The Effects of Passive Smoking on Laryngeal and

Tracheal Mucosa in Male Wistar Rats During Growth:

An Experimental Study
*,†,§Henrique Zaquia Leão, *,†,‡Claudio Galleano Zettler, †,§Eduardo Cambruzzi, §Marcelo Lammers,
§Paula Rigon da Luz Soster, ¶,**Fernanda Bastos de Mello, ††Guilherme Reghelin Goulart,
††,‡‡Deivis de Campos, and *,§Geraldo Pereira Jotz, *‡§¶**‡‡Porto Alegre, Brazil, †Porto Velho, Brazil, and
††Santa Cruz do Sul, Brazil

Summary: Cigarettes contain toxic and carcinogenic substances. In this context, cigarette smoking, and similar ac-
tivities, are associated with numerous pathologies, being considered a risk factor in up to 10% of the total number of
deaths in adults. Recent evidence suggests that the exposure of children to smoking in the early days of their devel-
opment causes many diseases. Using light microscopy, this study aims to analyze the possible histopathological effects
of an experimental model of chronic inhalation of cigarette smoke (passive smoking) on the laryngeal and tracheal
mucosa of young Wistar rats. A total of 24 young Wistar rats were studied for a period of 120 days. The animals were
divided into two groups: passive smoking (n = 16) and control (n = 8). The level of exposure to cigarette smoke was
evaluated from the urinary cotinine level. Although no cancerous lesions were identified, histopathological analysis in
the laryngeal and tracheal mucosa of all the animals in the experimental group showed that the proportion of moder-
ate and focal inflammation was higher in animals exposed to chronic inhalation of cigarette smoke (P = 0.041).
Histopathologic analysis revealed moderate and focal inflammatory lesions in the region of the infraglottic mucosa in
exposed animals, although without dysplastic or neoplastic lesions in the laryngeal and tracheal mucosa.
Key Words: Cigarette–Larynx–Trachea–Young rats–Histopathology.

INTRODUCTION young Wistar rats subjected to an experimental model of chronic

The ambient smoke (AS) released by burning tobacco is a mixture
of smoke directly exhaled by smokers (primary stream smoke)
and smoke released by the burning distal portion of the ciga-
rette (secondary stream smoke). AS can cause harmful effects
in people who inhale it. People exposed to AS are called passive
smokers.1–3
Among the diseases most frequently induced by cigarette
smoking in adults are oral, oropharyngeal, and esophageal cancer,4
with squamous cell carcinoma being the most common histo-
logical form. Cigarette smoke is thought to induce laryngeal
tumors in hamsters,5 and at least one study has shown an in-
crease of 1% to 9% in the incidence of adenomatous lung tumors.6
Our hypothesis is that during development, the mucosa in dif-
ferent glottal spaces and the tracheal mucosa show different
responses to the aggression promoted by cigarette smoke. There-
fore, this study aims to qualitatively analyze the possible
histopathological effects on laryngeal and tracheal mucosa in
Accepted for publication December 24, 2015.
Conflict of interest: There are no conflicts of interest on the part of the authors.
Financial support: Financial support was provided by the Universidade Federal do Rio
Grande do Sul.
From the *Post-graduate Program in Health Sciences, Universidade Federal de Ciências
da Saúde de Porto Alegre—UFCSPA, Porto Alegre, Brazil; †School of Medicine, Universidade
Luterana do Brasil—ULBRA, Canoas, Brazil; ‡Pathology Department, Hospital Santa Casa
de Misericórdia de Porto Alegre—ISCMPA, Porto Alegre, Brazil; §Morphological Sci-
ences Department, Universidade Federal do Rio Grande do Sul—UFRGS, Porto Alegre,
Brazil; ¶Pharmacosciences Department, Universidade Federal de Ciências da Saúde de Porto
Alegre—UFCSPA, Porto Alegre, Brazil; **Laboratory Animal Reproduction and Exper-
imentation Center (CREAL), UFRGS, Porto Alegre, Brazil; ††Biology and Pharmacy
Department, Universidade de Santa Cruz do Sul—UNISC, Santa Cruz do Sul, Brazil; and
the ‡‡Department of Basic Health Sciences, Universidade Federal de Ciências da Saúde
de Porto Alegre—UFCSPA, Porto Alegre, Brazil.
Address correspondence and reprint request to Geraldo Pereira Jotz, Rua Dom Pedro II
891 conj. 604. Cep: 90550-142, Porto Alegre, RS, Brazil. E-mail: geraldo.jotz@terra.com.br
Journal of Voice, Vol. 31, No. 1, pp. 126.e19–126.e24
0892-1997
© 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jvoice.2015.12.014
cigarette smoke inhalation.
We divided the larynx into three internal spaces with differ-
ent functions: the supraglottic, which separates the gastrointestinal
tract from air; the glottal, with voice function; and the infraglottic,
which connects to the trachea and is comparable to the adja-
cent tracheal lumen.7
METHODOLOGY
Induction of experimental model
This study was approved by the Ethics Committee on Animal
Use (CEUA) of the Universidade Federal do Rio Grande do Sul
under protocol n° 19.127 and the Ethics Committee on Animal
Use (CEUA) of the Universidade Federal de Ciências da Saúde
de Porto Alegre (UFCSPA), Brazil, under protocol 084/12.
A total of 24 male Wistar rats (Rattus norvegicus) were ob-
tained from the research in animals sector of the UFCSPA, RS,
Brazil. The animals were randomly divided into two groups:
Group F (passive smoking; n = 16) and group C (control; n = 8).
The number of animals for this type of experiment has been sta-
tistically validated.8 The induction of the animals to passive
smokers was conducted in accordance with previous experi-
mental protocols,5,9–11 as follows.
Exposure to smoke
The animals in group F were placed in plastic boxes, measur-
ing approximately 30 × 40 × 16 cm3 (four per box), with a wire
mesh platform to access food and water. The boxes containing
the animals were transferred to a laminar flow hood (CQ 800,
Union equipment, laboratorial), measuring 60 × 80 cm, with a
20 × 20 cm pyramid-shaped exhaust pipe.
The concentration of Carbon Monoxide (CO) in the exhaust
hood was adjusted according to previous protocols9,11 (CO
Henrique Zaquia Leão et al The Effects of Passive Smoking on Laryngeal and Tracheal Mucosa
concentration between 100 and 300 ppm). The hood had a single
overhead outlet, and the airflow was passive, with an average
temperature of 22 °C and average humidity of 60%.
The concentration of particulate smoke was measured with
a nephelometer (DustTrakII (Almont, Brazil), model 8532, serial
number 8.53E+09), with a tube including a 2.4 μg/m3/minute air
filter. This device was used from 5 minutes after onset of ex-
posure until cycle completion.
Over a period of 120 days, from the 12th day after birth, the
animals were exposed daily to the smoke generated by burning 16
cigarettes per day (four cigarettes per session, with four sessions
per day, at 8, 12, 16, and 20 hours). Each exposure session lasted
for 20 minutes, whereas cigarette combustion lasted for 10 minutes
on average. In the intervals between sessions, the animals that had
not yet been weaned were placed together with their mothers.
The smoke was administered in the absence of the mother until
the pups were weaned (at 21 days) to avoid contaminating the
breast milk. During exposure to the smoke environment, the boxes
containing group F were transferred to a laminar flow hood with
four cigarettes in combustion. The AS (in the hood) dissipated
passively through the extract vent to the exterior. We used com-
mercial filter cigarettes with the following composition: 10 mg
of tar, 0.8 mg of nicotine, and 10 mg of carbon monoxide.
The animals from group C were housed in two open boxes
in the same room as the animals exposed to secondhand smoke,
at a distance of 5 m from the hood. The animals from both groups
had free access to water and feed.
After 120 days of exposure, following all the protocols re-
quired by Resolution n° 1000 of the Federal Council of Veterinary
Medicine, all animals were sacrificed using the appropriate dose
use of ketamine (75–90 mg/kg) and xylazine (10 mg/kg), after
which the larynx and trachea were removed in block section.
Cotinine levels analysis
Exposure to cigarette smoke in animals was assessed by urinary
concentrations of cotinine (a biomarker for passive tobacco con-
sumption). Urine samples were collected at the end of the
exposure time (120 days) via suprapubic puncture, while they
were alive under anesthesia, immediately before the animals being
sacrificed. The cotinine levels were quantified using high-
performance liquid chromatography according to the protocol
established by Ceppa et al.12
Histological processing
After sacrificing the animals, the organs were fixed in 4%
paraformaldehyde (16 hours at 4 °C), dehydrated in ethanol,
diaphanized in xylene, embedded in Paraplast (Sigma Chemi-
cal Company, St. Louis, MO). After which, 5 μm semi-serial
sections were obtained using a microtome and plated onto poly-
lysine (Sigma) (Sigma Chemical Company, St. Louis, MO)
coverslips. The sagittal sections were then stained with hema-
toxylin and eosin (HE) for morphological analysis.
Pathological analysis
The histological slides were numbered and then analyzed by ex-
perienced pathologists who were blinded to the origin of the
images. Any identified alterations were classified according to
126.e20
TABLE 1.
Initial and Final Weight of the Control Group (C) and
Passive Smoking Group (F)
Initial Avarage (g) Final Avarage (g)
C 20.1 363.2
F 21.9 376.4
the severity observed (qualitative analysis). The presence or
absence of inflammation in the mucosa, dysplasia, and carci-
noma was assessed, as well as the degree of differentiation in
such lesions.
Statistical analysis
The statistical analysis was performed with the aid of GraphPad
Prism 5.0 software (GraphPad Software, Inc., USA). A t test for
independent samples was used to compare differences in the final
weight of the animals and cotinine urine concentrations between
the two groups (P < 0.05). In addition, because of the small
sample, the chi-square test of independence with the calcu-
lated significance was used to detect differences in the proportion
of light and focal inflammation between group F and group C.
RESULTS
At the end of the experiment, the analysis of animal weight
showed no significant differences between group F
(376.4 ± 52.8 g) and group C (363.2 ± 35.8 g) (P = 0.532)
(Table 1). Moreover, the analysis of the urinary cotinine con-
centration in the rats from group F (4.72 ± 0.89 ng/ml) was
significantly higher than the concentration in group C
(0.65 ± 0.12 ng/ml) (P = 0.0001) (Figure 1).
The exposure to the particulate smoke, measured by the neph-
elometer, showed an average exposure of 98.4 μg/m3 between
the 6th and 10th minute of exposure, 86.6 μg/m3, between the
11th and 15th minute of exposure, and an abrupt fall to 2.8 μg/
m3 in the last 5 minutes of exposure (Figure 2).
FIGURE 1. Cotinine levels (nanogram per milliliter of urine) in the
control and passive smoking groups.
126.e21 Journal of Voice, Vol. 31, No. 1, 2017

TABLE 2.
Demonstration That the Proportion of Moderate and Focal
Inflammation is Higher in the Smoker Group than in the
Control Group. Chi-square Test (P = 0.041)
Control Smoking
Group Group
Laryngeal and Tracheal Mucosa (n = 8) (n = 16)
Within the normal range 6 6
Moderate and focal inflammation 1 10
Tracheitis acute 1 –
FIGURE 2. Exposure to the particulate smoke.

inflammatory alterations were identified. Characteristic chronic

Of the animals in group C, 75% showed laryngeal and tra-
cheal mucosa within normal limits and 12.5% showed acute
tracheitis and moderate and focal inflammation. Of the animals
in group F, 37.5% showed mucous within the normal range and
62.5% had moderate and focal inflammation (Table 2). Thus,
according to the chi-square test, there is a statistically signifi-
cant association between the groups and the classification of
laryngeal and tracheal mucosa. According to the analysis, the
proportion of moderate and focal inflammation is higher in group
F than in group C (P = 0.041) (Table 2).
No animal showed any degree of dysplastic or neoplastic lesion.
In addition, macroscopic examination of the resected tracheal
block showed no alteration, thus no other area was subjected to
biopsy (Figure 3A–D).
The histological evaluation, which determined pathologic find-
ings in group F, was based on HE slides. In this group, only
inflammation was revealed on the lamina propria of the larynx
due to the presence of mononuclear inflammatory cells, which
included lymphocytes, plasma cells, and macrophages
(Figure 4A–C). The degree of inflammation, as determined by
the number of inflammatory cells in each sample, was mild to
moderate. Fibrosis was minimal and focal. In some areas, dis-
crete edema or degenerative changes were found in the
subepithelial tissue.
Acute inflammatory alterations, such as fibrinoid exsudate,
erosion, ulceration, neutrophilic infiltrate, and prominent vas-
cular alterations/hyperemia, were not present. The epithelial layer
exhibited zones of hyperplastic or regenerative response, without
nuclear or cellular atypias, suggesting dysplastic or neoplastic
process. No damage to cartilaginous or muscle tissue was noted.
The absence of epithelioid granulomas and nuclear/cytoplasmic
inclusion excluded any fungal and viral infection, respectively.

FIGURE 3. (A) Larynx—normal histological aspects: mucosa covered by pseudostratified ciliated epithelium with goblet cells (arrow), slide
consists of connective tissue and hyaline-type cartilage tissue from the thyroid cartilage. (B) Supraglottic region of the larynx—normal histologi-
cal aspects: lamina propria consists of seromucous glands and connective tissue (arrow). The mucosa, as well as other areas of the larynx, exhibits
a ciliated pseudostratified epithelium with goblet cells. The photomicrograph includes hyaline-type cartilage from the epiglottis. (C) Infraglottic
region—presence of moderate chronic inflammatory infiltrate in the lamina propria (arrow). (D) Infraglottic region—moderate lymphocytic in-
filtrate permeating seromucous glands of the lamina propria (arrow). Hematoxylin and eosin, 100×.
Henrique Zaquia Leão et al The Effects of Passive Smoking on Laryngeal and Tracheal Mucosa
FIGURE 4. (A) Laryngeal wall within normality (Group C: control), hematoxylin-eosine, 100×. (B) Chronic inflammation of the laryngeal mucosa
(Group F—passive smoking), hematoxylin-eosine, 100×. (C) Chronic inflammatory infiltrate due to lymphocytes and plasmocytes (Group F: passive
smoking), hematoxylin-eosine, 400×.
DISCUSSION
Passive smoking is an ongoing public health problem that
demands changes in habits and behaviors in populations world-
wide as well as laws to restrict smoking, especially indoors.
There are several techniques described in the literature to sim-
ulate secondhand smoke environments for use in animal
experiments and facilitate the study of the effects on the mucosa.
Such experiments involve the use of “smoking machines” where
cigarettes are connected to a suction pump so that the smoke
invades the experimental environment.6,13–15 By contrast, in our
study, cigarette combustion occurred in a closed hood where the
smoke was diffused passively, using the same model used in pre-
vious studies into the effects of passive smoking in rats.5,10
In humans, there is great individual variation in the urinary
excretion rates of nicotine and cotinine.16 Moreover, using an
animal model minimizes distortions arising from factors such
as mode of use, exposed area, concentration, and frequency of
exposure to smoke. Similarly, the choice of an animal model also
excludes any possible gender-and/or ethnicity-related differences.
Recently, an interesting study has demonstrated that the urinary
concentration of cotinine in human smokers varies widely de-
pending on the regular or intermittent daily consumption of
cigarettes. In this study, the values quantified were 1396 ± 69 ng/
mL for regular smokers (5–30 cigarettes per day) and 478 ± 44 ng/
mL for intermittent smokers (no control of the number of
cigarettes per day for 4–27 days per month).17 Thus, we recog-
nize that the concentration of urinary cotinine, measured in active,
albeit intermittent, human smokers, is many times higher than
the concentration obtained from the experimental model. This
may explain, at least in part, the fact that we found no severe
lesions in group F, even though they were regularly exposed to
environmental smoke.
The average level of cotinine found in the experimental group
was 7.25 times higher than that found in the control group, dem-
onstrating a significant exposure to environmental smoke in the
urine. Cotinine is a highly specific and highly sensitive marker
with a postexposure time of 3–4 days, measurable in urine.14,18
The level of urinary cotinine in passive smokers has been shown
to have a positive correlation with health risks in children and
is clearly the best biomarker for passive exposure to nicotine.18
The results of the present study prove that the animals were
126.e22
exposed to the combustion of cigarettes with varying effects on
the animals.
The exposure of rats for 120 days covers the animal’s devel-
opment period, which when compared with that in humans would
extend from childhood to adolescence.19 This ensures that the
exposure was limited to the animal’s development period. The
active exposure of young adolescents to smoking has been iden-
tified as an independent mechanism that generates carcinogens
that promote genetic alterations.14 Balansky et al, in their study
involving mice exposed to secondhand smoke after birth and con-
tinuously in early life, reported tumorigenesis in the lungs, liver,
and urinary system.14
In the present study, although mild and focal inflammatory
lesions were found, there was no apparent dysplasia or subse-
quent lesions. Although the inflammatory process is an important
aspect in the pathophysiology of laryngeal disorders, the main
objective of our study was to evaluate the carcinogenesis of the
effects of passive smoking in the laryngeal-tracheal structures.
After macroscopic and microscopic analysis of the specimens,
the presence of dysplastic lesions or carcinoma in the laryn-
geal and tracheal samples was not confirmed.
The localized inflammatory response in the infraglottic larynx
corroborates the findings of Jecker et al,20 who described a
growing population of immune cells in endolaryngeal mucosa,
with a reduction of the same cell type in the glottic region. Sim-
ilarly, another study21 has shown that long-term passive smoke
directly affects the auditory tube and middle ear mucosa.
We were surprised that no tumor was detected because, ac-
cording to the previously described estimate of animal life span,
we expected a more aggressive reaction, such as tumor lesions
in the tracheal mucosa. Haussmann et al22 reported lesions as
diffuse as squamous metaplasia of the pseudostratified epithe-
lium and squamous hyperplasia on the base of the epiglottis.
However, we found no dysplastic lesions and/or carcinogens in
the experimental group.
A careful study was made of the methods of smoke expo-
sure contained in the literature,5,6,9–12 based on which we chose
to place the animals in a laminar flow hood without mechani-
cal ventilation or extraction. In addition, the cigarettes with the
highest tar and nicotine levels available on the market (Rio Grande
do Sul, Brazil) were left to burn in that environment.
126.e23
All cigarette smoke exposure systems share similar charac-
teristics and consist of a cigarette smoke generator and an
exposure chamber where animals are confined and exposed to
smoke. We decided not to use a generator, but rather depend on
passive cigarette combustion, to ensure that the whole body of
the animal was exposed to the smoke.23
The study by Chen et al23 found small differences regarding
the biological effects of three types of exposure. It is known that
assessing the effect of conventional cigarettes or experimental
models used for animal is complex due to the inadequacy of using
live animals to represent disorders that affect humans.24
Passive smoking occurs through the exposure to smoke from
burning tobacco, and the consequent release of more than 4722
products, 44 of which are proven carcinogens.25 Furthermore,
studies show that with the combustion of the cigarette, approx-
imately 5000 residual elements are produced, and 98 of which
are considered carcinogenic.26
The period of greatest exposure of the animals to particulate
smoke was found to be between 8 and 12 minutes, peaking at
10 minutes, as shown in Figure 2. Thus, it can be inferred that
the period of greatest harmful action to the animals’ mucosal
tissues would be around the period of peak exposure to partic-
ulate smoke. Furthermore, exposure was found to be minimal
after 15 minutes of simultaneously burning 4 cigarettes. Objec-
tively, the animals were exposed to a peak of particulate smoke
in the form of a Gauss curve.
In our study, the inflammatory lesions observed and the
anatomopathological examination in the animals are consistent
with those found in humans. It should also be noted that despite
the daily passive smoking, there was no loss of animals during
the experiment, indicating that susceptibility to the develop-
ment of diseases is closely related with the animals’ immune
system. In relation to human beings, this suggests that not all
passive smokers develop smoking-related diseases, and the de-
velopment of any such diseases will depend on the comorbidities
associated with the animals and humans exposed to cigarette
smoke, as well described in the literature.27,28
Some studies report that smoking contributes to weight loss
and that cigarette smoke can alter the hypothalamic appetite reg-
ulation in the central nervous system,29–31 a fact not observed in
our study. However, another recent study5 shows that the animals
in the smoking group had a significant decrease in weight com-
pared with those in the control group.32 Conversely, other authors
have found no significant effect of cigarette exposure on body
weight. We believe that this may be explained by methodolog-
ical differences because the time the animals are exposed to smoke
can affect several biological parameters.
Despite the knowledge gathered over the past 60 years of re-
search, we still suffer the effects of passive exposure to cigarette
smoke, especially children and the elderly. Semple et al31 have
shown that individuals who do not smoke and live with smokers
are exposed to up to 10 times more pollution than those who
live in homes where there are no smokers, and 3 times more than
that recommended by the World Health Organization. It should
also be noted that the opposite is not true, ie, that people not
exposed to cigarette smoke develop laryngeal cancer and tra-
cheal, demonstrating the existence of a close relationship between
Journal of Voice, Vol. 31, No. 1, 2017
the immune system and the development or otherwise of certain
diseases.14
CONCLUSION
Histopathological analysis showed no degree of dysplastic or neo-
plastic lesion in any region of the mucosa of the larynx or trachea
of young Wistar rats submitted to chronic inhalation of ciga-
rette smoke. A higher moderate and focal inflammation was
observed in the smoking group, with obvious damage to the
infraglottic region.
Although inflammatory alterations are well established in the
literature, it is noted that even with high doses of tar and nic-
otine in an environment with poor ventilation and a high density
of particulate smoke, there was no tumor development in the
studied group, proving that cigarette smoke alone was not an
important trigger factor in the neoplastic process, suggesting that
other factors and associated comorbidities are also important in
the genesis of tumors.
Acknowledgments
The authors thank Antonio Generoso Severino and Clovis Tadeu
Bevilacqua Filho for their technical assistance and the students
Bruno Mastella, Cilomar Martins de Oliveira Filho, Pedro
Henrique Duarte Marks, and Victor Hugo Vargas Bittencourt for
their help during the experiment. The authors thank Tais Malysz
for her assistance with the production of images.
REFERENCES
1. Stark MJ, Rohde K, Maher JE, et al. The impact of clean indoor air
exemptions and preemption policies on the prevalence of a tobacco-specific
lung carcinogen among nonsmoking bar and restaurant workers. Am J Public
Health. 2007;97:1457–1463.
2. Stayner L, Bena J, Sasco AJ, et al. Lung cancer risk and workplace exposure
to environmental tobacco smoke. Am J Public Health. 2007;97:545–551.
3. Tse LA, Yu IT, Au JS, et al. Environmental tobacco smoke and lung cancer
among Chinese nonsmoking males: might adenocarcinoma be the culprit?
Am J Epidemiol. 2009;169:533–541.
4. Taybos G. Oral changes associated with tobacco use. Am J Med Sci.
2003;326:179–182.
5. Kosma RH, Alves EM, Barbosa-de-Oliveira VA, et al. Um novo modelo
experimental murino de enfisema: enfisema induzido pela fumaça do cigarro
em ratos Wistar. J Bras Pneumol. 2014;40:46–54.
6. Dalbey WE, Nettesheim P, Griesemer R, et al. Chronic inhalation of cigarette
smoke by F344 rats. J Natl Cancer Inst. 1980;64:383–390.
7. Jecker P, Ptok M, Pabst R, et al. Distribution of immunocompetent cells
in various areas in the normal laryngeal mucosa of the rat. Eur Arch
Otorhinolaryngol. 1996;253:142–146.
8. Silva JB, Gazzalle A, Mano LFM, et al. É possível validar estatisticamente
um dispositivo experimental com 10 ratos, de baixo custo, para pesquisa
em tabagismo passivo? Rev Bras Cir Plast. 2011;26:194–197.
9. Gairola CG, Drawdy ML, Block AE, et al. Sidestream cigarette smoke
accelerates atherogenesis in apolipoprotein E−/− mice. Atherosclerosis.
2001;156:49–55.
10. Duarte JL, Cardoso de Faria FA, Ceolin DS, et al. Efeitos da inalação passiva
da fumaça de cigarros sobre as pregas vocais de ratos. Rev Bras
Otorrinolaringol. 2006;72:210–216.
11. Valenti VE, Abreu LC, Saldiva PH, et al. Effects of sidestream cigarette
smoke exposure on baroreflex components in spontaneously hypertensive
rats. Int J Environ Health Res. 2010;20:431–437.
12. Ceppa F, El Jahiri Y, Mayaudon H, et al. High-performance liquid
chromatographic determination of cotinine in urine in isocratic mode. J
Chromatogr B. 2000;746:115–122.
Henrique Zaquia Leão et al The Effects of Passive Smoking on Laryngeal and Tracheal Mucosa
13. Hecht SS, Hoffman D. N-nitroso compounds and man: sources of exposure,
endogenous formation and occurrence in body fluids. Eur J Cancer Prev.
1998;7:244–246.
14. Balansky R, Ganchev G, Iltcheva M, et al. Potent carcinogenicity of cigarette
smoke in mice exposed early in life. Carcinogenesis. 2007;28:2236–2243.
15. Martins RHG, Gonçalves TM, Madeira SL, et al. Scanning electron
microscopy of the tongue, pharynx and larynx of rats exposed to cigarette
smoke. J Voice. 2014;28:287–289.
16. Benowitz NL, Hukkanen J, Jacob P III. Nicotine chemistry, metabolism,
kinetics and biomarkers. Hand Exp Pharmacol. 2009;192:29–60.
17. Shiffman S, Dunbar MS, Benowitz NL. A comparison of nicotine biomarkers
and smoking patterns in daily and nondaily smokers. Cancer Epidemiol
Biomarkers Prev. 2014;23:1264–1272.
18. Benowitz NL. Cotinine as a biomarker of environmental tobacco smoke
exposure. Epidemiol Rev. 1996;18:188–204.
19. Andreollo NA, Santos BF, Araújo MR, et al. Idade dos ratos versus idade
humana: qual é a relação? Arq Bras Cir Dig. 2012;25:49–51.
20. Jecker P, Ptok M, Pabst R, et al. Age dependency of the composition of
immunocompetent cells and the expression of adhesion molecules in rat
laryngeal mucosa. Laryngoscope. 1996;106:733–738.
21. Kong SK, Chon KM, Goh EK, et al. Histologic changes in the auditory tube
mucosa of rats after long-term exposure to cigarette smoke. Am J
Otolaryngol. 2009;30:376–382.
22. Haussmann H, Anskeit E, Becker D, et al. Comparison of fresh and
room-aged cigarette side stream smoke in a subchronic inhalation study on
rats. Toxicol Sci. 1998;41:100–116.
126.e24
23. Chen BT, Benz MV, Finch FL, et al. Effect of exposure mode on amounts
of radiolabeled cigarette particles in lungs and gastrointestinal tracts of F344
rats. Inhalat Toxicol. 1995;7:1095–1108.
24. Davis DL, Nielsen MT, eds. World Agriculture Series: Tobacco Production,
Chemistry and Technology. London: Blackwell Science; 1999.
25. Hoffmann D, Hecht SS. Chemical Carcinogenesis and Mutagenesis I;
Cooper CS & Grover P (Edt). London: Springer-Verlag; 1990:63.
26. Talhout R, Schulz T, Florek E, et al. Hazardous compounds in tobacco
smoke. Int J Environ Res Public Health. 2011;8:613–628.
27. Piccirilo JF, Vlahiotis A. Prognostigram. In: De Jong RB, ed. Prognosis
in Head and Neck Cancer. Taylor & Francis.; 2006:331–345.
28. Ferrier MB, De Jong RB. Oncologic. In: De Jong RB, ed. Prognosis in Head
and Neck Cancer. Taylor & Francis.; 2006:315–330.
29. Mineur YS, Abizaid A, Rao Y, et al. Nicotine decreases food intake
through activation of POMC neurons. Science. 2011;332:1330–
1332.
30. Chen BR, Hansen MJ, Jones JE, et al. Cigarette smoke exposure reprograms
the hypothalamic neuropeptide Y axis to promote weight loss. Am J Respir
Crit Care Med. 2006;173:1248–1254.
31. Semple S, Apsley A, Azmina Ibrahim T, et al. Fine particulate matter
concentrations in smoking households: just how much second hand smoke
to you breathe in if you live with a smoker who smokes indoors? Tob Control.
2014;051635.
32. Paiva SA, Zornoff LA, Okoshi MP, et al. Behavior of cardiac variables in
animals exposed to cigarette smoke. Arq Bras Cardiol. 2003;81:221–
228.