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Nutrient Interactions and Toxicity

Vitamin A Depletion Induced by Cigarette Smoke Is Associated with the


Development of Emphysema in Rats1,2
Ting Li, Agostino Molteni,* Predrag Latkovich,* William Castellani*
and Richard C. Baybutt3
Department of Human Nutrition, Kansas State University, Manhattan, KS 66506 and *Department of Pathology
and Pharmacology, Medical School of the University of Missouri at Kansas City, Kansas City, MO 64108

KEY WORDS:

smoking

vitamin A deficiency

lung

Cigarette smoke is the major cause of chronic obstructive


pulmonary disease, a leading cause of death in the United
States (1). In these patients, two respiratory conditions are
most closely associated with cigarette smoking, chronic bronchitis and emphysema.
Emphysema is a destructive disease of the pulmonary parenchyma characterized by large air spaces in the lungs, which
are associated with a smaller number of alveoli. In 1964 the
U.S. Surgeon General first reported a potential relationship
between smoking and emphysema, and the connection was
strengthened by numerous epidemiologic and animal studies
(2). Having established that cigarette smoking is the leading
cause of emphysema, the focus has shifted to determining how
smoke-induced emphysema develops. Among the proposed
mechanisms, the protease/antiprotease and oxidant/antioxidant hypotheses are popular. The major tenant of the protease/
antiprotease theory is that cigarette smoking results in an
imbalance between the antiprotease, -1 antitrypsin and elastase, resulting in increased activity of elastase. The active

trachea

inflammation

elastase catabolizes the matrix protein elastin, which results in


a reduction in the number of alveoli and the creation of larger
air spaces or areas of emphysema.
The basis for the oxidant/antioxidant hypothesis is that the
oxidants in the cigarette smoke deplete the antioxidant supply
in the lung and cause oxidative injury to the tissues, leading to
emphysema. During exposure to cigarette smoke, large
amounts of oxygen free radicals are generated; these radicals
could damage the lipid components of the cell membranes as
well as the matrix components of the lung (3). Destruction of
lung matrix, especially elastin, may lead to emphysema. The
precise chemical and biochemical mechanisms for the injuries
induced by cigarette smoke have not been thoroughly explored.
An alternative hypothesis for the development of cigarette
smoke-induced emphysema attributes the disease to the consequences of vitamin A deficiency. Vitamin A and related
retinoids regulate normal lung development, maturation and
maintenance of the alveolar epithelium (4). Benzo(a)pyrene
(BP),4 a chemical compound found in cigarette smoke, administered to rats, induces vitamin A depletion in the lung and
liver (5). Previously, we found that vitamin A deficiency per se

1
Presented in part in abstract form at Experimental Biology 2001, March/April
2001, Orlando, FL[Li, T., Molteni, A., Latkovic, P., Castellani, W. & Baybutt, R.C.
(2001) Cigarette smoke-induced vitamin A depletion in rats is associated with
the development of emphysema. FASEB J. 15: A628 (abs.)].
2
Supported by the Kansas State University Cancer Center, Kansas Agriculture Experiment Station (Contribution #03100-J), and United States Department
of Agriculture grant #99352007602.
3
To whom correspondence should be addressed. E-mail: baybutt@ksu.edu.

4
Abbreviations used: ALT, alanine amino transferase; AST, aspartate amino
transferase; BP, benzo(a)pyrene; CT, control; GGT, -glutamyl transferase; LDH,
lactate dehydrogenase; RA, retinoic acid; RAR, RA receptor; SM, smokeexposed rats; TPM, total particulate matter.

0022-3166/03 $3.00 2003 American Society for Nutritional Sciences.


Manuscript received 14 March 2003. Initial review completed 8 April 2003. Revision accepted 15 May 2003.
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ABSTRACT We showed previously that vitamin A deficiency per se causes emphysema. Benzo(a)pyrene, a
constituent in cigarette smoke, induces vitamin A depletion when administered to rats; therefore, we tested the
hypothesis that cigarette smoke induces vitamin A depletion, which is associated with the development of
emphysema. Male weanling rats were fed a purified AIN-93G diet and divided into two groups. The experimental
group was exposed to cigarette smoke from 20 nonfiltered commercial cigarettes/d for 5 d/wk, whereas the control
group was exposed to air. After 6 wk, tissues were collected for histological and biochemical analyses. Retinol
levels were measured in serum, lung and liver. The trachea, lung and liver were examined for histological changes.
Vitamin A levels decreased significantly in serum, lung and liver of smoke-treated rats. Histological examination
revealed the presence of interstitial pneumonitis along with severe emphysema. There was a significant inverse
relationship between vitamin A concentration in the lung and the severity of emphysema (r 0.69 and P 0.03).
Detachment or hyperplasia (and metaplasia) of the tracheal epithelium and liver vacuole formation also were
evident in the smoke-treated rats. The results of this research indicate that exposure to cigarette smoke induces
vitamin A depletion in rats, which is associated with the development of emphysema. J. Nutr. 133: 2629 2634,
2003.

LI ET AL.

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MATERIALS AND METHODS


Animals. Male Sprague-Dawley weanling rats (Harlan Sprague
Dawley, Indianapolis, IN) were housed individually in stainless steel
cages at room temperature, 24C under a 12-h light:dark cycle
(light 0600 1800 h) with a relative humidity of 50%. Animal care
and use were approved by the Institutional Animal Care and Use
Committee of Kansas State University. Rats were fed a standard AIN93G diet (10) and consumed food and water ad libitum. Food intake
was recorded daily and body weight was measured once each week.
Cigarette smoke exposure conditions. Rats were randomly distributed into two groups, those exposed to cigarette smoke (SM) or
those expose to air (CT, control). The smoke-exposure group was
exposed to 20 commercial nonfiltered cigarettes/d, 5 d/wk. The smoke
treatment was modeled after previously published studies (11). Rats
were placed in a plastic chamber that was 65 cm long, 50 cm wide,
and 45 cm high with three holes, two for holding the cigarettes at one
end of the chamber and another on the opposite side of the chamber
connected to a tube attached to a Leeson vacuum pump (model
#A6C17EB20.1; Labconco, Kansas City, MO). The rats were exposed
to 5 min of smoke followed by 5 min of air until all 20 cigarettes were
consumed, which took 11.5 h. CT rats were placed in another
chamber but exposed to air only. The extent of exposure of rats to
cigarette smoke was ascertained by measuring total particulate matter
(TPM) inhaled. Briefly, TPM was determined by using Pall Gelman
25-mm filters (Pall, Filterite Division, Timonium, MD) throughout
the area in which the rats were located in the smoke chamber. The
weight difference of the filter paper was determined after the full
treatment duration (20 cigarettes). The mean concentration of TPM
was 13.1 3.4 (SEM) g/L, with a total exposure of 467 120 g/d
for the SM group and 0.0 g/L and 0.0 g/d for the CT group (air
exposure). As a measure for the gaseous phase of the smoke, the
percentage of blood carboxyhemoglobin was determined to be 3.68
0.08% for the SM group and 1.36 0.04% for the CT group.
Serum and tissue collection. On the termination day, rats were
anesthetized with sodium pentobarbital intraperitoneally. Blood was
drawn from the abdominal aorta and the serum was separated and
stored at 70C until analysis. Liver, heart and lung were removed at
necropsy and weighed. A lobe of liver was collected and fixed with
10% buffered formalin for 1 wk. Another lobe of the liver was
immediately frozen in liquid nitrogen and stored at 70C until
analysis. The left lobe of lung was inflated with uniform pressure (9
cm H2O) with 10% buffered formalin and placed in the same solution. The right lung was frozen in liquid nitrogen immediately and
stored at 70C. A section of the trachea was also fixed in formalin
for histological analysis.
Morphological assessment. After the tissues were fixed in formalin, they were embedded with paraffin and cut in the sagittal plane.

The sections were stained by hematoxylin and eosin and examined by


light microscopy. Massons trichrome staining for collagen and
Weigerts staining for elastin were also applied to representative
samples from each group. A semiquantitative evaluation of histological damage to the lung, liver and trachea was carried out according
to previously published methods (12,13). Briefly, two pathologists,
unaware of the treatments, subjectively evaluated the tissues for
specific types of injury. Mean scores were assigned to each tissue.
Scores ranged from 10 (presence of definite damage) to 40 (very
severe damage). A score of 5 indicated some areas of the sections
had the specific damage but some areas were without damage. Tissue
with normal appearance received a score of 0. The means of the
assigned values for each group were reported.
Retinol analysis. Serum, lung and liver were analyzed for total
retinol concentration by the method of Ross (14). The samples were
extracted, saponified and quantified by HPLC analysis. The details
were published previously (6). The mean retinol recovery rate was
91% for liver, 93% for lung and 90% for serum.
Serum enzymes. Serum alanine aminotransferase (ALT), -glutamyl transferase (GGT), aspartic aminotransferase (AST) and lactate dehydrogenase (LDH) were measured by the Dade Dimension
R&L analyzer (Newark, DE) using the manufacturers reagent.
Statistical analysis. All data are presented as means SEM.
Treatment effects were analyzed using one-way ANOVA with the
general linear model procedure (SAS Institute, Cary, NC). Linear
regression analysis (SAS Institute) was used to determine the correlation between lung vitamin A concentration and the severity of
emphysema. In all analyses, P 0.05 was considered significant.
For the histological analysis, all statistical evaluations were performed with the Astute add-in for Microsoft Excel (DDU Software,
Leeds, UK). Probabilities were derived by Kruskal-Wallis one-way
ANOVA as determined by software. Probability estimates were derived by the Mann-Whitney U test from the statistic determined by
the software referenced to tables of critical values of the U test for
small sample size (15).

RESULTS
General observations. None of the rats died before the
termination day. Daily food intake did not differ between SM
and CT, nor did mean weight gain/wk (CT 34.75 6.05
g/wk; SM 31.07 5.33 g/wk). Initially, there was some
vasculitis and hemorrhaging in the lungs of SM rats at 4 wk,
but this was not observed at 6 wk (data not shown). Lung,
heart and liver to body weight ratios in SM rats were significantly greater than in CT rats (Table 1).
Histopathological evaluations. The lungs of CT rats killed
at wk 6 were essentially normal (Fig. 1A). Interstitial pneumonia and severe diffuse emphysema were observed histologically in the lungs of SM rats (Fig. 1B and C, respectively).
These changes were quantified in relation to the severity of the
lesion and summarized (Table 2). The inflammatory process
was characterized by alveolar septal thickening, septal infiltration by erythrocytes and chronic inflammatory cells (mostly

TABLE 1
Body and relative organ weights of rats exposed to cigarette
smoke (SM) or air (CT) for 6 wk1
Treatment Body weight Lung weight
g
CT
SM

244 12
224 7.3

Heart weight

Liver weight

g/100 g body
0.52 0.01
0.60 0.04*

0.37 0.01
0.41 0.01*

2.84 0.06
3.05 0.05*

1 Values are mean SEM, n 4, CT; n 6, SM. * Different from


control (CT), P 0.05.

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caused emphysema in rats (6). In addition, administration of


all-trans-retinoic acid (RA), an active metabolite of vitamin
A, reversed experimental elastase-induced emphysema by increasing the number of pulmonary alveoli (7). Furthermore,
postnatal treatment with RA increased the number of alveoli
relative to those in the lungs of control rats (8). Moreover, RA
was found to increase elastin in rat lung fibroblast culture (9).
Taken together, these studies suggest that vitamin A plays a
critical role in preventing the development of emphysema.
Thus, we hypothesized that cigarette smoke may induce vitamin A depletion, which in turn contributes to the development of emphysema.
The purpose of this study was to determine whether lung
vitamin A status is altered in response to cigarette smoke
exposure and whether this is associated with the development
of emphysema. We evaluated the histological appearance of
the lung and measured the levels of vitamin A (retinol) in the
lung and serum after smoke exposure in comparison with
air-exposed controls. Because the liver is the major site for
vitamin A storage, we also measured liver vitamin A levels
and evaluated the histological appearance of liver in response
to cigarette smoke exposure.

VITAMIN A DEPLETION AND DEVELOPMENT OF EMPHYSEMA

macrophages), with a scattered presence of these cells within


the alveolar space. A number of small caliber arteries also
showed mild vasculitis, characterized by thickening of the
artery wall (mostly the media), some periadventitial edema
with few scattered macrophages, and a modest reduction of the
arterial and arteriolar lumen. Pneumonitis was present along

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TABLE 2
Semiquantitative evaluation of the morphological changes
observed in the lung of rats exposed to cigarette
smoke (SM) or air (CT) for 6 wk1
Treatment

Pneumonia

Emphysema

Elastin deposition

CT
SM

2.5 1.4
15.0 3.2*

2.5 1.4
16.7 2.1*

10.0 0.0
5.8 0.8*

1 Values are mean SEM, n 4, CT; n 6, SM. Numerical values


were assigned according to the observers impression of the severity of
the tissue injury. * Different from control (CT), P 0.05.

DISCUSSION
In the present study, we investigated histological changes
caused by exposure to cigarette smoke and measured vitamin
A levels in the exposed rats. Our data demonstrated that
exposure to cigarette smoke: 1) induced vitamin A depletion

FIGURE 1 Representative histological sections of the lung of normal


and cigarette-exposed rats. (A) Tissue section of the lung of a control
rat. No abnormalities were found. (B) Section of an area in the lung of
a rat exposed to cigarette smoke for 6 wk. The alveolar septa were
thickened significantly with infiltration of inflammatory cells. Diffuse
interstitial pneumonia was present. RBC were spread in the septa and
the alveolar lumen. (C) Histological section of another area of the lung
of the same rat depicted in Fig. 1B. The alveolar wall was thinner than
normal. Emphysematous areas, dilation of many alveolar spaces and
destruction of the septa walls were evident. RBC and a few chronic
inflammatory cells were also present. Staining: trichrome; magnification: X400.

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with diffuse areas of emphysema in the lung of SM rats, which


involved the whole organ (Fig. 2). Elastin stain also was less
evident in SM rats relative to controls (Table 2).
Large areas of detached epithelium and hyperplasia of the
tracheal epithelium were seen in SM rats (Fig. 3B) compared
with CT rats (Fig. 3A). There also were a few areas of squamous metaplasia. The mean assigned score for detached epithelium and hyperplasia were 33.7 13.7 and 12.5 1.7 for
the SM rats, respectively, which was significantly higher than
the CT rats (1.3 1.3 and 1.3 1.3, respectively). Similar
conditions were noted in the large bronchi of the same rats. In
addition, inflammatory cells and cellular debris were observed.
The liver of SM rats showed moderate vacuolization of the
hepatocytes (8.3 1.7) that was greater (P 0.05) than the
mean assigned score for the CT group (1.3 1.3). Fibrosis was
not apparent, but mild deposition of collagen was observed in
the portal triad of these rats. Control rats did not have any
abnormalities, with the exception of minimal fatty infiltration
in one rat.
Serum, lung and liver levels of retinol. After 6 wk of
smoke exposure, retinol levels were significantly decreased in
SM rats in serum, lung and liver (Table 3). There was a
significant inverse correlation (r 0.69, P 0.03, n 10)
between the retinol concentration in the lung and the severity
of the emphysema in all rats (Fig. 4).
Biochemical analysis of serum. After 4 wk, serum GGT
and AST were significantly higher in the SM group compared
with the CT group (data not shown), but returned to control
values after 6 wk. The values for ALT and LDH for the SM
group did not differ from controls throughout the study.

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LI ET AL.

in the lung, serum and liver; 2) resulted in pulmonary inflammation and emphysema with vitamin A concentration in the
lung significantly inversely related to the severity of emphysema; 3) reduced elastin staining in the lung; 4) caused detachment or hyperplasia of tracheal epithelium; and 5) increased vacuole formation (fatty infiltration) in the liver.
To our knowledge, this study provides the first evidence
that cigarette smoke inhalation by rats decreases lung, serum
and liver retinol levels. Because food intakes did not differ
between the groups, the depletion appeared to be caused by
cigarette smoke per se. These finding are supported by others
(5,16) who administered BP, a constituent of cigarette smoke,
to rats, causing vitamin A deficient lungs and livers. In addition, others have found that intraperitoneal administration of
tobacco extract or a tobacco constituent, N-nitrosonornicotine, decreased the hepatic pool of vitamin A (17). Furthermore, exposure to cigarette smoke induces the production of
the lung and liver cytochrome P450 isoforms, CYP1A1 and
CYP1A2 (18,19), which increase the catabolism of RA
(18,20) and may lead to vitamin A depletion. Although any of
these may have depleted vitamin A levels, the precise mechanism remains to be determined.
The vitamin A depletion in our smoke-exposed weanling
rats is consistent with other smoke exposure studies. When
adult ferrets were exposed to cigarette smoke, retinoid catabolism increased, resulting in significantly lower levels of RA in
the lung (21,22). Despite this increased catabolism, lung retinol concentrations were not significantly altered in that
study. Although we did not measure RA in the present study,
the decreased tissue retinol concentrations suggest an increased retinoid catabolism. The reason why significantly
lower levels of retinol were not observed in the ferret study is
not known, but may be due to differences in the age of the
animals. Adult ferrets have larger liver stores of vitamin A and
may have compensated more effectively for losses in the lung.
The amount of smoke exposure may be another important
determinant for the smoke-induced vitamin A depletion.
When weanling guinea pigs were exposed to a low dose of 6
cigarettes/d for 6 wk, the levels of retinol in the lung increased

FIGURE 3 Representative histological sections of the trachea of


normal and cigarette smoke-exposed rat. (A) Trachea section of a
control rat. No abnormalities were observed in the surface epithelium
(black arrows). (B) Section of the trachea of a smoke-exposed rat. The
epithelium of the trachea showed marked hyperplasia and mild metaplasia (black arrows). Inflammatory cells were present and areas of
detached epithelium were observed (white arrow). Staining: trichrome;
magnification: X400.

(23). In contrast, when we exposed weanling rats to a higher


dose of 20 cigarettes/d for 6 wk, lung retinol decreased. Studies
are currently underway to evaluate the dose-response effect of
TABLE 3
Retinol concentrations of serum, lung and liver of rats
exposed to cigarette smoke (SM) or air (CT) for 6 wk1
Treatment

Serum

Lung

mol/L
CT
SM

4.4 0.7
2.3 0.3*

Liver
nmol/g

27.9 2.1
21.8 1.1*

104.3 6.6
60.4 13.0*

1 Values are mean SEM, n 4, CT; n 6, SM. * Different from


control (CT), P 0.05.

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FIGURE 2 Representative histological section of the lung of a rat


exposed to cigarette smoke. The lung section shows large area of
alveolar space enlargement, thinning of alveolar wall in other areas and
extensive destruction of septa wall. Staining: hematoxylin and eosin;
magnification: X100.

VITAMIN A DEPLETION AND DEVELOPMENT OF EMPHYSEMA

FIGURE 4 Correlation between lung vitamin A concentration and


emphysema evaluation score of a rat exposed to cigarette smoke for 6
wk (r 0.69, P 0.03).

be caused by excessive neutrophil elastase activity (38) or by


metalloproteinases (39). Retinoic acid inhibits the activity of
human leukocyte elastase (40) and metalloproteinase activity
(41). During systemic infection or inflammation, increased
neutrophil elastase activity is regulated by 1-proteinase inhibitor (1-PI), which contributes most of the functional
antielastase protection of the alveolar wall (42). Retinol and
retinaldehyde, but not RA, increased 1-protease inhibitor in
corneal epithelial cells (43). Further work is necessary to
determine whether these effects would be replicated in the
lung.
Metaplasia or detachment of the tracheal epithelium were
also detected in smoke-exposed rats in the present study. This
finding was consistent with those of others (44,45) who observed metaplastic areas in cultured tracheal epithelium after
exposure to cigarette smoke. In addition, vitamin A supplemented in the diet has been shown to reverse tracheal squamous metaplasia (46). Furthermore, the cigarette smoke constituent BP impairs uptake of retinol by tracheal cells (47).
The detachment of tracheal epithelium may be due to decreased cell adhesion, which has been previously found in
cultured tracheal epithelial cells of vitamin A deficient hamsters (48).
Vacuolization of hepatocytes in smoke-exposed rats, as
found in this study, is a typical indicator of steatosis (fatty
infiltration) or multivesicular lysosomes, usually representing a
toxic response. Liver injury alters vitamin A status by activating the hepatic stellate cells, the major storage site of vitamin
A, and depleting its vitamin A content (49). This vacuole
formation is found in a variety of liver diseases and is associated with lower hepatic vitamin A levels (50). In our previous
study in which rats consumed a diet without vitamin A, we
also noticed numerous vacuoles in the hepatocytes in the
vitamin A deficient rats (6).
In summary, the results of this study provide evidence that
cigarette smoke induces vitamin A depletion in the serum,
lung and liver, and that the compromised status of vitamin A
is associated with the development of emphysema, as shown by
an inverse correlation between lung vitamin A and the development of emphysema. Further studies are underway to determine whether improved vitamin A status could prevent or
slow the development of emphysema in rats.
ACKNOWLEDGMENTS
The authors thank Sung I. Koo, Denis Medeiros and Weiqun
Wang for their critical review of the manuscript. We are grateful for
the assistance of other Kansas State University and Truman Medical
Center faculty, staff, graduates and undergraduates for their technical
assistance.

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