Procedures for gross separation into basic, acidic, phenolic, and neutral
fractions and for further processing of these fractions vary from laboratory to
laboratory.  The criteria upon which identification is based also vary.  The
most reliable identifications are based upon an ultraviolet absorption spec-
trum and/or a fluorescence spectrum in good agreement over the entire range
\tith that of an authentic sample and include one or more of the following:
Rf value observed in a paper chromatogram 111) ; order of elution from
alumina; mass spectrometry.
COMPOUNDS OF THE PARTICULATE PHASE
  OTHER THAN HIGHER POLYCYCLICS
This brief summary is based largely on the comprehensive review by
Johnstone and Plimmer of the Medical Research Council at Exeter Uni-
irraity. England ( 24 I.  It should be noted that water constitutes 27 percent
Ilf the particulate phase.  Th e major groups of compounds included are
.ho\tn in Table 1.
ALIPHATIC AND ALKYCLIC HYDROCARBONS
Almost all of the possible hydrocarbons, C, through C,, saturated and
urr+aturated, straight-chain and branched-chain, have been reported to be
prcaen, in tobacco smoke. Intermediate, normally liquid paraffins are pres-
ent. All the C,, through C,, n-a lkanes have been identified, as well as the
CZ: and C,!,-c',, isoparaffins.
T4BL.E I.--Major classes of compounds in the particulate phase of cigarette
                    smoke
Percent in Sumber 01
particu-   comwmd
late* phase
-
7.7-12.8 1      25
5.3-8.3       18
  8. 5      21
  4. 9
  0.44    El
1. G-3. 8      45
Toxic action on lung
Some irritant
Possible irritation
Some irritant
Some irritant
Some carcinogenic
Irritant and possibly cocarcinokxnic
TERPENES AND ISOPRENOID HYDROCARBONS
isoPrene, the basic unit of the terpenes and of higher terpenoids has been
!dcntified in
mprrthadiene. cigarette smoke (34) as have its dimers, dipentene and 1,8-p-
and shown t  The triterpene squalene, consisting of six isoprene units
        o b
hilit! of its be* e present in smoke (47) is of interest because of the possi-
     mg cyclized to polycyclic compounds and because of its ready
51

CHa     CE4      CHa
                                          C&
HaC
                            CH:      CHa      CHa
Squalene
reaction with air to form hydroperoxides (which would be destroyed during
attempted isolation ) ; a hydroperoxide derived from cholesterol has been
shown to be carcinogenic i cancer-causing) : at least under certain conditions
of administration I 12) .  Phytadienes. products of the dehydration of the
diterpene alcohol phytol, are also present in smoke and subject to air oxida-
tion to hydroperoxides.
   C&             Crt     CH:
                                CHIOH
HsC
Phytol
ALCOHOLS ANT) ESTERS
A wide variety of mono- and dihydric alcohols, both aliphatic and aro-
matic, are present in tobacco smoke.  Solanesol, a primary alcohol con-
taining 9 isoprene units, has been found in both tobacco and tobacco smoke;
20 g. of pure material was isolated from 10 lbs. of flue-cured aged tobacco
(0.44 percent).  Grossman et al i 13) found that pyrolysis of solanesol at
500" C. gives isoprene, its dimer dipentene, and other terpenoid products and
concluded that the alcohol is the source of terpenoid compounds which are
important factors in the flavor of tobacco smoke.
Ethylene glycol and glycerol have been found present in smoke, but it
is not clear from the literature whether they are present in smoke from un-
treated tobacco or arise from addition of these humectant substances to
tobacco to improve moistness.
Many common esters, such as the ethyl esters of the C2, C,, and C, fatty
acids, are present in smoke.  Higher fatty- acids are found both as free acids
and as esters.
STEROLS
Stigmasterol, p-sitosterol, and r-sitosterol have been isolated from to-
bacco smoke.  Indeed the sterol fraction is reported (29) to constitute
approximately 0.15 percent of whole tar.  The sterols are of interest as
possible precursors of polyc)-clic aromatic hydrocarbons and because of the
evidence, noted above. that sterol hydroperoxides can be carcinogenic.
          ALDEHYDES AND KETONES
Most common aldehydes of low molecular weight (acetaldehyde, pro-
pionaldehyde, acetone, methyl ethyl ketone, etc.) have been found present
52

-4 CHlOH
HIC  I
5oo"= Ad +
      Isoprene
I-&C `-.2Hz
Dipentene
(major product)
Solanesol
    \
6
  (3
HsC' CHa
   C&
&C /`hII.,
CH,
I
H:C
J&C
HIC
CHa
in tobacco smoke, as have such dicarbonyl compounds as glyoxal and di-
acetyl. Dipalmityl ketone exemplifies ketones of high molecular weight
isolated from tobacco smoke.
0
                           16'
                      C&
Dipalmityl ketone
ACIDS
A large number of volatile and nonvolatile acids of low molecular weight
are present in tobacco smoke.  Fatty acids of chain length C,, to C,, are
reported to constitute 1 percent of the whole tar and the bulk of these acids
are present in the free form (46).  Unsaturated fatty acids and keto acids
`e.g., pyruvic acid) are also present.
53

PHENOLS AND POLYPHENOLS
Since the phenols and polyphenols present in tobacco leaf play an im.
portant role in the curing and smoking quality of tobacco, a great deal of
investigative work has been done on the estimation, separation, and ident&
cation of complex tobacco phenols such as rutin and chlorogenic acid.  The
presence of simple phenols in tobacco smoke was established as early as
1871.  The phenol content of smoke became of increasing importance with
OH
HO        H? \ -
      0 CH- CH~O~co,H
                              -        ir   HOi
                                                   OH
Rharnnoae
        Rutin                  Chlorogenic acid
the demonstration that phenol and substituted phenols can function as
cocarcinogens; that is, they promote the appearance of skin tumors in mice
following application of a single initiating dose of a known carcinogen (4).
Furthermore, the smoke from one cigarette contains as much as 1 mg. of
phenols (7). In add t i ion to simple alkylphenols, naphthols, and the poly.
phenols, resorcinol and hydroquinone are also present.
ALKALOIDS, NITROGEN BASES, AND HETEROCYCLICS
Pyridine, nicotine, nornicotine, and other substituted pyridine bases con.
stitute some 8-15 percent of whole tar; nicotine and nornicotine constitute
about 7-8 percent of the total tar.  The companion bases are products of
the pyrolysis of the alkaloids present in tobacco leaf.  Quinoline and three
poly-cyclic heterocyclic compounds have also been identified in smoke (45)
and will be discussed later since the three polycyclic compounds are carcino-
genic.  `4 pentacyclic compound related to xanthene, namely 1,8,9peri-
naphthoxanthene. has been identified in smoke (45).
1,8,9-Perinaphthoxanthene
AMINO ACIDS
Although tobacco leaf contains a number of amino acids, relatively few
have been found present in smoke; among these are glutamine and glutamic
acid.
54

INORGANIC COMPONENTS
It is estimated that the main-stream smoke from one cigarette contains
about 150 1j.g. of metallic constituents. which are mainly potassium (90
tIercent I _ sodium (5 percent I, and traces of aluminum, arsenic, calcium. and
copper.  Arsenic is reported to be present to the extent of 0.3-1.4 pg. in
the smoke of one cigarette.  Th e inorganic compounds are most likely
chlorides. but metals themselves may be present.
Apparently bery-ilium is present in tobacco in trace quantities. but is not
1 olatilized in the smoking process ( 4s ) .  Nickel is present in cigarettes in
trace amounts and may occur in main-stream smoke to a small extent,
l:robably as the chloride (31 t .  Spectrographic analysis has shown the
presence of chromium in smoke at a level of less than 0.06 ;tg. per cigarette.
This level appears too low to represent a hazard 148).
IVONCARCINOGENIC AROMATIC HYDROCARBONS
The aromatic h>-drocarbons present in tobacco smoke have received
an enormous amount of attention since some of them are carcinogenic.
Toncarcinogenic hydrocarbons of smoke containing one to three rings
include benzene. toluene and other alkplbenzenes, acenaphthene, acenaph-
thylene. flnorene. anthracene. and phenanthrene. Hydrocarbons of estab-
lished carcinopenicity to mice all contain from four to six condensed rings.
Ifowever. no less than 27 hydrocarbons containing four or more `condensed
rings which have been tested for carcinopenicity with negative results have
heen isolated from tobacco smoke tar.  As methods of separation and
identification improve, it is almost certain that additional hydrocarbons will
be found present in smoke, because almost every conceivable ring system
has been demonstrated to be present and the number of possible alkylated
polycyclics is very large indeed.
CARCINOGENIC HYDROCARBONS AND HETEROCYCLICS
           IN TOBACCO SMOKE
In 1925-30 Kennaway et al. in seeking to identify the active substance
in high-boiling fractions of coal tar distillates of established carcinogenicity
to mice, discovered that dibenzo(a,h)anthracene (for formula, see Table
21 prepared by synthesis evokes skin cancer when applied to the skin of
mice (11). The hydrocarbon was recognized as different from the carcino-
gen of coal tar because its fluorescent spectrum did not match the character-
istic three-banded spectrunr of the tars.  In 1933 Cook and co-workers i 11)
isolated the coal tar constituent responsible for the characteristic fluorescence
and identified it as benzota) pyrene.
the carcinogens now known.      It is one of the most potent of all
55

TABLE 2.--Carcinogenic Polycyclic Compounds Isolated From Cigarette
                  Smoke
Compound
1. Benzo(s)pyrene
2. Dibenzo(s,i)pyrene
3.  Dibenao(s,h)snthrscene
4. Benao(c)phenanthrene
5.  Dibens(s,j)acridine
6,  Dibenz(a,h)acridine
7.  7H-Dibenzo(c,g)carbszole
structllre
    ' I
     / /
(Id??
: 1; >
w
I> `I
\ '  \
        /
Carcino-
genicity   Amount reported,
      rg/KMM cigarettes
++++
++++
++
    16
(ave. of 10 reports)
0.02-10
(2 reports)
(1 retort)
ot-       not stated
+
  2.7
(1 report)
  0.1
(1 report)
  0.7
(1 report)
56

Since the discovery of carcinogenic hydrocarbons, a large number of
polycyclic hydrocarbons and heterocyclic analogs have been tested for car-
cinogenicity to mice and to rats in many laboratories, both by application
to the skin and by subcutaneous injection.  Bioassays in different labora-
tories, often on independently prepared samples, are remarkably consistent
and place a series of hydrocarbons in the same relative order of potency.
A compilation (and its supplement) prepared by J. L. Hartwell (16) of the
IKational Cancer Institute lists 2108 compounds of which 481 were reported
to cause malignant tumors in animals. All but one of the polycyclic hydro-
carbons listed in Table 2 as having been identified in tobacco smoke have
already been documented in the Hartwell report and can be assigned a
rating as very potent ( + + + + ), potent ( + + + ) , moderately carcino-
genic I, + + ), or weakly carcinogenic ( + ) (31). Many other such com-
pounds studied are reported in the Hartwell survey and in another by :Irthur
D. Little, Inc. (31). The rating assigned to dibenzo (a,;) pyrene is based
on experiments with over 10,000 inbred mice in which one subcutaneous
injection in the groin of 0.5 mg. of hydrocarbon in tricaprylin produced
50 percent sarcomas at the injection site in 14 weeks and 98 percent tumors
in 24 weeks (20). Benzo(a)pyrene is one of the two most potent of the
seven carcinogens detected in tobacco smoke and it is present in much larger
quantity than any of the other carcinogens listed. Two polycyclic hydro-
carbons isolated from tobacco smoke but not yet adequately tested for
carcinogenicity are: benzo (j ) Auoranthene and dibenzo (a,l) pyrene.
Identification of benzo (a)pyrene is reported in 19 separate investiga-
tions; the amount given in the table per 1000 cigarettes (70 mm. long,
Neighing about 1.0 g. each) is the average of 10 values selected on the
basis of the quality of criteria used for identification (31). Compounds
1, 2, 3, 4, and benzo (j) fluoranthene were identified in one laboratory over
a period of years and are listed together in a review by Van Duuren (44).
Isolation of the three heterocyclic carcinogens (5,6,7) is reported by Van
Duuren (45).
Because of losses in the process of fractionation and purification, the
amount of carcinogens reported in a given investigation may be less than the
amount actually present.  Wy d n er and Hoffman (50) investigated this
point by adding a known amount of radioactive C"-1abelled benzo(a)pyrene
to a smoke condensate and applied the usual procedure for isolation of
benzo(a)pyrene, which involved, in the last stages, chromatographing twice
on silica gel and four times on paper. The activity of the benzo(a) pyrene
finally isolated indicated a loss of 3540 percent of carcinogen during proc-
essing. Th e amount of benzo(a) pyrene given in Table 2 thus should be
multiplied by a factor of 1.5 to give the estimated true amount.  Probably
the amounts of the other carcinogens in smoke are also at least 1.5 times the
reported amounts.
Relatively little work has been done on the components of smoke produced
with cigars and pipes. Table 3 summarizing a comparative study made in
one laboratory (5) indicates that the amount of benzo(a)pyrene, the only
carcinogen in the group studied, increases sharply from cigarettes to cigars
to pipes.
                                          57

TABLE 3.-Polycyclic hydrocarbons isolated from tobacco smoke
              [,,g. pm 1000 g. of tobxcm consumrd~
COCARCINOGENS
Assays of tobacco smoke tars for carcinogenicity are done by applying a
dilute solution of tar in an organic solvent with a camel's hair brush to the
backs of mice beginning when the animals are about six weeks old. Applica.
tion is repeated three times a week for a period of a year or more. The results
of a number of such assays present a puzzling anomaly: the total tar from
cigarettes has about 40 times the carcinogenic potency of the benzo( a) pyrene
present in the tar. The other carcinogens known to be present in tobacco
smoke are, with the exception of dibenzo(a,i) pyrene, much less potent than
benzo (a) pyrene and they are present in smaller amounts. Apparently, there.
fore, the whole is greater than the sum of the known parts (27, 33,49).
One possible or partial explanation of the discrepancy is that the tar con.
tains compounds which, although not themselves carcinogenic, can enhance
the cancer-producing properties of the carcinogens.  Berenblum and Shubik
(3), reporting on cocarcinogenesis. described the potentiating effect of croton
oil, which itself is noncarcinogenic except in certain strains of mice (4a), on
the action of hydrocarbon carcinogens. Phenol is reported to have a similar
potentiating effect (4. 50) and, as noted above. cigarette smoke contains
considerable phenolic material. Long-chain fatty acid esters (39) and free
fatty acids (19) have been shown to function as cocarcinogens, and sub.
stances of both types occur abundantly in tobacco smoke. It is possible that
the potentiatinp action of croton oil is due to the presence of fatty acids and
their esters. A further observation of possible importance is that some poly
cyclic hydrocarbons. though very weak or inactive as carcinogens, are capable
of initiating malignant growth under the influence of a promoter. Thus
henz (a) anthracene, identified in cigarette smoke, is verv weak or inactive in
initiating malignant growth by itself. but initiates carcinogenesis under the
influence of croton oil as promoter (15).
If more were known about the possible cocarcinogenicity of the many
inactive components of tobacco smoke, some of the apparent discrepancy
between isolation and bioassay data might disappear. It is possible that some
of the carcinogenicity of smoke is due to hydroperoxides formed from un-
saturated smoke components and destroyed in the isolation procedures.
Furthermore both sets of data are far from precise; for example, one esti-
mate of the amount of the highly potent dibenzoi a,i)pyrene per 1000
cigarettes (Table 2) is 0.02~~. and another is 1Opg.
However. it is not necessary to wait for an exact balance of the two sets
of data to draw a conclusion from each.  The isolation experiments, taken
58

alone, indicate that cigarette smoke contains a number of identified chemicals
which are carcinogenic to mice.  The bioassavs suggest that cigarette smoke
probably contains components which. actin g in a manner as yet undescribed,
are involved in the induction of tumors in mice.
Assessment of all conceivable synergistic effects presents a gigantic problem
for exploration.  Tobacco smoke contains considerable amounts of phenols
and fatty acids, both of which, as previously mentioned, enhance the activity
of known carcinogens. Cellulose acetate filters now in use remove `XL80
percent of acidic constituents of tobacco smoke.
MECHANISM OF THE FORMATION OF CARCINOGENS
Most of the carcinogenic compounds identified in cigarette smoke tar are
not present in the native tobacco leaf but are formed by pyrolysis at the high
burning temperature of cigarettes.  Van Duuren (4.4) reports formation of
benzo(a) pyrene and pyrene on pyrolysis of stigmasterol, a smoke com-
HO
Stigma&sol
Benro(a)pyrene      Pyrene
ponent. Similar pyrolysis of pyridine or of nicotine gives dibenzo( a,j)
acridine and dibenzo (a,h) acridine, both of which are carcinogenic (Table
2). Pyrolysis of nontobacco cigarettes made from vegetable fibers and
spinach resulted in formation of benzo( a jpyrene (50).
Hurd and co-workers (22) by careful experimentation have elaborated
plausible mechanisms for the formation of polycyclic aromatics by pyrolysis
of materials of low molecular weight at temperatures in the range 800-900" C.
Postulated radical intermediates are:
(a) CHz=C=kH - CH~-C+ZH
(b) EH-cH=I~H - ~H=cHGH
tc) CH=CH~H=CH
These radicals can arise from propylene, toluene, picoline, or pyridine.  A
variety of polycyclic hydrocarbons can be generated by reaction of these
radicals with themselves or with other small radicals present in the heating
zone. For example, dimerization of (b ) should give benzene.
                                          59

Jt thus appears that the pyrolysis of many organic materials can lead to
the formation of components carcinogenic to mice. Cigarette paper con.
sists essentially of cellulose. Pyrolysis of cellulose has been shown to produce
henzo(a)pyrene. The observation (2') that treatment of tobacco with
copper nitrate decreases the benzo (a) pyrene content of the cigarette smoke
suggests a possibility for improvement by the use of additives or catalysts.
The fact that side-stream smoke contains three times more benzo (a) pyrene
than main-stream smoke has been cited (50) as evidence that more efficient
oxidation could conceivably lower the content of carcinogenic hydrocarbons.
THE G.4S PHASE
The gas phase accounts for 60 percent of total cigarette smoke.  Hobbs
et al. ( 34, 35`1 found that 98.9 mole percent of the gas phase is made up of
the following seven components:
Yitrogen ____------- --------- ________. 73 mole percent
Oxygen---- --------_____------_______ 10
Carbon-dioxide ____- -- _______-__----_ -  9.5
Carbon-monoxide--------------------- 4.2
Hydrogen---------------------------~ 1.
Argon------------------------------. 0.6
Methane----------------------------- 0.6
98. 9
The approximately one percent of the gas phase not accounted for by the
seven major constituents contains numerous compounds, no less than 43
of I\ hirh have been identified as present in trace amounts.  Some of these
are listed in Table 4 (1).
TABLE 4.-Some gases found in cigarette smoke
(1
(PPm)
  100
  NX)
5. cinl
2d
  0. 5
Unknown
l-one
Sonc
sonr
Irritant
Irritant
Irritant
Irritnnt
Irritant
Irntant
Irritant
Irritant
r."known
i;;itant
Repiratory enzyme poison
Unknown

EFFECTS ON CILIARY ACTIVITY*
An important line of investigation was opened up by the report by Hilding
(1s) that cigarette smoke is capable of inhibiting the transport activity of
ciliated cells such as found in the respiratory tract.  It has been suggested
( 10. 17 I that failure of ciliary function to provide a constantly moving
stream of mucus enables environmental carcinogens to reach the epithelial
cells.  Kensler and Battista t 28) describe development of a method of
bioassay for inhibition of ciliary transport activity involving exposure of
the trachea of a rabbit to the test material. The smoke from a regular
cigarette was found to inhibit transport activity by 50 percent after exposure
to two or three puffs.  Several commercial filter cigarettes gave essentially
the same result. The fact that these filters lower the phenol content by
70 to SO percent and trap about 4.0 percent of the particulate phase suggested
that neither phenolic nor particulate materials are responsible for the inhibi-
tion noted.  The next trial was with an absolute filter. that is, one which
removes the entire particulate phase and gives nonvisible gas. The obser-
vation that such treatment did not significantly alter the inhibitory effect
of the puff established that components of the gas phase are responsible for
inhibition of ciliary transport activity.  Assays of known components of
the gas phase showed the followin g compounds to possess such activity:
hydrogen cyanide, formaldehyde. acetaldehyde. acrolein, and ammonia, al-
though no one of these occurs at levels high enough to produce the effect
noted for smoke.
Activated carbons differ markedly in their adsorption characteristics.
Carbon filters previously employed in cigarettes do not have the specific
power to scrub the gas phase.  It has been reported that a filter containing
special carbon granules removes gaseous constituents which depress ciliary
activity (28) .
PESTICIDES AND ADDITIVES
Before 1930 practically the only insecticides used in the growing of to-
bacco were lead arsenate and paris green (the mixed acetate-arsenite salt of
copper).  Analysis of 6 brands of American cigarettes purchased in 1933
showed a range of 7.5-26.4 parts of As,O, per million, with an average value
of 13.9 ppm. (6).  Cogbill and Hobbs (S) found that main-stream smoke
of Cigarettes containing 7.1 pg. of arsenic per cigarette contains 0.031 pg. per
puff.  This amount would be equivalent to 0.25 pg. of arsenic per cigarette
(8 puffs), and hence a smoker consuming 2.5 packs of such cigarettes per
day might inhale 12.5 pg. of arsenic per day.  By comparison, analysis of the
atmosphere of New York City over a 12-year period indicated an average
content of 100-400 pg. of arsenic per 10 cubic meters, which is an approxi-
mate daily intake per person (38).
Extensive Federal efforts to discourage the use of arsenicals for the control
of tobacco hornworms on the growing tobacco crop resulted in a sharp de-
`This tapir is disrussel~ more fully in ~haptrr IO.
61

cline in the arsenic content of cigarettes after 1950. Thus, the average
arsenic content of 17 brands of cigarettes analyzed in 1958 was 6.2 ppm. of
As,O, (14).
It seems unlikely that the amount of arsenic derived even from unfiltered
cigarettes is sufficient to present a health hazard.
Chemicals recommended by the Department of Agriculture for the control
of tobacco insects are: malathion, parathion, Endosulfan, DDT, TDE, end&,
dieldrin, Guthion, aldrin, heptachlor, Diazinon, Dylox, Sevin, and chlordane
(42a). Trace amounts of TDE and endrin have been detected in commercial
cigarettes and cigarette smoke.  Guthion and Sevin residues were detected
in main-stream cigarette smoke at levels approximating 0.3 percent and l
percent of that added to cigarettes prior to smoking.  Tobacco treated with
Guthion and Sevin at the recommended levels showed no measurable con-
tamination of main-stream cigarette smoke (4b). (For discussion of car-
cinogenicity of tobacco pesticides, see Chapter 9.)
Cigarette manufacture in the United States includes use of additives such
as sugars, humectants, synthetic flavors, licorice, menthol, vanillin, and rum.
Glycerol and methylglycerol are looked on with disfavor as humectants be-
cause on pyrolysis they yield the irritants acrolein and methylyglyoxal.
Additives have not been used in the manufacture of domestic British cigarettes
since the Customs and Excise Act of 1952, Clause 176, and probably longer,
inasmuch as Section 5 of the Tobacco Act of 1842 imposed a widespread
prohibition on the use of additives in tobacco manufacture.
SUMMARY
Of the several hundred compounds isolated from the tobacco leaf, two
groups are specific to tobacco.  One of these groups includes the alkaloid
nicotine and related substances. The other includes compounds described as
isoprenoids. Cigarette smoke is an heterogeneous mixture of gases, uncon-
densed vapors, and particulate matter. In investigating chemical composition
and biological properties, it is necessary to deal separately with the particulate
phase and gas phase of smoke.
Components of the particulate phase other than the higher polycyclics
include aliphatic and alicyclic hydrocarbons, terpenes and isoprenoid hydro-
carbons, alcohols and esters, sterols, aldehydes and ketones, acids, phenols
and polyphenols, alkaloids, nitrogen bases, heterocyclics, amino acids, and
inorganic chemicals such as arsenic. potassium, and some metals.  Seven
polycyclic compounds isolated from cigarette smoke have been estahlished to
be carcinogenic. They are shown in Table 2. The over-all carcinogenic
potency of tobacco tar is many times the effect which can be attributed to
substances isolated from it. The d'ff
                           1 erence may be associated in part with
the presence in tobacco smoke of cocarcinopens, several of which have been
identified as smoke components.
Components of the gas phase of cigarette smoke have been shown to pro-
duce various undesirable effects on test animals or organs, one of which is
suppression of ciliary transport activity in trachea and bronchi.
62

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  714-422 O-64-6                                   63

  deciliated islands in the respiratory epithelium.  Ann Othol 65: 116
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18. Hilding. A. C.  On cigarette smoking. bronchial carcinoma and ciliary
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  New Eng J Med 251: 1155-60, 1956.
19. Holsti. P. Tumor promoting effects of some long chain fatty acids in
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20. Homburger, F., Tregier, A.  Modifying factors in carcinopenesis. Progr
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   4524-6, 1962.
22. Hurd, C. D., Macon, A. R., Simon. J. I.. Levetan, R. V.  Pyrolytic forma-
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   Chem Sot 84: 4509-15, 1962.
23. Hurd, C. D., Simon, J. I. Pyrolytic formation of arenes. 3. Pyrolysis
  of pyridine: picolines and methylpyrazine.  J Amer Chem Sot 84:
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24. Johnstone, R. A. W., Plimmer, J. R.  The chemical constituents of to-
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   144 (14) : 26-31, Apr 5, 1957.
27. Kennaway, E., Lindsey, A. J.  Some possible exogenous factors in the
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   557-61, 1958.
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   13.Duvatriene -I.:<-dials from tobacco.  J Org Chem 27: 396%95,
    1962.
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  temperature of cigarettes.  T 1 o )acco 144: (81 18-22, Feb 22, 1957.
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    1958.
-44. Van Duuren, B. L.  Some aspects of the chemistry of tobacco smoke.
  In: James. G., Rosenthal, T. eds. Tobacco and health. Springfield,
   Ill., Thomas. 1962.  Chapter 3, p. 33-47.
45. Van Duuren, B. L. The polynuclear aromatic hydrocarbons in cig-
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  components of cigarette smoke condensate.  J Org Chem 23: 473-5,
    1958.
47. Van Duuren, B. L., Schmitt, F. L. Isolation and identification of
  squalene from cigarette smoke condensate.  Chem Industr, 1006-7,
   1958.
18. Williams, J. F.. Garmon, R. G.  Beryllium in cigaret tobacco.  Tobacco
  Sci 5: 25-7, 1961.
49. Wynder, E. L., Fritz, L.. Furth, Iv.  Effect of concentration of benzopy-
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   1961.
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  The role of higher polycy-clic hydrocarbons.  Cancer 12: 1079-86,
   1959.
65

Chapter 7
Pharmacology and
Toxicology of
Nicotine

Contents
Page
GENERAL PHARMACOLOGIC ACTION OF NICOTINE ON
SERVE CELLS  ....................
EFFECTS ON THE CENTRAL NERVOUS SYSTEM  ...
CARDIOVASCULAR EFFECTS.  .............
GASTROINTESTINAL EFFECTS.  ............
DISTRIBUTION AKD FATE.  ..............
CHRONIC TOXICITY  ..................
SUMMARY  .......................
REFERENCES.  .....................
Figure
FIGURE I.  Summary diagram  of routes for the metabolism
of nicotine in mammals . . . . . . . . . . . . . . . . .
69
69
70
71
71
73
74
75
72

Chapter 7
GENERAL PHARYACOLOGIC ACTION OF NICOTINE ON
             NERVE CELLS
The pharmacology and chronic toxicity of nicotine. in dosage comparable
to the amounts that man may absorb from smoking or other use of tobacco.
are pertinent to an evaluation of health hazard.
The most notable action of nicotine involves a direct effect on sy-mpathetic
and parasympathetic ganglion cells ( 18 I.  This usually occurs as a transient
excitation, followed by depression. or even paralvsis with effective doses.
The ganglia are rendered more sensitive to acetylcholine initially and thus
make preganglionic impulses more effective.  Paralysis is associated with
diminished sensitivity of ganglia to acet\lcholine and concomitant reduction
in the intensity of postganglionic discharges.  Similar effects occur at the
neuromuscular junction, resulting in a curariform action in skeletal muscle
with adequate doses I 161.  In the central nervous system, as in ganglia,
primary stimulation is succeeded b! depression. Furthermore. nicotine like
acetylcholine discharges epinephrine from the adrenal glands and other
chromaffin tissue (20) ;  it also releases antidiuretic hormone from the
posterior pituitary by stimulating the supraopticohypophyseal system 13 ) .
Nicotine also augments various reflexes by excitation of chemoreceptors in
the carotid body ilO).
The pharmacological response of the whole organism at any one time
therefore, representing as it does the algebraic sum of stimulant and de-
pressant effects resulting from many direct. reflex, and chemical mediator
influences on autonomic nervous transmission and excitabilitv of virtually all
organ systems, defies accurate description. The wide variation in smoking
habits leads to every conceivable pattern of fluctuating blood levels of nico-
tine during the day.  This su ggests strongly that nicotine-sensitive cells may
be shifting continuously from excitation to depression. Such activity prob-
ably accounts for the unpredictable effects observed in different individuals
and in the same individual at different times.  Using the classic pharma-
cological approach, it is therefore virtually impossible to make reliable state-
ments regarding the effect of smoking on the many organ systems.  In order
to characterize the biological effects of nicotine in man, it thus becomes neces-
sary to place heavy reliance on symptoms and signs derived from clinical and
epidemiological studies.
EFFECTS ON THE CENTRAL NERVOUS SYSTEM
The action of nicotine on central nervous system functions has recently
been reviewed ( 20 1.  Very little of the reported work involves human
69

experimentation, and most of it is with doses much larger than are ass,,.
ciated with the act of smoking.  It suffices to note here that moderate doses
of nicotine elicit marked increases in respiratory, vasomotor, and emetic
activity, and still larger doses lead to tremors and convulsions, both in ani.
mals and man.  The amounts absorbed even in heavy smoking may produce
transient hyperpnea through carotid and aortic arch reflexes (5). The
increase in blood pressure which is commonly observed is partly central in
origin.  Nausea and emesis are more pronounced in the novice smoker but
may occur even in heavy smokers with excessive use of tobacco.  Electra.
encephalographic (EEG) studies in the intact rabbit (21) indicate that nice.
tine, in doses of 0.5 to 3.0 milligrams per kilogram, produced an "arousal
reaction" involving the hippocampus.  In a later stage of the same reaction
there appeared a discharge pattern similar to that noted in convulsions.
Lesions in the septum abolished the "arousal reaction," chlorpromazine and
evipan abolished the discharge pattern.  None of the congeners of nicotine,
including lobeline, produced similar patterns.
Knapp and Domino ( 12) found that concentrations of nicotine (10 to
20 pg/kg), a level commonly reached in man by smoking, produced EEG
arousal patterns in four species of animals, the rabbit, cat, dog, and monkey,
after neopontine transection.  Th ese effects did not appear to be related to
fluctuations in blood pressure or to catecholamine or serotonin levels.
In a study of electrical activity (as measured by electroencephalogram)
in 25 human subjects before and after smoking one cigarette, Lambiase and
Serra (15 ) noted an 80 percent depression in voltage and an acceleration in
frequency of the alpha rhythm which remained unchanged in form during
the recordings.  These alterations were more consistent in subjects over 35
years of age and were attributed to carbon monoxide and nicotine resulting
in cerebral anoxia and/or release of epinephrine. Hauser et al. (9), who
studied the EEG changes on cigarette smoking in healthy young adults, ob-
tained highly variable responses usually toward an increase in the dominant
alpha frequency of 1 or 2 cycles per second.  Some subjects showed sim-
ilar changes when puffing a glass cigarette stuffed with cotton and others
when puffing specially prepared nicotine-free cigarettes.  They concluded
that the effects noted were more likely to represent a psycho-physiologic
response to the act of smoking than to any substances present in cigarette
smoking. Bickford (1) arrived at a similar conclusion. Wide gaps of
information exist in this area and it is not meaningful to attempt inferences
concerning correlations of electrical events in the central nervous system
and subjective effects of smoking from the type of evidence currently
available.
CARDIOV.-\SCULAR EFFECTS
The cardiovascular effects of nicotine are described in Chapter 11, Cardio.
vascular Diseases.
70

GASTROINTESTINAL EFFECTS
hlost but not all experimental and clinical evidence supports the popular
\ iew that smoking reduces appetite ( 6: 17 p. 271 I.  This reduction has been
attributed both to direct effects on gastric secretions and motilit! and to
reflexes arising from local effects on the taste buds and mucous metnbranes
in the mouth. The unpredictable and temlborary elevation of blood sugar
is probably too small to contribute significantly f 17. p. 326). Nicotine
effects on the hypothalamus. comparable to the appetite reduction produced
II! other stimulants like amphetamine. and psychological mechanisms may
pIa!- significant roles ( 23 1.  Hunger contractions are inhibited but gastric
movements of digestion do not appear to he influenced sigtlificantlv 1)~
                                                      
moderate smoking (4 I.
Nausea, often associated with \-omiting. is 1,~ far the most common
31 tnptom related to the gastrointestinal tract.  This effect probably origi-
nales centrally in the tnedullarv emetic chemoreceptor trigger zone ( 14).
It is now generally agreed that nicotine stimulates peristalsis but the
mechanism is a cotnplex one. probahl! in\-&+ local. central and reflex
actions. Schnedorf and Ivy 121 I found \tide individual variation in gastro-
intestinal passage time in medical student smokers and non-smokers but
#rained the impression that
-                smokin g tends to augment motility of the colon.
These effects are probably related to actions on the parasympathetic gan$ia
in the bowel. The summative effects of all of these pharmacological actions
nn the whole intestinal tract do not produce a consistent pattern.  Excessive
smoking may be associated \+ith diarrhea. constipation. or alternating pat-
terns between the two extremes.  The only consistency is that s\mptoms
attributable to nicotine effects on the gastrointestinal tract are very common.
DISTRIHUTION AND FATE
Nicotine is actively and rapidly metabolized by man and other mammals,
the metabolites being in large measure excreted in the urine.  If any tissue
storage occurs, it is in such small quantity as to elude current analytical
Mnics. Nicotine is a rather unstable molecule \\hich in neutral or alka-
lifte conditions undergoes a varietv of changes.  A review of the current
`oncepts of the known and sugiested pathways for the metabolism of
nicotine is shown in Figure 1 (18).  The main intermediate appears
to be I - j -cotenine which yields y- (3.pyridyl) --/-methylamino butyric
acid. Cotenine has 10~ toxic& and lacks the potent pressor activity of
nicotine.
r) ogs receiving 150 tnp/kg.`day orally for 108 days exhibited no weight
"W or other objectire simns (2) .
irltrrvals for 6 da `s       Man has ingested 500 mg orally at g-hour
          ) witChout untoward effects.  Ro evidence has been pre-
ynted that the other known metabolites of nicotine carry an\- significant
"stemic toxicity.

         SUMMARY DIAGRAM OF ROUTES
   FOR THE METABOLISM OF NICOTINE IN MAMMALS
(Some hypothetical intermediates are shown in brackets.)
N,<WLiW          Ryhprroxkie          wydr<,xy"icdi"r*            Ilydruxycotinlne
      y-(a-l'yr,dyl)-y-mrLhylnmino),ulyreld~.~~d~                        "~Stdh~lC"tl"ill~
                                                  CH)
      y-(3-l'yiidyI)-y-n,~~rllyiR",i"~~bntyr,r Acid       cutinine             Cotinlnn Mrthon1um Ion
                                [VI

CHROItlC TOXICITY
Evaluation of the chronic toxicity of tobacco smoke may be considered
in several categories: la) the systemic toxicity of nicotine or its congeners,
I b I the systemic toxicity of other constituents of smoke or tobacco, carbon
monoxide and other compounds, (c) specific organ toxicity in certain sus-
ceptible individuals, such as those with Buerger's disease and allergic re-
sponses, (d I local effect of irritants on mucous and pulmonary membranes
by tars. phenols, the oxides of nitrogen, and others.  The latter three types
of potential toxicity are discussed in Chapter 9. Cancer, and Chapter 10.
Non-Neoplastic Respiratory Diseases.
It might appear that the least difficult problem in this group of variables
would be to assess the chronic toxicity of nicotine since we are dealing with
a comparatively simple organic compound of known composition and re-
action. Whereas there is a voluminous literature of studies involving
chronic exposure to nicotine or tobacco smoke in many animal species (17,
pp. 501-504), most of these are poorly designed and controlled and are of
little value for extrapolation to man.  For example, in the best nicotine
experiments involving life span studies, the daily dose of nicotine was near
the maximal tolerated dose (just subconvulsive J . which is greatly in excess
of any human smoking exposure.  Even though some authors I 11) observed
weight loss and degenerative vascular changes in rats under these severe
conditions. others 122i noted some weight loss but no histologic change.
In life span experiments in rats, with tobacco smoke in amounts approxi-
mating human smoking exposure, very little systemic toxicity was noted
18, 13).  Even though animal experimentation is inadequate, especially in
long-term effects of nicotine on large animal species. existing data permits
a tentative conclusion that the chronic systemic toxicity of nicotine is quite
low in small to moderate dosage.
The clinical literature is devoid of human data concerning chronic expo-
sure to nicotine alone, and the general statements regarding the chronic
toxicity of nicotine for man represent inferences drawn from chronic expo-
sure to tobacco in various forms, including industrial poisoning.  Repeated
exposure to tobacco in excessive amounts is reported to induce amblyopia,
arrhythmias, digestive disturbances, cachexia and a wide variety of other
signs and symptoms.  But the effects of excessive dose are of little concern
here.  The question is whether prolonged exposure to nicotine, in the quan-
tities absorbed systemically from smoking or other tobacco use, produces
toxic effects whidh result in unpleasant symptoms, dangerous signs, specific
degenerative disease, or shortening of the life span.  Unfortunately even a
tentative answer to this question must be obtained indirectly and by making
certain assumptions.  Inasmuch as nicotine is systemically absorbed from
all routes of administration, smoking, chewing, snuffing, or "snuff dipping,"*
it appears logical to assume that if the amounts of nicotine absorbed in the
various methods of use are of the same order of magnitude, any toxic effects
observed should also be in this order of magnitude.  There appears to be
general agreement that this is so.  Calculations indicate that the nicotine
"A small amount of snuff is placed in the groove between the teeth and thp lower lip
Or beneath the tonwe and held there from 30 minutes to several hours.
            r
73

absorbed (50-60 mpi from 6 cigars uninhaled equals that from 30 ciga.
rettes inhaled ( 19). Chewing tobacco may yield 8 to 87 mg in 6 to 8 hours
(24') ; in chewing snuff. 20-60 mg of nicotine (7).
The following variables play a role in the amount of nicotine absorbed
(17, p. 8, :
To sum up, the rate and amount of absorption of nicotine by the
smoker depend to a greater or less extent upon the following factors:
  1. Length of time the smoke remains in contact with the mucous
     membranes ;
2. pH of the body fluids with which the smoke comes in contact:
3. Degree and depth of inhalation;
4. Degree of habituation of the smoker ( ?) ;
5. Nicotine content of the tobacco smoked;
6. Moisture content of the tobacco smoked;
7. Form in which tobacco is smoked (cut [cigarettes] or uncut
   ] cigars] ) ( ?) ;
8. Length of butt;
9. Use of holder or filter;
10. Alkalinity or acidity of the tobacco smoke ( ?) ;
11. Agglomeration of smoke particles (more important in cigarette.
  smoking).
There is no acceptable evidence that prolonged exposure to nicotine creates
either dangerous functional change of an objective nature or degenerative
disease. The minor evidences of toxicity, nausea, digestive disturbances and
the like, are similar in kind and degree with all forms of use.
The fact that the over-all death rates of pipe and cigar smokers show little
if any increase over non-smokers is very difficult to reconcile with a concept
of high nicotine toxicity.  In view of the mortality ratios of pipe and cigar
smokers, it follows logically that the apparent increase in morbidity and
mortality among cigarette smokers relates to exposure to substances in smoke
other than nicotine. Unfortunately, th ere are no useful mortality statistics
in those who cheu, snuff. or "dip" tobacco, and the literature regarding in-
dustrial exposure is so confusing that little help is available here. The type
of projection made above. however unsatisfactory, is not inconsistent with
the animal toxicity data as well as the fact that nicotine undergoes very rapid
metabolism to substances of low toxicity.  The evidence therefore supports
a conclusion that the chronic toxicity of nicotine in amounts ordinarily ob-
tained in common forms of tobacco use is very low indeed.
SUMMARY
The pharmacological effects of nicotine at dosage levels absorbed from
smoking (l-2 mg per inhaled cigarette) are comparatively small; the
response in any point in time represents the algebraic sum of stimulant and
depressant actions from direct, reflex, and chemical mediator influences on
the several organ systems.  Th e predominant actions are central stimulation
and/or tranquilization which vary with the individual, transient hyperpnea,
74

peripheral vasoconstriction usually associated with a rise in systolic pressure,
suppression of appetitite. stimulation of peristalsis and. with larger doses.
nausea of central origin which may be associated w-ith vomiting.
Nicotine is rapidly metab-olized by man and certain other mammals. The
primary pathway through ( - I cotenine to r- ( 3-pyridyl ) -y-methylamino-
butyric acid is described in detail.  The known metabolites have verv- low
toxicity.
The rapidity of degradation to non-toxic metabolites, the results from
chronic studies on animals, and the low mortality ratios of pipe and cigar
smokers when compared with non-smokers indicate that the chronic toxicitv
of nicotine in quantities absorbed from smoking and other methods of to-
bacco use is very low and probably does not represent a significant health
problem.
REFERENCES
1. Bickford. R. G. Physiology and drug action: An electroencephalo-
  graphic analysis. Fed Proc 19: 619-25. 1960.
2. Borzelleca. J. F.. Bowman. E. F.. McKennis. H.. Sr. Studies on the
  respiratory and cardiovascular effects of ( - I cotenine.  J Pharma-
  co1 Exp Ther 137: 313. 1962.
3. Burn. J. H.. Truelove, L. H.. Burn. I.  The antidiuretic action of nico-
  tine and of smoking.  Brit Med J 1: 403-6, 1915.
4. Carlson. A. J., Lewis. J. H. Contributions to the physiology of the
   stomach.  14. The influence of smoking and of pressure on the abdo-
  men (constriction of the belt) on the gastric hunger contractions.
  Amer J Physiol 34: 149-54, 1914.
5. Comroe. J. H.. Nadel. J.  The effect of smoking and nicotine on respira-
   tion.  In: James. G., Rosenthal. T. eds. Tobacco and health. Spring-
  field. Thomas, 1962.  Chapter 17, p. 233-43.
6. Effect of smoking on appetite and on peripheral vascular disease.
  [Queries and minor notes] JAMA 119: 534, 1942.
7. Gaede. D.  Sur wirkung des schnupftabaks. Naunyn Schmiedeberg
  Archiv Exp Path, 130-45: 1944.
8. Haag, H. B., Weatherby, J. H., Fordham, D., Larson. P. S.  The effect
  on rats of daily-life span exposure to cigarette smoke.  Fed Proc 5:
  181, 1946.
9. Hauser, H., Schwarz. B. E., Roth. G., Bickford, R. G.  Electroencephalo-
graphic changes related to smoking. Electroenceph Clin Neuro-
10. H physiol 10: 576, 1958.
  eymans. C.. Bouchaert. J. J.. Dautrebande. L. Sinus carotidien et
  reflexes respiratories. 3. S ensibilite des sinus carotidiens aux sub-
  stances chimiques. Action stimulante respiratorie reflexe du sulfure
  de sodium, du cyanure de potassium, de la nicotine et de la labeline.
  Arch Int Pharmacodyn 40: 54-91, 1931.
`1. Hueper, W. C.  Experimental studies in cardiovascular pathology. 7.
  Chronic nicotine poisoning in rats and dogs.  Arch Path (Chicago)
  35: 846-56, 1943.
75