21 to 35 years of age, received six
series of nicotine injections spaced 30 min apart. Each series of
injections consisted of 10 2+g/kg injections spaced 1 min apart.
Subjects reported a pleasant sensation after the first series of
injections, but this response was not observed thereafter. Heart rate
and blood pressure values remained above baseline, but there was
little increment with successive injections, despite nicotine blood
level increases which were similar to those observed after the first
series of injections, In contrast, skin temperature fell progressively
during the period of nicotine dosing, gradually returning to baseline
at the end of the study. These data indicated rapid development of
tolerance to subjective effects and heart rate and blood pressure
responses, but tolerance was not complete because heart rate and
blood pressure remained above baseline. Henningfield (1984) also
assessed subjective responses of human subjects after i.v. injections
with nicotine at lo-min intervals. The subjective response of "liking"
the effects of nicotine was lost after five or six injections. Benowitz
and coworkers (1982) studied the effect of a 30-min infusion of
nicotine at a rate of 1 to 2 ug/kg/min. Shortly after initiation of
infusion, heart rate and blood pressure increased, but the increase
did not continue even though plasma nicotine concentrations
continued to rise during the continuous infusion. Maximal cardiovas-
cular changes were seen within 5 to 10 min, whereas maximal
plasma nicotine levels were not reached until 30 min. These findings
indicate that tachyphylaxis to the effects of nicotine may develop in
humans within 5 to 10 min, the time required to smoke one cigarette.
In contrast to heart rate, skin temperature (reflecting cutaneous
vascular tone) declined and rose in association with changes in blood
nicotine concentrations, showing no evidence of tolerance.

The above studies indicate rapid development of tolerance to some
(but not all) actions of nicotine in people. These studies were
performed with cigarette smokers who had abstained from smoking
the night before the study. Since significant quantities of nicotine
persist in the body even after overnight abstinence, there is probably
some persistence of tolerance. Experimental data supporting this
conclusion were obtained in a study of cardiovascular responses to
infused nicotine in smokers following either an overnight or 7-day
tobacco abstinence (Lee, Benowitz, Jacob 1987). Heart rate and blood
pressure responses were significantly greater after more prolonged
abstinence. However, within 60 to 90 min, the blood concentra-
tion-effect relationship in subjects after brief abstinence approxi-
mated that observed after prolonged abstinence. Thus, a significant
level of tolerance persists throughout the daily smoking cycle, but is
lost with prolonged abstinence. Tolerance, at least after abstinence
for one week, is rapidly reestablished with subsequent exposure.

48


Animal Studies

Many studies demonstrate that acute tolerance or tachyphylaxis
develops very quickly to actions of nicotine. Barrass and coworkers
(1969) demonstrated that pretreatment of mice with a single i.v. dose
(0.8 mg/kg) of nicotine resulted in an increase in the LD,, (dose
which is lethal to 50 percent of animals) for nicotine. Maximal
protection was seen 5 min after the injection, but this protection
diminished steadily over the next hour. Tachyphylaxis develops to
the effects of nicotine on locomotor activity. Stolerman, Bunker, and
Jarvik (1974) noted that pretreating rats with a 0.75-mg/kg dose of
nicotine 2 hr before challenge doses of nicotine (0.25 to 4.0 mg/kg)
resulted in a shift of the nicotine dose-response curves, indicating
reduced sensitivity. The ED,, values (doses that are effective in
producing the measured response in 50 percent of animals) for
nicotine-induced decreases in locomotor activity were nearly 2.4-fold
greater in nicotine-pretreated rats than in saline-pretreated animals.
Nicotine pretreatment also results in tachyphylaxis to the effects of
nicotine on body temperature (hypothermia) in cats (Hall 1972),
water-reinforced operant responding in rats (Stitzer, Morrison,
Domino 1970), discharge of lateral geniculate neurons of cats
(Roppolo, Kawamura, Domino 1970), repolarization of sartorius
muscle in frogs (Hancock and Henderson 19721, blood pressure
elevation in rats (Wenzel, Azmeh, Clark 19711, contraction of aortic
strips in rabbits (Shibata, Hattori, Sanders 1971), respiratory stimu-
lation in cats (McCarthy and Borison 19721, and gastrointestinal
contraction in squid (Wood 1969) and guinea pigs (Hobbiger, Mitchel-
son, Rand 1969). More recent studies have demonstrated that
pretreatment with as little as one dose of nicotine will attenuate
nicotine-induced elevations of plasma corticosterone (Balfour 1980)
and adrenocorticotropic hormone (ACTH) (Sharp and Beyer 1986)
levels in rats (see also Chapter III).

The interval between the pretreatment and challenge doses of
nicotine is a critical factor that determines whether tachyphylaxis is
observed. Aceto and coworkers (1986) examined the effect of iv.
nicotine infusion on heart rate and blood pressure in the rat.
Tolerance did not develop when the interval between pretreatment
and challenge doses was 30 min; marked tolerance was detected
when the interval was reduced to 1 min. However, Stolerman, Fink,
and Jarvik (1973) observed that after a single intraperitoneal dose of
nicotine to rats, acute tolerance to a second dose did not become
maximal until 2 hr after the initial injection.

Mechanisms of Tachyphylaxis

Although tachyphylaxis has been described for a wide variety of
nicotine's effects, very little is known about mechanisms. A nicotine

49


metabolite may play a role in the development of tachyphylaxis.
Barrass and colleagues (1969) argued that nicotine metabolites may
block nicotine receptors and thereby antagonize nicotine's lethal
effects. This argument was made because pretreatment with nic-
otine-N'-oxide protected mice from the lethal effects of large doses of
nicotine. LD,, values were increased approximately ninefold by
pretreatment with nicotine-N'-oxide. These authors hypothesized
that this protection may involve conversion of nicotine-N'-oxide to
hydroxynicotine. Their results indicated that injection of a reduction
product of cotinine, believed to be hydroxynicotine, gave immediate
protection, whereas maximum protection was not seen until 40 min
after injection of nicotine-N'-oxide. Thus it appears that metabolism,
possibly to hydroxynicotine, is required for the protective action of
nicotine-N'-oxide.

Another hypothesis is that tachyphylaxis is the result of desensiti-
zation of nicotine receptors. Desensitization of the receptor involves
a conformational change that results in increased affinity of the
nicotinic receptor for agonists coupled with decreased ability of the
receptor to transport ions (Weiland et al. 1977; Sakmann, Patlak,
Neher 1980; Boyd and Cohen 1984). Desensitization of nicotinic
receptors at the motor end-plate was first described by Katz and
Thesleff (1957) and has since been studied by a large number of
investigators, using either skeletal muscle or the electric organs of
the eel, Torpedo californica. Although tachyphylaxis has been
commonly suggested as being due to desensitization of brain
nicotinic receptors, the role of desensitization in tachyphylaxis to
specific behavioral effects of nicotine has not been studied. This is
because concentrations of nicotinic receptors in specific areas of the
brain corresponding to the behavioral effects being measured are not
high enough to use available methods.

Chronic Tolerance
Human Studies

Chronic tolerance to tobacco and nicotine has not been studied
systematically in human subjects, but it is clear, as noted previously,
that some tolerance does develop. Tolerance is not complete;
symptoms of nicotine toxicity such as nausea appear when smokers
increase their normal tobacco consumption by as little as 50 percent
(Danaher 1977).

These findings are consistent with the observations that smokers
increase their tobacco consumption and intake of nicotine with
experience. Such escalating dose patterns may be observed for
several years after initiation of either cigarette smoking or smok-
eless tobacco use. Cigarette smokers may achieve such increases by
augmenting the number of cigarettes smoked and by increasing the
amount of nicotine extracted from each cigarette. For users of

50