FIGURE 7.-Weakly acidic compounds in cigarette smoke

14-56


TABLE 16.-Major phenols in cigarette smoke

Phenol

   j@$arette
Nonfilter         Filter

Remarks'

Phenol
cJ-cresvl
m-++.hSO1
2&Dimethylphenol
Catechol
34fethylc&cbol
U&ethylcatechol
Hydroquinone
Reaorcinol
Eugenol
Isoeugenol
Scopoletin
Chlorogenic Acid
Rutin
,E-Naphthol

5CL130
al-40
40-70
1625
160-500
15-25
1525
5&m
154%
3-10
a-20
140-280
N.D.
N.D.
OS2

1050           1
7-20            1
15-W           1
612            1
6fxm           2
lo-20           2
10-20           2
N.D.2
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.

`Remarks: 1  = Tumor promoting agent on mouse skin
      2  = Cacarcinogen on mouse skin;
      -  - Inactive or not tested.
2N.D. - Quantitative data not determined.
SOURCE: Keith, C.H. (5.9, ?dwie, G.P. (61).

vapor pressure, no selective reduction by filter tips was observed for
catechols (Table 16).

Cyclopentanediones found as constituents of the weakly acidic
portion of tobacco smoke are considered important flavor compounds
in tobacco smoke. Their concentrations are highest in the smoke of
Oriental tobaccos, less in Burley and the least in flue-cured varieties
(921) (26). It appears that these compounds are not toxic.

Carboxylic Acids
A considerable number of carboxylic acids are present in tobacco and
tobacco smoke. More than 50 of these have been identified thus far in
smoke, accounting for 4 to 7 percent of the particulate matter. The
composition of the fraction of volatile carboxylic acids (CI to CS) is a
determining factor in the flavor of tobacco varieties. Oriental tobaccos,
for example, have a high proportion of ,&methylvaleric acid and also
contain hydroxyderivatives of vale& and /?-methylvaleric acid. Flue-
cured tobaccos are often high in acetic acid, whereas benzoic acid
predominates in Burley tobaccos. The non-volatile fatty acids in
tobacco range from G--CL with highest concentrations of palmitic acid
(CE), &-acids, stearic, oleic, linoleic and linolenic acids. These range
from 0.01 to 0.7 percent in dry tobacco leaf and from 1 to 3 percent in
the tar. The highest fatty acid concentrations are found for Turkish
tobacco and its smoke.

14-57


TABLE I?.-Free fatty acids in cigarette smoke

Palmitic
Stearic
Oleic
Linoleic
Linolenic

Acid

g/l g Tobacco smoked'

  Turkish 1  Bright  Maryland  Burley    Blend



    2s4      197     1M      5.5      152
     90      14      4.3      33       75
    108      39      32      21       53
    146     113      52      50       96
    329     310      66      52      240

Total (mg)
Wet TPM (mg)
5 fatty acids
70 of TPM (wet)

0.96     0.73     0.30     0.21      0.62
37.2     37.6     26.4     20.1      323


2.6      1.95     1.14     1.05      1.9

Woisture content of the toIwx%a varied between 11.5 and lZ.oR,
SOURCE: Hoffman, D. (4&J.

Transfer rates of unchanged fatty acids from tobacco into main-
stream smoke can be up to 20 percent, especially for the saturated
fatty acids of C&G8 chain length. Lower transfer rates are observed
for the CIS unsaturated fatty acids-oleic, linoleic, and linolenic acid.
Comparative concentrations of the major fatty acids in the smoke of
various cigarettes are presented in Table 1'7.

Although high concentrations of fatty acids play a role as tumor
promoters in model studies with BaP it appears that these fatty acids
are of lesser importance in tobacco carcinogenesis. About two dozen
hydroxy-y-lactones of G to G-acids have been identified in tobacco
smoke. They probably arise from tobacco leaf carbohydrates by
thermal degradation (81). y-La&ones have not been fully examined for
their biological significance in tobacco carcinogeneis. However, several
of these compounds are known alkylating agents and as such induce
sarcomas in rats (54).

Metallic Constituents

Minerals and other inorganic compounds in the tobacco plant derive
from soil, fertilizers, or agricultural sprays. The most prominent metal
ions in tobacco are Ca + + , Mg + + , K + , and Na + . During combus-
tion, the bulk of metallic constituents remain in the ashes, but some
compounds are vaporized or transferred into the smoke stream. With
the growing sophistication of analytical techniques, the list of trace
amounts of metals is increasing. Presently, 76 metals, including Bi, Si,
As, Se, and Te, excluding the post-uranium metals, have been detected
in cigarettes. Of these, 30 have been identified in the smoke (Table 18)
(`3%

14-58


TABLE l&-Metals in cigarette smoke particulate

                                          Metals for which

WC%)

good quantitative
data are not
  available

K
Na
Zll
Pb
Al
CU
cd
Ni
%I
Sb
Fe
AS
Te
Bi
Hg
Mn
La
SC
CT
Ag
se
co
ce
AU

70
1.3
0.36
0.24
0.22
0.19
0.12'
0.030
0.070
0.052
0.042
0.012'
0.006
0.004
O.o(L1
0.003
0.0018
0.0014
0.0014
0.0012
0.001
O.lWO2
O.WO2

Si
Ca
Ti
Sr
Tl
Pd

Ggarettes other than the University of Kentucky Keference cigarette
*Levela expresrd in Wmu of mdioaaivity
SOURCE: Norman. V. (6s).

With respect to tobacco carcinogenesis, special interest has focused
on As and Ni. The continued trend toward replacement of arsenical
sprays with other pesticides has been reflected in progressively lower
arsenic contents of leaf and smoke. Between 1940 and 1950, arsenic
values in the dry leaf of up to 50 to 60 ppm were reported for U.S.
tobaccos (31). The last published data for U.S. tobaccos range between
0.5 and 0.9 ppm (28). Between `7 and 18 percent of the total arsenic in
tobacco reappears in the mainstream smoke of cigarettes. Studies with
"As-labelled cigarettes have shown that, depending on the individual's
smoking patterns, 2.2 to 8.6 percent of the arsenic in cigarette tobacco
is transferred into the respiratory tract. About 50 percent of the
inhaled arsenic is eliminated within 10 days, primarily in urine; the
remainder is either deposited in body tissues or is exhaled or otherwise
eliminated (41).

All forms of nickel (metal, oxide, sulfide, salts, and carbonyl) tested
in the experimental animal were found to be carcinogenic. In nickel
factories, primarily in those converting nickel sulfide to nickel oxide,

14-59


workers have a high risk for cancer of the nasal cavity and cancer of
the lung. In cigarette tobacco, 2.0 to 6.2 pg Ni per cigarette were
reported; other tobacco products contained between 0.5 and 8.5 pg per
gram. In South Africa, nickel values of 52 and 88 pg per gram of Swazi
snuff were reported as a possible contributing factor in the high
incidence rate of cancer of the nose and in accessory sinuses in male
Bantus (5). During smoking, 10 to 20 percent of the nickel in the
tobacco is transferred into the mainstream smoke (62). In one study,
tentative evidence indicated that most of the nickel transferred into
the mainstream smoke (~10) is present in the gas phase (~8 percent)
(90). This and a model study suggest that nickel is present in the gas
phase of tobacco smoke as nickel carbonyl. Ni(CO)r is highly carcino-
genic in the respiratory tract of rats. It induces epidermoid carcinomas
and adenocarcinomas of the lung (89).

Several forms of cadmium are carcinogenic in the experimental
animal. Two studies suggest that occupational exposure to cadmium
oxide may increase the risk of prostate cancer (45). In mainstream
smoke, concentrations are 9-70 ng Cd per cigarette (&). It has been
suggested that a heavy smoker retains about 1.5 cog of Cd per day and
that he may accumulate up to 0.5 mg through inhalation.

Radioactive Compounds

Two types of radioactive compounds have been reported in tobacco and
tobacco smoke. These are the a-particle emitting elements of the
disintegrating radium and thorium series and the ,&emitters. In the
latter group, potassium-40 is the most abundant in tobacco products
(100). A sample of 100 U.S. and Canadian cigarettes was found to
contain 2,120 and 2,295 pCi of MK-derived p-activity, respectively. The
P-activity from @K in the mainstream smoke of 106 cigarettes was 15.9
and 9.4 pCi, corresponding to a transfer rate of 0.75 percent and .41
percent, respectively. "OK is a soft emitter with EL, of 1.3 meV.
The presence of radioelements =Ra, 210Pb, and ZlOPo in tobacco

products (e.g., from fallout, natural background) have been of special
interest and concern (69). The general range of 2lOPo in 1 g of U.S.
tobacco leaf varies from 0.15 to 0.45 pCi. In the smoke of one U.S.
cigarette, 2lOPo values of between 0.03 and 0.07 pCi were reported. The
average 21OPo content was ~0.036 PCi per cigarette or ~2.6 pCi of
2lOPo per 1 g smoke condensate with a 210Pb: 21OPo ratio of 0.66 + 0.23
(42). %2Pb has a half-lifetime of 22 years and decays by emission of
two &particles to 2%Po; the latter decays by a-emission with a half-
lifetime of 138.4 days. Preliminary studies indicate that most of the
2ioPb is concentrated in the nonvolatile and insoluble portion of the
particulate matter of cigarette smoke (58).

Analysis of human tissues demonstrated that the lung, blood, and
liver of smokers contain a higher concentration of 21OPo than do those
of nonsmokers. It has been calculated that a smoker's intake of 2*OPo is

14-60


reflected within several days by the observed excess burden of 3-10
pCi of 21OPb and 2lOPo in the lungs. Based on the measured
concentration of 2lOPo in epithelial samples, Little and Badford
estimated a maximum radiation dose of ~200 rem per 25 years to the
lower lobe bifurcations of the lung (56); however, others have
estimated a far lower effective radiation dose (14, 70).

After multiple intratracheal installations of 2*OPo in Syrian golden
hamsters, a dose-dependent increase was observed in epidermoid
carcinoma and adenocarcinoma in the peripheral lung fields (55).
Simultaneous and multiple intratracheal instillation of benzo(a)pyrene
(total dose 4.5 mg) and 21OPo (total dose 50,006 pCi) on the same carrier
induced twice the number of tumors expected from the additive effect
of either carcinogen alone (59).

Agricultural Chemicals

As in the case of arsenical pesticides, a significant reduction in the use
of chlorinated hydrocarbon insecticides on tobacco has occurred during
the last decade. This is reflected in the reduction of such insecticide
residues as DDD, DDT, endrin, and endosulfan on tobacco (Figure 8).
Whereas in 1968 70.2 percent of all U.S. flue-cured auction-marketed
tobaccos contained more than 10 ppm of DDT, in 1972 there was no
tobacco of the same type containing levels above 10 ppm of DDT. In
the latter year, 73.1 percent of the tobaccos marketed contained only
0.1 to 0.49 ppm of DDT (I?`). DDD values declined from levels of 2 10
ppm in 97.6 percent of the 1968 crop to levels no higher than 0.1 to 0.49
percent in 63.9 percent of the tobaccos marketed in 1972. Again, there
was no tobacco with levels of DDD above 10 ppm in 1972. Similar
reductions of insecticide residues on tobacco were reported for endrin,
dieldrin, and endosulfan (17, 30). A further gradual decrease of these
pesticides in tobacco is expected. During smoking, 11 to 18 percent of
DDT and DDD are transferred without change of structure from
tobacco into the mainstream smoke of cigarettes. DDE, DDM, and 4,4'-
dichlorostilbene (Figure 8), an immediate decomposition product of
DDT and DDM resulting from elimination of HCL and molecular
rearrangement, are also detected in mainstream smoke (39). One study
showed that levels of chlorinated hydrocarbon insecticides in adipose
tissues of smokers were not elevated above those in nonsmokers (18).
Other pesticide residues found on some U.S. tobaccos are parathion (up
to 0.03 ppm), carbaryl (up to 1.5 ppm), endosulfan (up to 2.9 ppm), and
toxaphene (0.7 to 3.4 ppm) (30).

Some of the chlorinated hydrocarbon insecticides and the isomeric
impurities present in the technical preparations, e.g., o,p'-DDD, are
possible or known carcinogens in experimental animals. One of the co-
carcinogens is 4,4'dichlorostilbene, formed by pyrolysis from DDT and
DDD (No). As discussed earlier, the carbaryl residue on tobacco may
give rise to a carcinogenic nitrosamine. Similarly, maleic hydraside and

14-61


Cl
0
0

Hk-cc13
0
0


Cl

cl
Q
0

kc-CH Cl 2
Q
0


Cl

DD-f                 DDD

0
0

C=CCI2
0
0

Cl

Cl
0
0

C=CHCl

Q
0

Cl

DDE                DDM

Cl

Gldnn

Cf
0
0

HC = CH
0
0

    Cl

02N -0

PSMhNNl

Malee Hydrazlde (MH)

FIGURE S.-Residues of agricultural chemicals in tobacco and
cigarette smoke.

14-62


its soluble salts have been mentioned. Present evidence is not
uniformly clear as to whether pure MH is mutagenic or carcinogenic,
though the weight of the evidence suggests it is mutagenic. (22, 32).

Tobacco Additives

Tobacco products are refined by the addition of additives, humectants,
tobacco casings, and flavor-enhancing compounds. The most widely
used humectants are propanediol, glycerol, diethylene glycol, triethyl-
ene glycol, and D-sorbitol (100). Humectants amount to 2 to 4 percent
of the original tobacco weight for cigarettes. Analyses of 18 U.S.
cigarette brands showed ranges of 0.46 to 2.24 percent of propylene
glycol and 1.7 to 3.15 percent of glycerol in the tobaccos (15). Smoke
analyses demonstrated that in filter cigarettes 9.9 percent and in
nonfilter cigarettes 12.6 percent of the propylene glycol in tobacco
reappear unchanged in the mainstream smoke. The glycerol transfer
rate into the mainstream smoke of filter and nonfilter cigarettes was
12 and 14 percent, respectively. The smoke of humectant-treated
cigarettes had increased amounts of acetaldehyde and acetone (53).
Transfer of humectants into the mainstream smoke is probably
significantly greater in pipe smoking than in cigarette smoking
because of the former's higher puff frequency (60).

The use of humectants in tobacco products has raised concern as to
their effects on smoke toxicity. Formation of volatile aldehydes and
ketones, including acrolein, from combustion of such humectants
would add to the ciliatoxicity of tobacco smoke. The glycols, especially
diethylene glycol, are suspected to influence the smoker's risk for
bladder cancer (44).

Pipe tobaccos may contain up to 30 percent of casing agents. These
are primarily sugars, starches, humectants, and plant extracted
isoprenoids. These casing agents influence the flavor of the tobacco
smoke, as well as the burning rate of the tobacco, and thus affect
smoke toxicity. When cigarette tobacco contained 5 percent or higher
levels of sugar additives, the resulting smoke was higher in furfural,
nicotine, and tar content than the smoke from an identical cigarette
without the sugar casing (86).

The flavor of cigarette smoke is also affected by the curing, aging,
and blend of tobaccos used. Considerations such as acreage yield and
tobacco prices during the last decade have resulted in changes of leaf
aroma affecting the tobacco blends and thus the smoke flavor. More
importantly, however, the trend toward low-tar, low-nicotine ciga-
rettes and toward a reduction of undesirable volatile smoke compounds
has brought about major changes in the smoke flavor of cigarettes.
The use of rolled stems and reconstituted tobacco sheet admixed with
leaf lamina and the use of effective filter tips are major factors
inducing changes in smoke flavor. All of these developments have led
to increased use of flavor additives, especially for low-tar, low-nicotine

14-63


TABLE 19.~Harmful constituents of cigarette smoke particulate

matter

I. Compounds judged most likely to contribute to the health
  hazards of smoking':
  Nicotine 5G2.500 Irg/cig         "Tar"2 %0-35,000 pg/cig
II. Compounds judged as probable contributors to the health
  hazards of smoking:
  Cresols (all 3 isomers)          Phenol 9-202 pg/cig
     63-97 pgkig
III. Compounds judged aa suspected contributor to the health
  hazards of smoking:
  DDT       04.77 &cig     Endrin O-0.06 pg/cig
  Hydmquinone   33 pg/cig       Nickel compounds 04.53 &cig
  Pyridine      25-213 @zig

`Vahes from May 1978 FTC lint

3'ar" contains the polynuclear aromatic hydrocarbona which are "generally saeepted IYI being responsible for a
substantial portion of the carcinogenic activity of the total "tar". "Tar" also contab ,%uxpbthylamine, a known
human bladder carcinogen for which there ia no known safe level of human expcuue.
SOURCE: U.S. Public He&h Service (93).

cigarettes. In fact, these new cigarettes require flavor corrections by
additives in order to be acceptable to the consumer. Tobacco extracts
as well as nontobacco flavors, such as licorice, coca, fruit, spices, and
floral compositions, are used. More recently, suggestions for synthetic
flavor additives for cigarette tobaccos are increasing in the patent
literature. At present., the selection of tobacco flavor additives from
the GRAS (Generally Regarded As Safe) List or from natural extracts
and the screening of their smoke decomposition products for toxicity or
other biological activity are not required by law and are done
voluntarily by manufacturers.

Toxic and Carcinogenic Agents-A Summary
The report of an expert panel on the "harmful constituents of cigarette
smoke" classified the harmful and possibly harmful smoke compounds
into the following categories: (1) contributors, (2) probable contribu-
tors, and (3) suspected contributors to the health hazard of smoking
(93).

The constituents of the particulate matter are listed according to
this classification in Table 19. Since 1970, when the harmful smoke
constituents were so defined, much progress has been made toward the
identification of toxic and especially of tumorigenic agents in cigarette
smoke. The identified tumorigenic agents and their quantities in
cigarette smoke are listed in Table 20. The majority of co-carcinogenic
agents in cigarette smoke remain to be identified.
The increased risk for cigarette smokers of cancer of the esophagus,
kidney, and urinary bladder suggests the possibility that cigarette

14-64


TABLE 20.-Known tumorigenic agents in cigarette smoke
       particulates

Compound        M/cig        Compound         &ig

I. Tumor Initiators

Benzo(akwene
Other P&H'
Dibetv.&)acridine
Other Am Arenea
Urethane

0.01-0.05
0.30.4
0.0&0.01
0.01-0.02
0.035

II. ~catinoge~

Pyrene
Other PAHZ
1-Methylindoles
%Methylcarbazoles
4. PDichlomstilbene
Catechol
Alkylcatechols

N'-Nitrosonomicotine
YN-Methyl-N-nitros-
amino)-l+pyridylj-
1-butanone
N'-N&rosoanatabine
Polonium-210
Nickel Compound
Cadmium compounds
FNaphthylamine
PAminobiphenyl
&Toluidine

0.14-3.70

0.11-0.42
  +3
O.O3XW?pCi
  k5.8
0.01Jl.07
0.0014022
0.001-0.002
0.16

`For detaila see Tshle 15
`For details see Table 15
~ncentmtiom unknown
SOURCE: U.S. Public He&b Service (9~).

smoke contains unidentified organ-specific carcinogens besides the
known trace amounts of carcinogenic aromatic amines and N-nitrosa-
mines.

14-65


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Physiological Responses to Cigarette Smoke

Previous editions of this report have examined acute and chronic
effects of cigarette smoke. Starting with epidemiological evidence and
buttressed by clinical and pathological findings, the role of cigarette
smoke has been implicated in numerous disease processes in humans.
. Since smoke is such a complex mixture of elements, experimental
work in humans must be augmented by animal studies in order to
define the specific role of particular smoke components. Inhalation
studies (33) must be designed to closely mimic smoke exposure in the
human population and provide data relating to: (1) understanding the
physiological or biochemical mechanism of action of whole cigarette
smoke or individual smoke components, (2) understanding of pathogen-
esis and early identification of endpoints which are predictive in
nature, and (3) screening potentially less hazardous cigarette models to
differentiate their relative influence on physiological or pathological
endpoints.

Bioassays must be designed with appropriate exposure modes, since
cigarette smoke-related diseases in man are usually chronic and
involve a history of prolonged interaction between smoke components
and target tissue.

Animal Smoke Inhalation Exposure Methodology
Smoke Generation

Exposure systems for tobacco smoke can be classified as active or
passive, depending upon the system used for generating cigarette
smoke.

Active exposure systems require the animal to generate the smoke
by drawing air through a lighted cigarette to simulate what happens to
the human smoker. McGill, et al. (30) used a water-reward system to
train baboons to puff on lighted cigarettes and to inhale cigarette
smoke. Once the animals were trained to take puffs of a specific
duration, it was possible to control the animal's smoking behavior by
manipulating the water reward per puff. The effectiveness of this
system was shown by the fact that the animals remained in good
health throughout the period of training and were able to achieve
blood carboxyhemoglobin levels similar to those of human smokers.
However, since most experimental animals will not cooperate as well
as baboons, passive devices in which smoke is generated by a machine
are commonly used. Passive exposure systems can then be further
classified as continuous or intermittent. A continuous system is one
which smokes a series of cigarettes at one time by using one or two
rotating discs or turrets to position the cigarettes at a smoking port
where the puff is usually drawn by a vacuum pump. By designing the
system so that a cigarette on one turret is being smoked while a

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cigarette on the other turret is being rotated into position, it is possible
to generate a nearly continuous stream of smoke (24).

In the intermittent system, smoke is generated either by applying
positive pressure to a chamber containing a cigarette and forcing
smoke out through the cigarette (36) or by a cam-activated plunger
which draws a puff of smoke and injects it into a holding tube (4
where it is allowed to stand. The smoke generated by the piston is a
closer approximation of the human smoke generation process than
earlier mechanical smokers. It can be more accurately controlled as to
puff volume, duration, and frequency and thus is the currently
preferred system.

Methods of Inhalant LIelivery

A great number of different exposure systems are available for
tobacco smoke inhalation experimentation. Since the goal of much of
the inhalation research currently being done is intended to simulate
human experience, some degree of compromise is usually involved in
selecting an inhalation system. The basic systems for delivering
tobacco smoke inhalants include:

(1) complete chamber exposure-the entire animal is exposed to the
inhalant (6,36).

(2) partial chamber exposure-only the nose of the animal is
exposed to the inhalant (29).

(3) face mask or mouth piece exposure-the inhaled smoke is
delivered to the nose or mouth through a mask or mouthpiece, with a
means of allowing expired smoke and air to be exhausted (8,35).

(4) tracheal exposure-the inhalant is delivered directly into the
trachea via a cannula inserted into a permanent tracheotomy (12).

The decision to use a particular exposure system is made after
considering factors such as selection of a suitable animal model; the
ability to control exposure levels, including delivery of smoke as a
bolus in a fresh air stream; system wash-in and wash-out times; the
ability to sample inhalant and/or test gases from the system inlet or
outlet; and the ability of the exposure system to deliver smoke to the
experimental animal while offering the least alteration of normal
respiratory function.

Lhsimetry

Administration of experimental inhalants via the pulmonary route
requires a description of the concentration, duration, and pattern of
inhalant exposure. Unfortunately, there is no simple relationship '
among these variables that will determine the dose delivered to a
specific site of interest in the experimental animal. Prime attention
must be given to the definition of real-life human exposure conditions
so that appropriate parameters can be incorporated into the experi-

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ment, although as noted by Nettesheim, et al. (33), the investigator
determines the smoke exposure conditions but the animal determines
smoke uptake or dose.

Periodic measurements to determine the amounts of cigarette smoke
components received by experimental animals can be just as complex
and equally as important as the endpoints used in the characterization
and evaluation of the effects of tobacco smoke exposure.

Among the indicators which have been used for monitoring smoke
uptake are blood levels of nicotine (go), urinary nicotine and cotinine
(II), and tracers such as decachlorobiphenyl (6, 7) and W-dotriacon-
tane (15). Each of these indicators has problems associated with it, such
as the need for lengthy extractions for nicotine and cotinine and the
requirements for homogenation of tissue samples prior to determining
decachlorobiphenyl content.

Blood carboxyhemoglobin (COHb) levels have often been given to
indicate that animals have inhaled the smoke, since carbon monoxide
absorption occurs primarily in the lungs. In a study of total particular
matter (TPM) deposition in the lungs of small mammals, Binns, et al.
(6) also examined COHb levels to determine the correlation between
these tests. They found that TPM could only be predicted from COHb
levels within fairly wide levels in a particular species and showed no
clear relationship when comparing different species.

Limiting Factors in Smoke Exposure

The major factor limiting the size of the dose in cigarette smoke
inhalation studies is the acute toxicity of carbon monoxide and nicotine
(33). In developing exposure regimens, it is important to consider acute
toxicity of these two substances as well as the irritant nature of smoke
when it is delivered to animals in high concentrations (7).

Excessive carbon monoxide buildup in blood, which can alter the
transport of oxygen of the experimental animal, is a common problem
in continuous exposure systems. To prevent toxicity of smoke, such
systems require excessive dilution or intermittent exposure, which can
lead to exposures of animals to smoke of different chemical and
physical properties. Although the same situation is true for acute
toxicity of nicotine, its half life is much shorter than that of carbon
monoxide.

Intermittent systems have also been found to be advantageous in
smoke exposure studies. These systems operate on a puff-hold-purge
cycle with a holding period which can be adjusted to prevent major
chemical and physical changes in the smoke. Rylander (38) has
reviewed some of the contradictory results which occurred with varied
smoke exposure conditions and has stressed the need to monitor smoke
dilution, exposure duration, and selective absorption of volatile water-
soluble smoke constituents.

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Selected Animal Studies

Since Cahan and Kirman (12) published a method of delivering smoke
to dogs in a controlled manner, the dog has been widely used as an
animal model. While their report was primarily a technique paper, the
authors noted increases in hematocrits and cardiac hypertrophy along
with pulmonary fibrosis and emphysema in the smoking group.

A further description of pulmonary morphologic changes induced by
smoking was published by Frasca, et al. (22). Their electronmicroscopic
findings included a complete loss or marked reduction in the number of
capillaries and a marked thickening of the septa due to increased
amounts of collagen in the lung parenchyma. They also found large
numbers of macrophages in both the pleura and parenchyma, occurring
singly and in clumps. Many of these macrophages contained crystal-
line-like structures in membrane-bound inclusions.

Male cynomolgus monkeys trained to smoke an average of I2
cigarettes a day for 5 days a week over 6 months showed no changes in
their epithelia of large airways but did exhibit aggregation of a large
number of macrophages in the alveoli (8). These macrophages were
clumped, pigmented with black/brown granules, and had foamy
cytoplasm. Pulmonary physiological changes were limited to increases
in pulmonary resistance, while tidal volume, respiratory rate, dynamic
compliance, and nitrogen washout were normal throughout the test
period.

Park, et al. (35) found that pulmonary mechanics and arterial blood
gases of dogs which smoked eight cigarettes per day showed no
significant differences until after 11 months of smoking, when
functional residual capacity fell slightly and respiratory resistance
rose. They attributed these changes, in part, to the smaller lung size of
the smoking dogs. As in earlier studies, an increased number of
alveolar macrophages were harvested from the lungs of smokers.
Functional changes in macrophages included an increased initial latex
uptake and a decreased bacteriosuppressive activity in smoking dogs.

Cardiovascular Studies

Chronic changes in cardiovascular functions due to tobacco smoke have
not been extensively investigated in intact animals. A study by Ahmed,
et al. (I) compared hemodynamics and left ventricular microscopic
structural changes after beagle dogs smoked seven cigarettes per day
or were given an equivalent intramuscular dose of nicotine daily for 22
months. They reported that both experimental groups had smaller left
ventricular ejection fractions and lower left ventricular dP/dt values,
both of which reflect a deficit in the contractile function of left
ventricular muscle. Mean aortic blood pressure was elevated in both
groups, indicating an increased peripheral resistance. Since the left

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ventricular contractility indices were still lower after acute phleboto-
my, it appeared that the left ventricular function was compromised
independently of the increased afterload. The only histological change
was an increased amount of collagen in the interstitium.

Armitage (2) administered puffs of smoke to anesthetized or spinal
eats and demonstrated transient increases in blood pressure. By
comparing these pressure changes with those observed when intrave-
nous injections of nicotine were given, he was able to obtain an
estimation of the pharmacologic "dose" of nicotine-like substance(s)
contained in a puff of smoke. The study demonstrated that the source
of the pressor response was in the particulate phase of the smoke
although it may not have been nicotine per se, since smoke from low-
nicotine cigarettes caused increased blood pressure similar to smoke
from a cigarette with a standard nicotine level.

The role of tobacco smoke in altering myocardial oxygen partial
pressure (MPo2) was studied by Rink (37) in a series of experiments in
open-chested cats with implanted oxygen electrodes. Intravenous
injections of nicotine or intratracheal puffs of smoke resulted in
transitory increases of blood pressure and slight increases in MPoz. It
was postulated that the effect of lower oxygen availability due to CO
in tobacco smoke was overshadowed by the actions of nicotine in
increasing myocardial blood supply.

The preceding studies have all indicated the adaptive nature of the
animal or organ system under study. While compensatory mechanisms
may serve to minimize the acute or chronic insult of tobacco smoke or
its specific components, the underlying assumption has been that the
system is "normal" or "healthy" and thus able to respond.

To examine the effect of tobacco smoke on an impaired cardiovascu-
lar system, Belle& et al. (5) produced myocardial infarcts in dogs by
ligating the anterior descending branch of the left coronary artery.
After allowing four days for recovery, ventricular fibrillation thresh-
old (VFT) was determined in control and smoking dogs with and
without infarcts. As expected, VFT was lower in dogs with myocardial
infarcts. In both control dogs and in dogs with acute myocardial
infarction, inhalation of cigarette smoke decreased VFT for up to 90
minutes after exposure. The authors noted that the effects of
myocardial infarction and cigarette smoke on the VFT were additive.

Exercise Tolerance

To investigate smoke-related impairments in physical exertions,
animals have been subjected to exercise programs involving swimming
or running on a treadmill before and after smoking. Hrubes and Battig
(26) trained rats to swim to the point of exhaustion. As the animals
became adapted to the program, endurance times rose from 5 to `7 or 8
minutes, but after acute smoke exposure, the endurance times fell to 5
minutes.

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Reece and Ball (36) examined electrocardiographic, blood enzyme,
and hematological data on dogs which ran on a treadmill for 10
minutes a day for a year. In the smoking group, electrocardiographic
change indicated cardiac enlargement, suggestive of left ventricular
hypertrophy. Of the enzymes studied, postexercise lactate concentra-
tions rose after smoke exposure began, reflecting a deficiency in
oxygen transfer, transport, or utilization, all of which occur with
carbon monoxide exposure. Other enzymes altered during smoke
exposure included glutamic oxaloacetic transaminase and creatine
phosphokinase. While there was no histopathological basis for these
changes, the authors noted the potential for the combination of
hypoxia and nicotine to inhibit the production of certain enzymes.

Toxicity of Specific Smoke Components
Since the list of harmful constituents in cigarette smoke was published
in 1972 in the report The Health Consequences of Smoking, there has
not been a notable increase in knowledge regarding the pathophysio-
logical role of many specific smoke components.

Rylander (38) reviewed experimental work dealing with aerosol and
volatile components of smoke and listed three requirements for
determining relative toxicity: (1) realistic dilution of the smoke as
drawn from cigarettes, (2) selective absorption of volatile, water-
soluble compounds from the smoke, and (3) realistic exposure duration.

These same criteria should apply to examination of specific
components of tobacco smoke. Many studies such as those which
determined LDa levels or reported results of continuous exposures
were considered not to represent smoke-related results.

Nicotine

In an early study to determine how nicotine in cigarette smoke could
cause an increase in heart rate, Burn and Rand (10) administered
nicotine to isolated rabbit atria. By comparing normal and reserpine-
treated atria, they found that nicotine caused increases in rate and
amplitude of contraction by releasing epinephrine and norepinephrine
from stores in the heart. Interest in the role of nicotine in
cardiovascular diseases processes has continued from that time, aided
in part by the availability and ease of administration of pure nicotine
solutions.

Ilebekk and Lekven (27) used a continuous infusion of nicotine to
examine the mechanical efficiency of the left ventricle during the
administration of approximately 2.1 mg of nicotine over a 5-minute
period, They found that nicotine increased cardiac contractility and
elevated left-ventricular-systolic and enddiastolic pressures. Thus,
even though peripheral vasoconstriction occurred, stroke volume was
increased by nicotine during these short-term studies.

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By comparing chronic smoke exposure and daily intramuscular
injections of nicotine, Ahmed, et al. (1) were able to demonstrate that
left ventricular performance did deteriorate over the course of 22
months. Ahmed reported that aortic blood pressure rose in both test
groups, so that nicotine appeared to be involved in the increased
peripheral resistance. Since both the smoking and nicotine groups
exhibited similar interstitial fibrosis in the middle layers of myocardial
tissue, nicotine appears to have a cardiotoxic effect which has
previously been ascribed to carbon monoxide.

The association between nicotine and hypertension is not as clearcut
as the two preceding reports may suggest. Fisher, et al. (21)
investigated the role of nicotine in atherosclerosis and experimental
hypertension in rabbits and found nicotine had no effect on either
disease process over a 90day period. While others had reported no link
between nicotine and atherosclerosis, the authors noted that the dose
of nicotine may not have been optimal to allow comparison with
previous work in the area of hypertension.
A report by Hansson and Schmiterlow (25) examined the distribution
of nicotine in various tissues and noted that the metabolism of nicotine
in isolated tissue slices was oxygen-dependent. In a study of nicotine
conversion rates in intact rata, Miller, et al. found that, while plasma
nicotine clearance rates were independent of peak plasma levels (31),
dose-dependent differences of nicotine distribution in tissues resulting
from changes in regional perfusion may have effected total plasma
clearance of nicotine. It thus appears likely that selective oxygen
availability as well as plasma nicotine levels may influence nicotine
catabolism in experimental animals.

Carbon Mon0xid.e

When pregnant rats were maintained in a CO atmosphere that
produced carboxyhemoglobin levels averaging 15 percent saturation,
their offspring exhibited reduced birth weights, decreased weight
gains, and lower brain protein levels than air-breathing controls (19).
While this study might be criticized for using continuous rather than
intermittent exposures, the data do suggest a highly sensitive indicator
Of CO toxicity.

Additional study of carbon monoxide toxicity also pointed out
another case of relative susceptibility, again using the rat bioassay.
When comparing tracheal pressure, blood pressure, and heart rate
responses in guinea pigs and rats exposed to 2.84 percent carbon
monoxide, Mordelet-Dambrine, et al. (3.2) noted that rats appeared to
be more sensitive, since they had lower survival times. These
differences may be due to differences in CO sensitivities, or they may
be due to anesthetic variables that are hard to quantitate across
species.

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To avoid anesthetic problems, Cramlet, et al. (13) used conscious dogs
that were chronically instrumented to provide continuous cardiovascu-
lar data with cannulae for blood sampling from left and right atria
while the dogs inhaled carbon monoxide. Measurements were made
when COHb reached 10, 26, and 30 percent saturation. The only
significant cardiac changes were heart rate increases at 26 and 36
percent saturation; arterial oxygen saturation was reduced at all
levels. The authors concluded that cardiac compensation was adequate
to prevent tissue hypoxia up to 30 percent COHb in healthy dogs.

In an effort to study the effects of carbon monoxide in dogs with
impaired hearts, DeBias, et al. (18) produced myocardial infarcts by
injecting latex spheres into the left coronary artery. Control and
infarcted dogs were exposed to carbon monoxide continuously for 14
weeks with serial electrocardiograms and hematologic evaluation.
Although COHb averaged 14 percent in exposed animals, the animals
remained in good health throughout the study.

Repeating the same protocol in cynomologus monkeys, DeBias, et al.
(17) found hematocrit, RBC, and hemoglobin levels altered by 3 weeks
of exposure to 100 ppm CO, with recognizable electrocardiographic
changes. The authors concluded that the sensitivity to CO was species-
related as well as dose-related.

Carrying these results one step further, the DeBias group (16)
examined the effect of carbon monoxide on ventricular threshold in
cynomologus monkeys. Animals with and without myocardial infarcts
produced by latex bead injections into the coronary artery were
exposed to 100 ppm CO for 6 hours. This CO level produced COHb
values of 9.3 percent compared to 1.1 percent in air-breathing animals.
It was noted that infarcted and CO-breathing animals both had lower
ventricular fibrillation thresholds, and that the effects were additive.

The lack of chronic studies on CO effects in animals and humans
suggests that such studies be undertaken to fill this void in our
knowledge, especially as it relates to smoking and related diseases.

Nitric Oxide

While nitric oxide is found in cigarette smoke in concentrations of zero
to 600 pg/cigarette (39), blood levels for humans, monkeys, and rats
have only recently been reported (23). Their data indicate that a
consistently low level of NO was maintained in the blood of both
smokers and nonsmokers. The lack of a significant difference between
smokers and nonsmokers sugggsts that a mechanism exists in
mammals to rapidly detoxify NO, and that exogenous NO appears to
have little effect on its steady state in blood.

Examining the role of NO at the cellular level, Arnold, et al. (3)
exposed tubes containing rat and bovine tissue to the gas phase of
cigarette smoke, nitric oxide, and room air and determined changes in
guanylate cyclase activity. This enzyme is involved in the formation of

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