Current Cancer Drug Targets, 2008, 8, 703-708 703
1568-0096/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.
Budesonide and Phenethyl Isothiocyanate Attenuate DNA Damage in
Bronchoalveolar Lavage Cells of Mice Exposed to Environmental
Rosanna T. Micale1, Francesco D’Agostini1, Vernon E. Steele2, Sebastiano La Maestra1 and Silvio
1Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy; 2National Cancer
Institute, Rockville, MD 20852, USA
Abstract: Chemoprevention by dietary and pharmacological means provides a strategy for attenuating the health risks re-
sulting from cigarette smoking and in particular from passive exposure to environmental cigarette smoke (ECS). We
evaluated the ability of the glucocorticoid budesonide and of the natural agent phenethyl isothiocyanate (PEITC) to affect
DNA damage in bronchoalveolar lavage (BAL) cells of CD–1 mice exposed to ECS, starting within 12 h after birth and
continuing until the end of the experiment. After weanling, based on a preliminary subchronic toxicity study, groups of
mice received daily either budesonide (24 mg/kg diet) or PEITC (1,000 mg/kg diet). After 2 weeks of treatment, all mice
were sacrificed and subjected to BAL, mainly recovering pulmonary alveolar macrophages. Evaluation of single-cell
DNA strand breaks was made by using the alkaline–halo test, a modification of the comet assay. The analysis of 481 BAL
cells yielded the following results (expressed as nuclear spread factor): (a) Sham-exposed mice: mean 0.84 (lower-upper
95% confidence intervals 0.74-0.94); (b) ECS-exposed mice: 2.77 (2.46-3.09); (c) ECS-exposed mice treated with PEITC:
1.15 (1.05-1.26); (d) ECS-exposed mice treated with budesonide: 1.37 (1.25-1.49). Thus, exposure to ECS resulted in a
significant increase of DNA damage as compared with sham, and both PEITC and budesonide significantly attenuated this
damage. In conclusion, the analysis of sentinel cells collected by BAL, a semi-invasive technique that is commonly used
in humans for diagnostic purposes, showed that the investigated chemopreventive agents are able to revert the DNA dam-
age produced by passive exposure to cigarette smoke.
Keywords: Budesonide, phenethyl isothiocyanate, environmental cigarette smoke, bronchoalveolar lavage cells, alkaline–halo
Cigarette smoke (CS) is a major risk factor for lung can-
cer, cancer at several other sites, and a variety of chronic
degenerative diseases . An obvious way to prevent these
diseases is to refrain from smoking or to quit smoking. The
epidemiological evidence demonstrates the effectiveness of
this approach. In fact, in recent years there was a trend to an
attenuation of the epidemic of lung cancer in the population
of several western countries where anti-smoke campaigns
were successful. For instance, the proportion of Americans
who smoke has declined 50% since 1965, and the U.S. mor-
tality rate attributable to smoking declined about 35% be-
tween 1987 and 2002 . A complementary preventative
strategy is to favor the intake of dietary and/or pharmacol-
ogical agents aimed at lowering the risk of developing CS-
related diseases. This strategy, referred to as chemopreven-
tion, is commonly used for the prevention of cardiovascular
diseases and deserves more and more interest in the preven-
tion of cancer, notwithstanding the difficulties in evaluating
efficacy in humans.
Chemoprevention of CS-related diseases has some criti-
cal targets, such as (a) addicted current smokers who are
unable to stop smoking, (b) individuals who are passively
exposed to CS, and (c) former smokers who quitted smoking
*Address correspondence to this author at the Department of Health Sci-
ences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy;
in recent years. In fact, while the risk of developing cardio-
vascular diseases decreases rapidly in ex-smokers, the risk of
developing cancer is reduced significantly with time, but
more time is needed after smoking cessation to reach the
baseline levels of never smokers . Indeed, ex-smokers
today comprise approximately 50% of all new lung cancer
cases . Involuntary smoking (or passive smoking) was
categorized by IARC in Group 1, being evaluated to be car-
cinogenic to humans . This kind of smoking results from
inhalation of environmental cigarette smoke (ECS), or sec-
ondhand smoke, which is a mixture of sidestream cigarette
smoke (SCS), released from the smoldering distal part of the
cigarette, and that portion of mainstream cigarette smoke
(MCS) that is exhaled by actively smoking individuals. ECS,
which is diluted with ambient air and undergoes ageing
processes, contains many free radicals and redox-active
The goal of the present study was to evaluate whether the
oral administration of chemopreventive agents can attenuate
DNA damage in bronchoalveolar lavage (BAL) cells of mice
that were exposed to ECS since birth. We started exposure at
birth because it has been suggested that exposure to tobacco
carcinogens at an early age may be an independent risk fac-
tor for lung cancer, and young age constitutes a critical pe-
riod during which tobacco carcinogens can induce fields of
genetic alterations that make the early smoker susceptible to
the damaging effect of continuing smoking . From the
fetal life to the neonatal life there is a striking increase of
oxidatively generated DNA damage and bulky DNA adduct
704 Current Cancer Drug Targets, 2008, Vol. 8, No. 8 Micale et al.
levels . These genomic alterations are accompanied by
overexpression of a number of genes, mainly having adap-
tive functions, which tend to attenuate oxidative stress and
DNA damage . When exposure of mice starts at birth,
MCS becomes a potent carcinogen  and ECS induces a
variety of molecular, biochemical and cytogenetic alterations
detected in post-weanling mice .
BAL cells are mainly composed of pulmonary alveolar
macrophages (PAM), which have been proposed as surrogate
cells in molecular epidemiology and chemoprevention of
cancer related to CS and other inhalable carcinogens .
Also due to their very high number, accounting in humans
for approximately 50–100 PAM per alveolus and a total of
23 x 109 PAM filling the alveolar spaces in one individual
, these cells play a crucial sentinel role in the lower res-
piratory tract. PAM are subjected to a strong first-pass effect
after exposure to CS. Moreover, they have a quite intense
sweeping activity and are equipped with the inducible ma-
chinery capable of metabolizing inhaled carcinogens both in
the sense of activation  and of detoxification . We
evaluated DNA damage in BAL cells of variously treated
mice by using the alkaline-halo test, a variant of the single-
cell gel electrophoresis (comet assay), in which the electro-
phoretic step is omitted. This test measures osmotically
driven radial migration of single-stranded DNA fragments
from the nuclear cage [13-16].
Of the two chemopreventive agents tested, whose doses
where selected in a preliminary subchronic toxicity study,
budesonide is a synthetic glucocorticoid, a family of com-
pounds that are effective chemopreventive agents in animal
models. In particular, budesonide inhibited the formation of
lung tumors in mice treated with CS compounds, such as
benzo[a]pyrene [17-21] and vinyl carbamate . Budeson-
ide also modulated the expression of a number of genes in
the lung tumors developed in benzo[a]pyrene–treated mice
. Phenethyl isothiocyanate (PEITC) is a naturally occur-
ring isothiocyanate contained in watercress (Nasturtium offi-
cinale), which has been shown to inhibit lung tumors in-
duced in mice and rats by the tobacco–specific nitrosamine
[24-28]. PEITC blocks the metabolic activation of NNK,
resulting in increased urinary excretion of detoxified me-
tabolites . Moreover, PEITC inhibited DNA alterations
and other intermediate biomarkers in ECS–exposed rats 
and modulated the expression of a number of genes in both
unexposed and ECS–exposed rats .
As reported herein, the analysis of almost 500 BAL cells
provided evidence that exposure of mice to ECS from birth
to the post–weanling period, for a total of 7 weeks, causes
evident DNA alterations. Administration with the diet of
either budesonide or PEITC, during the last 2 weeks of ex-
posure, considerably attenuated ECS–related DNA damage.
MATERIALS AND METHODS
Swiss CD–1 albino mice, either pregnant or post–
weanling, were purchased from Harlan Italy (San Pietro al
Natisone, Udine, Italy). Each dam and its litter were housed
individually in Makrolon cages on sawdust bedding and
maintained on standard rodent chow (MIL, Morini, S. Polo
d’Enza, Italy) and tap water ad libitum. After weanling, the
mice were divided by gender and housed in separate cages
(8-10 mice/cages). The cages were kept in a cabinet where
filtered air was circulated. The temperature of the animal
room was 23 ± 2°C, with a relative humidity of 55% and a
12–h day/night cycle. Housing and all treatments of mice
were in accordance with NIH guidelines and with our institu-
Subchronic Toxicity Study with PEITC and Budesonide
This study evaluated the maximum tolerated dose (MTD)
of PEITC and budesonide, both purchased from Sigma
Chemical Co. (St. Louis, MO). A total of 180 post–weanling
mice were used. They were divided into 9 groups, each com-
posed of 20 mice (10 males and 10 females), treated as fol-
lows: (A) untreated mice (controls); (B), (C), (D), and (E)
mice treated with PEITC at the doses of 125, 250, 500, and
1,000 mg/kg diet, respectively; (F), (G), (H), and (I) mice
treated with budesonide at the doses of 0.3, 0.6, 1.2, and 2.4
mg/kg diet, respectively. This range of doses was chosen
based on available literature data. The mice were inspected
daily for general appearance and weighed at weekly intervals
for 6 weeks. The MTD was assumed as the maximum dose
of each chemopreventive agent that did not induce apparent
signs of toxicity and did not reduce the body weight gain by
more than 10% as compared with control mice.
Exposure to ECS
Eighteen litters of mice, including a total of 197 new-
borns, were exposed to ECS, starting within 12 h after birth
and continuing throughout the weaning period and for 2 ad-
ditional weeks after weaning, for a total of 7 weeks. Other 6
litters, including a total of 71 mice, were kept in filtered air
during the same period of time and served as sham-exposed
ECS was generated by burning Kentucky 2R4F reference
cigarettes (Tobacco Research Institute, University of Ken-
tucky, Lexington, KY), having a declared content of 9.2 mg
tar and 0.8 mg nicotine each, with a 23 mm butt remaining
after smoking. The cigarettes were kept for 48 h before use
in a standardized atmosphere, humidified with a mixture of
70% glycerol and 30% water. A whole-body exposure of
mice to ECS was achieved by using a smoking machine
(model TE-10c, Teague Enterprises, Davis, CA), adjusted to
produce a combination of SCS (89%) and MCS (11%), mim-
icking a high-dose exposure to ECS. Burning 5 cigarettes at
one time yielded on an average a total suspended particulate
of 63.3 mg/m3 in the exposure chambers. Exposure was
daily, 6 h/day divided into two rounds with a 3-h interval
(8:00–11:00 a.m. and 2:00–5:00 p.m.).
Treatment of ECS–Exposed Mice with PEITC or
After weaning, 79 ECS-exposed mice were kept un-
treated, while 61 ECS-exposed mice received daily PEITC
with the diet, and 57 ECS-exposed mice received daily
Budesonide and Phenethyl Isothiocyanate Attenuate DNA Current Cancer Drug Targets, 2008, Vol. 8, No. 8 705
budesonide with the diet, at the doses indicated under “Re-
Collection of BAL Cells
Two weeks after weaning, 8 male mice per each one of
the 4 experimental groups (sham, ECS, ECS + PEITC, and
ECS + budesonide) were deeply anesthetized with diethyl
ether and killed by cervical dislocation. All other mice were
kept alive for a study on long-term ECS-related alterations,
which is now in progress. BAL was immediately performed
by lavaging the lungs with three 2 ml aliquots of cold (4°C)
0.15 M NaCl infused via a cannula inserted in the trachea
. The cells, pooled from the mice composing each ex-
perimental group, were washed and centrifuged (500 x g for
10 min at 4°C) twice with sterile Ca2+/Mg2+-free phosphate
buffer saline solution, composed of 8 g/l NaCl, 1.15 g/l
Na2HPO4, 0.2 g/l KH2PO4, 0.2 g/l KCl (PBS, pH 7.2), and
9.1 x 104 cells/?l were resuspended in 100 μl PBS. Survival
of cells, evaluated by means of the trypan blue dye exclusion
method (0.4% in PBS), was approximately 90% in all 4
Performance of the Alkaline–Halo Test
The alkaline-halo test was performed as described by
Sestili and Cantoni . Briefly, the cells were resuspended
at a concentration of 2.0 ? 104 cells/100 μl in 1.5% low-
melting agarose in PBS, pH 7.2 containing 5 mM ethyle-
nediaminetetraacetic acid (EDTA), and immediately sand-
wiched in duplicate between an agarose-coated slide and a
coverslip. After complete geling, the coverslips were re-
moved and the slides were immersed in a lysis buffer (2.5 M
NaCl, 100 mM EDTA, 10 mM Tris, 1% sarkosyl, 5% di-
methylsulfoxide, 1% Triton X100 and 0.02 M NaOH), pH
12.5, for 20 min on ice. The slides were then incubated for
15 min in an alkaline hypotonic buffer (0.1 M NaOH/1 mM
EDTA), pH 12.5, washed with 0.4 M Tris-HCl, pH 7,5, and
stained for 5 min with 10 μg/ml ethidium bromide.
Microscopic Analysis and Image Processing
The ethidium bromide-labeled DNA was visualized using
an Olympus BX51TF fluorescence microscope equipped
with a digital camera (Olympus Camedia C-4040). For each
coded spot, with exclusion of the outer area, images of at
least 100 randomly selected nuclei were acquired and sub-
mitted to an automated image analysis system (ImageJ soft-
ware, NIH Image program,
http://rsb.info.nih.gov/nih-image/). The slides were numeri-
cally coded before reading to minimize operator bias. In the
alkaline-halo assay, the level of the DNA damage is ex-
pressed as nuclear spreading factor (NSF), which represents
the ratio between the area of the halo (obtained by subtract-
ing the area of the nucleus from the total area, i.e., nucleus +
halo) and that of the nucleus. This analysis was performed on
digitally stained images with a computer-generated pseudo-
color scale, which stains the low fluorescence regions (halos)
Fig. (1). Distribution of NSF values among the 481 analyzed BAL cells, as related to exposure to ECS and treatment with either PEITC or
budesonide. The horizontal lines indicate the median NSF levels within each one of the 4 experimental groups. The 4 photographs show
examples of a negative halo test (Sham) and of positive halo tests of varying intensity (either ECS or ECS + PEITC or ECS + Budesonide).
706 Current Cancer Drug Targets, 2008, Vol. 8, No. 8 Micale et al.
in red and the high fluorescence regions (nuclei) in bright
The overall statistical significance of the differences in
body weights and NSF values, as related to treatments, was
evaluated by ANOVA, followed by post-hoc Scheffé test for
the analysis of comparisons between groups.
No loss of body weight was detected, at all doses tested,
in the post–weanling mice receiving either budesonide or
PEITC with the diet for 6 weeks. In particular, the body
weight in the 9 experimental groups (mean ± SE within each
group of 20 mice) ranged between a minimum of 21.4 ± 0.39
g and a maximum of 21.9 ± 0.62 g at the start of the experi-
ment, and between 30.1 ± 1.10 g and 32.3 ± 0.73 g after 6
weeks, without any significant difference. Therefore, the two
agents were used at the maximum dose tested, i.e. 24 mg/kg
diet for budesonide and 1,000 mg/kg diet for PEITC.
Fig. (1) shows the distribution of results among the 481
BAL cells examined from the 4 experimental groups. Exam-
ples of halo tests are also shown. It is evident at a glance that
NSF values were consistently higher in BAL cells from
ECS-exposed mice, as compared with sham-exposed mice.
Administration of either PEITC or budesonide to ECS-
exposed mice attenuated NSF values. In particular, the me-
dian NSF values in sham, ECS, ECS + PEITC, and ECS +
budesonide were 0.69, 2.41, 1.05, and 1.23, respectively.
Quantitative data and statistical analysis are reported in
Table 1. The ANOVA demonstrated that statistically signifi-
cant differences occur among the 4 experimental groups (P <
0.0001). The post-hoc Scheffé test showed that both the in-
crease of ECS vs. sham (3.3-fold) and the decrease of either
ECS + PEITC (2.4-fold) or ECS + budesonide (2.0-fold) vs.
ECS are statistically significant (P < 0.0001 in all cases).
Inhibition of ECS-induced DNA damage by PEITC was so
strong that the difference between ECS + PEITC and sham
was not significant, while a slight residual DNA damage was
detected in ECS-exposed mice treated with budesonide (P <
Previous studies performed in our laboratory showed that
several chemopreventive agents, both natural and synthetic,
are able to modulate a variety of intermediate biomarkers in
tissues of ECS-exposed rats and mice. Focussing on the res-
piratory tract, the investigated end-points included bulky
DNA adducts, oxidatively generated DNA damage, cell pro-
liferation, apoptosis, multigene expression, expression of
specific oncogenes and tumor suppressor genes, proteome
profiles, and cytogenetic alterations [29-37]. In all these
studies, however, the treatment regimen with chemopreven-
tive agents started before the period of exposure to ECS. In
contrast, in the present study administration of either PEITC
or budesonide started when the mice had already been ex-
posed to ECS for 5 weeks. Apart from the high doses that
need to be used in animal models, this experimental design
aims at mimicking the situation of an individual who is pas-
sively exposed to ECS early in life, when, as previously dis-
cussed, the organism is particularly vulnerable to the action
of genotoxic agents.
Therefore, the finding that both PEITC and budesonide
were successful to attenuate and, in the case of PEITC, to
abolish DNA alterations in BAL cells is noteworthy and
highlights the great efficacy of these chemopreventive
agents. Since it is unrealistic to hypothesize that a drug or a
dietary component may suppress an established DNA dam-
age, the observed patterns suggest that, during the two weeks
of chemopreventive treatment, there was a rather extensive
renewal of the population of BAL cells, and that the two
agents were able to inhibit further DNA damage in newly
recruited cells. PAM, which also in mice constitute the large
majority of BAL cells , are very long-lived cells (months
to years), although PAM resulting from the recent migration
of monocytes from the blood to alveoli, across the inter-
stitium, have a shorter lifespan than resident PAM . Ac-
cordingly, the turnover of PAM is warranted by their intense
removal from the alveolar spaces via the mucociliatory esca-
lator, accounting for a daily removal of 0.75 x 106 cells in
rats  and 24-120 x 106 cells in humans . It has been
evaluated that the sojourn of free PAM in the alveoli of
mice, under basal conditions, is 7 days , a figure that is
compatible with our results.
The meaning of the alkaline-halo test as an indicator of
DNA damage is not completely clear. The radial diffusion of
ethidium bromide-stained single-stranded DNA breaks is
consistent with the occurrence of DNA damage [13-16]. The
classical comet assay, involving an electrophoretic separa-
tion of DNA fragments, is not a good measure of apoptosis
. In contrast, the halo test efficiently detects cells under-
going apoptosis but is not able to distinguish between apop-
tosis and genotoxic damage. It is noteworthy that CS is a
Evaluation of 481 BAL Cells by Means of the Alkaline-Halo Test
No. of BAL
95% Confidence Intervals
Sham 96 0.84 0.74 – 0.94
ECS 113 2.77 2.46 – 3.09
P < 0.0001 vs. Sham
ECS + PEITC 129 1.15 1.05 – 1.26
P < 0.0001 vs. ECS
ECS + budesonide 143 1.37 1.25 – 1.49
P < 0.05 vs. Sham;
P < 0.0001 vs. ECS
*NSF, nuclear spread factor (see Materials and Methods).
Budesonide and Phenethyl Isothiocyanate Attenuate DNA Current Cancer Drug Targets, 2008, Vol. 8, No. 8 707
potent inducer of apoptosis in PAM and bronchial epithelial
cells [32-34, 38], and that isothiocyanates, such as PEITC
and benzyl isothiocyanate, and glucocorticoids, such as
budesonide and dexamethasone, are usually inducers rather
than inhibitors of apoptosis . Apoptosis is a double
edged sword, since it represents a protective mechanism in
carcinogenesis but its inhibition may reflect the ability of a
chemopreventive agent to counteract certain upstream sig-
nals, such as genotoxic damage, redox imbalances, and other
forms of cellular stress that trigger apoptosis .
The proinflammatory effects of ECS and the antiinflam-
matory mechanisms of both PEITC and budesonide may
contribute to interpret the results obtained, since inflamma-
tion plays a prominent role in the carcinogenesis process,
particularly in the promotion phase [44, 45].
In conclusion, the DNA damage produced in BAL cells
of mice exposed to ECS throughout the weaning period can
efficiently be reverted by administering drugs, such as
budesonide, or dietary principles, such as PEITC. BAL cells
were previously shown to be suitable for the detection of
ECS-related molecular and cytogenetic alterations in rodents
and for evaluating their inhibition by pre-treatment with
chemopreventive agents [32-34, 37, 38]. These data bear
relevance also because BAL is a semi-invasive technique
that is commonly used in humans for diagnostic purposes. A
phase II clinical trial showed that the oral administration to
heavy smokers of a chemopreventive
acetylcysteine, results in significant decreases of oxidatively
generated DNA damage and bulky DNA adduct levels in
BAL cells after 6 months of treatment . Due to its rela-
tive simplicity and capability to analyze in parallel large
numbers of samples, the method used in the present study is
proposed for evaluating the ability of chemopreventive
agents to attenuate CS-related DNA damage in animal mod-
els and in humans.
This study was supported by U.S. National Cancer Insti-
tute contract N01-CN-53301. We thank Prof. G. Ravera for
assistance in statistical analysis.
BAL = bronchoalveolar lavage
CS = cigarette smoke
ECS = environmental cigarette smoke
MCS = mainstream cigarette smoke
MTD = maximum tolerated dose
NNK = 4–(methylnitrosoamino)–1–(3–pyridyl–1–
PAM = pulmonary alveolar macrophages
PEITC = phenethyl isothiocyanate
SCS = sidestream cigarette smoke
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Received: January 10, 2008
Revised: August 18, 2008 Accepted: September 10, 2008