Influence of FHIT on benzo[a]pyrene-induced tumors
and alopecia in mice: Chemoprevention by
budesonide and N-acetylcysteine
Roumen Balansky*†, Francesco D’Agostini*, Gancho Ganchev†, Alberto Izzotti*, Barbara Di Marco*, Ronald A. Lubet‡,
Nicola Zanesi§, Carlo M. Croce§, and Silvio De Flora*¶
*Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy;†National Center of Oncology, Sofia 1756, Bulgaria;‡National
Cancer Institute, Rockville, MD 20898; and§Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210
Edited by Webster K. Cavenee, University of California at San Diego School of Medicine, La Jolla, CA, and approved March 29, 2006 (received for review
February 20, 2006)
The FHIT gene has many hallmarks of a tumor-suppressor gene and
is involved in a large variety of cancers. We treated A?J mice and
(C57BL?6J ? 129?SvJ)F1 (B6?129 F1) mice, either wild-type or
FHIT?/?, with multiple doses of benzo[a]pyrene (B[a]P) by gavage.
B[a]P caused a time-related increase of micronuclei in peripheral
blood erythrocytes. Both A?J and B6?129 F1mice, irrespective of
their FHIT status, were sensitive to induction of forestomach
tumors, whereas B[a]P induced glandular stomach hyperplasia and
a high multiplicity of lung tumors in A?J mice only. Preneoplastic
lesions of the uterus were more frequent in FHIT?/?mice. B6?129
F1mice underwent spontaneous alopecia areata and hair bulb cell
apoptosis, which were greatly accelerated either by FHIT heterozy-
gosity or by B[a]P treatment, thus suggesting that FHIT plays a role
in the pathogenesis of alopecia areata. The oral administration of
either budesonide or N-acetyl-L-cysteine (NAC) inhibited the occur-
rence of this inflammatory skin disease. In addition, these agents
prevented B[a]P-induced glandular stomach hyperplasia and de-
creased the size of both forestomach tumors and lung tumors in
A?J mice. Budesonide also attenuated lung tumor multiplicity. In
B6?129 F1mice, NAC significantly decreased the proliferating cell
nuclear antigen in lung tumors. Both budesonide and NAC inhib-
ited B[a]P-induced forestomach tumors and preneoplastic lesions
of the respiratory tract in B6?129 F1mice. In conclusion, heterozy-
gosity for FHIT affects susceptibility of mice to spontaneous alo-
pecia areata and B[a]P-induced preneoplastic lesions of the uterus
and does not alter responsiveness to budesonide and NAC.
alopecia areata ? lung tumors ? stomach tumors
tumor-suppressor gene (1). FHIT is altered by deletion or
and gastric cancer (2–4). The murine FHIT gene is similar in
sequence, location, and fragility to its human homologue (5),
which suggests that FHIT mutant mice might provide a model to
study the role of the FHIT pathway in the development of cancer
and possibly of other diseases. FHIT-deficient mice, either
FHIT?/?or FHIT?/?, were established by inactivating one FHIT
allele in mouse embryonic stem cells. These mice displayed an
elevated frequency of ‘‘spontaneous’’ tumors and chemically
induced tumors (6, 7). The FHIT-deficient mouse model has also
been used to prevent tumor development by gene transfer (8, 9).
One objective of the present study was to compare the
susceptibility of (C57BL?6J ? 129?SvJ)F1 (B6?129 F1) mice,
to benzo[a]pyrene (B[a]P), a prototype of genotoxic and carci-
nogenic polycyclic aromatic hydrocarbon (PAH). B[a]P diol
epoxide has recently been shown to down-regulate FHIT ex-
pression, presumably as the result of different signaling pathways
triggered by specific DNA lesions (10). In parallel, we used A?J
mice as a control, because this mouse strain has been extensively
he FHIT gene is thus far the only example of a gene at a
constitutive fragile region, and it shows many hallmarks of a
used in the lung tumor assay (11), a medium-term bioassay for
lung tumorigenesis. A?J mice carry the pulmonary adenoma
susceptibility 1 (Pas1) locus, a major locus affecting predispo-
sition to lung cancer in mice (12) and producing the EcoRI-
generated 0.55-kb K-ras fragment associated with high suscep-
tibility to lung tumor development (13).
Another major goal of this study was to assess the protective
effects of the two chemopreventive agents budesonide and
N-acetyl-L-cysteine (NAC). Budesonide is a glucocorticoid, a
family of compounds that are effective cancer chemopreventive
In particular, the studies performed by Wattenberg and col-
leagues (15–17) showed that budesonide, given either in the diet
or by aerosol, inhibits the formation of B[a]P-induced lung
tumors in A?J mice at all stages of tumor development, from
hyperplasia to cancer. The expression of a number of genes was
modulated by budesonide in the lung tumors developed in
B[a]P-treated A?J mice (18). The protective effect of oral
budesonide against lung tumors induced by intraperitoneal
B[a]P was also observed in A?J mice, either wild type or those
carrying germ-line mutations in P53 and?or Ink4A?Arf genes
(19). Moreover, budesonide delayed the appearance of lung
tumors in strain A mice treated with vinyl carbamate and
decreased their growth and progression to carcinomas (20).
NAC works as an analogue and a precursor of intracellular
L-cysteine and reduced glutathione (GSH). The safety of this
drug in humans is supported by ?40 years of clinical use, mainly
as a mucolytic agent but also as an antidote against acute
intoxication (21, 22). A number of experimental studies in
animal models performed during the last 20 years and phase II
clinical trials, along with mechanistic considerations, provide
evidence for the potential ability of this thiol compound to
inhibit oxidative, genotoxic, and carcinogenic effects (reviewed
in refs. 21 and 22). As an indicator of safety at the molecular
level, oral NAC did not substantially change the baseline ex-
pression of multiple genes but attenuated the alterations induced
by cigarette smoke in both mice (23) and rats (24). Among other
protective effects, dietary NAC attenuated B[a]P-induced pa-
thology of the liver and forestomach hyperplasia and papillomas
in P53 heterozygous TG.AC (v-Ha-ras) bitransgenic mice (25).
An incidental discovery during the progress of the reported
study herein was that B6?129 F1mice spontaneously developed
areas of alopecia, whose formation was greatly accelerated in
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: B[a]P, benzo[a]pyrene; Fhit, fragile histidine triad; MN, micronucleated;
NAC, N-acetyl-L-cysteine; NCE, normochromatic erythrocytes; PCNA, proliferating cell nu-
¶To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
May 16, 2006 ?
vol. 103 ?
no. 20 ?
FHIT?/?mice. NAC and, even more strikingly, budesonide
inhibited both the spontaneous alopecia in FHIT?/?mice and
the B[a]P-induced alopecia in wild-type mice. Moreover, in A?J
and?or B6?129 F1mice, these chemopreventive agents signifi-
cantly attenuated the induction of preneoplastic lesions and
tumors by B[a]P.
Body Weights and Survival. No treatment significantly affected the
body weight gain either in A?J or B6?129 F1 mice (data not
shown). Irrespective of the mouse strain, FHIT status, and
treatment, almost all mice (95.1%) were still alive at the end of
the experiment. The number of mice surviving within each
experimental group can be inferred from the incidence data
reported in Tables 1 and 2.
Cytogenetic Monitoring. The results relative to the periodic cyto-
genetic monitoring in the peripheral blood of the variously
treated mice are shown in Fig. 1. In control mice, which were
treated with corn oil, the levels of micronucleated (MN) nor-
mochromatic erythrocytes (NCE) were ?2-fold higher in A?J
mice than in B6?129 F1mice. Both A?J and B6?129 F1mice
responded to the multiple administrations of B[a]P by increasing
the frequency of MN NCE. This effect started to be statistically
significant after 10–12 days of B[a]P administration and reached
a plateau after ?3 weeks. The maximum increase in B[a]P-
treated mice, compared with controls, was ?2.2 in A?J mice,
?3.0 in wild-type B6?129 F1mice, and ?3.2 in FHIT?/?B6?129
F1 mice and thereafter tended to decline by the end of the
experiment. Irrespective of the mouse strain and FHIT status,
neither budesonide nor NAC affected the frequency of B[a]P-
induced MN NCE.
Spontaneous and B[a]P-Induced Alopecia Areata. A number of
B6?129 F1 mice developed evident alopecia areas. In Fig. 2,
examples are shown in which the lesions are distinguished
according to their localization on the muzzle (A), head (B), neck
(C), and back (D). Some mice had single localizations, whereas
other mice had multiple localizations of variable intensity. Fig.
2 also shows the time course induction of alopecia related to
FHIT status and treatments. The mice were checked for alopecia
0, 29, 43, 57, 72, 101, 116, 134, 148, 176, 198, 205, and 276 days
Table 1. Yield of forestomach tumors and glandular stomach hyperplasia in mice related to strain, Fhit status, and treatment
Forestomach tumors, total Forestomach tumors, ?1 mm
hyperplasia incidence, %Incidence, %
mean ? SEIncidence, %
mean ? SE
B[a]P ? Bud
B[a]P ? NAC
B[a]P ? Bud
B[a]P ? NAC
B[a]P ? Bud
B[a]P ? NAC
2.5 ? 0.46*
3.3 ? 0.50*
2.9 ? 0.42*
0.1 ? 0.08
3.9 ? 0.71*
1.9 ? 0.46*§
1.6 ? 0.15*¶
0.1 ? 0.05
3.3 ? 0.41*
0.9 ? 0.24*¶
1.7 ? 0.42*§
2.4 ? 0.47*
2.8 ? 0.39*
2.7 ? 0.40*
2.0 ? 0.44*
1.3 ? 0.40*
1.3 ? 0.34*
2.0 ? 0.19*
0.7 ? 0.20¶
0.8 ? 0.25¶
Statistically significant differences:*, P ? 0.001 and †, P ? 0.05 compared with controls; ‡, P ? 0.01; §, P ? 0.05; and ¶, P ? 0.001, compared with mice treated
with B[a]P only. Bud, budesonide.
Table 2. Yield and characteristics of lung tumors in mice related to strain, Fhit status, and treatment
Treatment of mice
budesonideB[a]P ? NAC
Multiplicity, mean ? SE
Diameter, mm, mean ? SE
Multiplicity, mean ? SE
Diameter, mm, mean ? SE
P53 in tumors, %
PCNA in tumors, %
Apoptotic cells in tumors, %
Multiplicity, mean ? SE
Diameter, mm, mean ? SE
P53 in tumors, %
PCNA in tumors, %
Apoptotic cells in tumors, %
0.2 ? 0.12
1.5 ? 0.29
10.4 ? 1.23*
1.2 ? 0.03
0.3 ? 0.19
1.8 ? 0.58
11.3 ? 3.55
29.2 ? 3.25
5.5 ? 1.04
0.4 ? 0.15
1.5 ? 0.39
11.0 ? 1.71
27.7 ? 1.79
4.9 ? 0.62
5.9 ? 0.71*†
0.9 ? 0.04†
0.5 ? 0.21
1.2 ? 0.17
8.0 ? 1.00
23.3 ? 1.45
6.4 ? 0.57
0.4 ? 0.16
1.3 ? 0.23
11.1 ? 2.22
24.2 ? 1.69
5.2 ? 0.91
9.4 ? 1.06*
1.0 ? 0.03†
0.3 ? 0.27
1.2 ? 0.17
8.5 ? 2.19
20.8 ? 2.02‡
5.9 ? 0.65
0.3 ? 0.13
1.1 ? 0.24
9.0 ? 1.39
21.0 ? 2.46‡
4.4 ? 0.71
B6?129 F1wild type
Statistically significant differences:*, P ? 0.001 and §, P ? 0.05 compared with controls; †, P ? 0.01 and ‡, P ? 0.05 compared with
mice treated with B[a]P only. NTA, no tumor available.
www.pnas.org?cgi?doi?10.1073?pnas.0601412103Balansky et al.
after the first B[a]P administration. Within wild-type mice, the
control animals started loosing their agouti hair after 101 days,
and all control mice were affected by alopecia areas after 176
days. B[a]P strongly accelerated this process until it affected
100% of mice after only 57 days, with statistically significant
differences compared with controls. Coadministration of budes-
onide significantly decreased the frequency of alopecia, not only
compared with B[a]P-treated mice from 57 days onward, but
even compared with untreated controls from 134 days onward.
Although less strikingly than budesonide, NAC also attenuated
B[a]P-induced alopecia areata after 57–72 days. After 200 days,
all mice, irrespective of FHIT status and treatment, suffered
from alopecia areata. Almost all FHIT?/?mice were still af-
fected after 276 days.
As is evident in Fig. 2, untreated FHIT?/?mice were much
more prone than untreated wild-type mice to developing ‘‘spon-
taneous’’ alopecia throughout almost the whole duration of the
experiment, with significant differences related to FHIT status
after 29–148 days. In mutant mice, B[a]P did not further affect
alopecia formation compared with controls. The chemopreven-
tive agents tested exerted strong protective effects on induction
of alopecia compared with either controls or B[a]P-treated mice.
In fact, both budesonide and NAC significantly inhibited alo-
pecia after 29–198 days.
As assessed by immunohistochemical analysis of apparently
‘‘healthy’’ skin fragments not affected by alopecia that were
collected from five mice per experimental group, virtually all
cells in the basal and granular layers of epidermis, as well as in
the hair bulbs and sebaceous glands, were positive for fragile
histidine triad (Fhit). Only 0.1–0.4% of cells were positive for
either mutated or inactivated P53, without appreciable differ-
ences related to FHIT status of mice and treatment with B[a]P
and chemopreventive agents. Hair bulb cells were analyzed for
the detection of apoptosis by the TUNEL method. In wild-type
B6?129 F1mice, the proportion of apoptotic cells was signifi-
cantly enhanced after treatment with B[a]P (15.3%) compared
with control mice (1.2%). The effect of B[a]P was significantly
attenuated by cotreatment either with budesonide (4.3%) or
NAC (9.4%). By comparison with wild-type mice, the back-
ground apoptotic frequency was much higher in untreated
FHIT?/?mice (13.5%) and was not significantly increased by
B[a]P administration (16.8%). Both budesonide (3.8%) and
NAC (5.7%) remarkably decreased apoptosis in hair bulb cells,
with significant differences compared not only with B[a]P-
treated mice but even with untreated mice.
Forestomach Tumors and Glandular Stomach Hyperplasia. Table 1
shows the incidence and multiplicity of macroscopically visible
forestomach tumors and the incidence of microscopically de-
tectable hyperplasia of the glandular stomach epithelium in the
variously treated mice.
B[a]P induced a high incidence and multiplicity of total
forestomach tumors in both A?J and B6?129 F1mice, irrespec-
tive of FHIT status. In A?J mice, budesonide and NAC did not
affect induction of tumors by B[a]P, but both agents were
successful in decreasing the size of forestomach tumors to a
significant extent. In fact, the diameter of these tumors (mean ?
SE) in A?J mice was 2.9 ? 0.28 mm in B[a]P-treated mice, 1.9 ?
0.15 in mice cotreated with budesonide (P ? 0.01, compared
with mice treated with B[a]P only), and 2.2 ? 0.17 in mice
cotreated with NAC (P ? 0.01). However, in B[a]P-treated
B6?129 F1 mice, both budesonide and NAC significantly de-
creased tumor multiplicity, which was more than halved in both
wild-type and FHIT?/?mice receiving either chemopreventive
agent. Moreover, both budesonide and NAC significantly de-
creased the tumor incidence in B[a]P-treated FHIT?/?mice.
The protective effect of the chemopreventive agents tested
was also clearly evident when comparing only forestomach
tumors whose diameter was ?1 mm, which are more likely to be
true tumors rather than preneoplastic lesions. In fact, as shown
in Table 1, the B[a]P-induced tumor multiplicity was reduced
1.5-fold by both budesonide and NAC in wild-type mice (non-
significant), whereas it was significantly reduced by budesonide
(2.9-fold) and NAC (2.5-fold) in FHIT?/?mice. In any case,
multiplicity of B[a]P-induced forestomach tumors was not sig-
nificantly different in wild-type mice and in FHIT?/?mice
cotreated either with budesonide or NAC.
The histopathological analysis revealed that, in addition to fore-
stomach tumors, B[a]P induced a significant increase of glandular
stomach hyperplasia (3.5-fold) in A?J mice. Both budesonide and
irrespective of the mouse strain and FHIT status, B[a]P increased
the incidence of microscopically detectable keratoses and hyper-
plasias of the forestomach (data not shown).
earlier in the experiment, A?J mice were much more susceptible
to the induction of lung tumors than B6?129 F1mice. Compared
with controls, the increase of lung tumor incidence in B[a]P-
treated B6?129 F1mice was statistically significant in FHIT?/?
mice only. However, the difference between wild-type and
FHIT?/?mice was modest and not statistically significant. As
various time intervals in A?J, wild-type, and FHIT?/?B6?129 F1mice, which
with budesonide (Bud) or NAC.
Frequency of MN NCE in peripheral blood samples collected after
Balansky et al.
May 16, 2006 ?
vol. 103 ?
no. 20 ?
assessed in B6?129 F1mice, 64.3% of B[a]P-induced lung tumors
were solid adenomas, 23.8% were mixed adenomas, and 11.9%
were papillary adenomas. The histopathological type was af-
fected neither by FHIT status nor by administration of either
In A?J mice, budesonide significantly decreased lung tumor
multiplicity, and both budesonide and NAC significantly de-
creased the lung tumor size. The low incidence and multiplicity
of lung tumors in B[a]P-treated mice were not significantly
changed in B6?129 F1mice cotreated with the chemopreventive
agents, regardless of FHIT status. Both budesonide and NAC
decreased the mean diameter of tumors, but, due to the larger
variability in size compared with tumors in A?J mice, these
differences were not statistically significant.
Approximately 10% of cells composing lung tumors in B[a]P-
treated B6?129 F1mice harbored the P53 protein in the nucleus,
21–29% had detectable proliferating cell nuclear antigen
(PCNA), and 4–6% were apoptotic. Interestingly, PCNA was
significantly decreased in the tumors from both wild-type and
FHIT?/?mice cotreated with NAC (Table 2). The immunohis-
tochemical analysis of 25 lung tumors induced by B[a]P in
B6?129 F1mice showed mixed areas of positivity and negativity
for Fhit protein in each tumor, without appreciable differences
related to either FHIT status or treatment (data not shown).
In addition to lung tumors, the histopathological analyses
performed in B6?129 F1mice revealed the presence of other
of severe emphysema in both wild-type (10.5%) and FHIT?/?
(8.7%) mice treated with B[a]P only, and one case was detected
in FHIT?/?controls (5.0%). These differences were not statis-
tically significant. Whereas no hyperplasia of the bronchial
in B[a]P-treated mice, both wild type (29.4%; P ? 0.01) and
FHIT?/?(30.4%; P ? 0.01). The B[a]P-induced hyperplasia of
the bronchial epithelium was significantly inhibited either by
NAC in both wild-type (no case; P ? 0.01, compared with mice
treated only with B[a]P) and FHIT?/?(one case; P ? 0.05) mice
or budesonide (no case in either wild-type or FHIT?/?mice; P ?
0.01). In addition, whereas no hyperplasia of alveolar walls was
wild-type (52.8%; P ? 0.001) and FHIT?/?(73.9%; P ? 0.001)
mice. A protective effect toward this B[a]P-induced damage was
only observed in FHIT?/?mice cotreated with budesonide
(21.7%; P ? 0.01).
Histopathological Alterations in Other Organs. After complete nec-
ropsy and histopathological analysis of macroscopically suspect
lesions of B6?129 F1mice, only a single case of cystic hyperplasia
of the uterus was detected in B[a]P-treated wild-type mice. In
contrast, preneoplastic lesions of the uterus (cystic hyperplasia and
leiomyomas) were detected in 21.2% of FHIT?/?mice (P ? 0.001),
irrespective of treatment with the chemopreventive agents. In
addition, liver hemangiomas were detected in 4.5% of these mice.
The present study evaluated the susceptibility of A?J mice and
B6?129 F1 mice, either wild-type or FHIT?/?, to a variety of
pathological conditions, either ‘‘spontaneous’’ or induced by
B[a]P. Moreover, the chemopreventive effects of budesonide
and NAC were investigated.
During the progress of this study, B6?129 F1mice underwent
spontaneous alopecia areata, which was greatly accelerated
either by FHIT heterozygosity or treatment with B[a]P. In
parallel, the proportion of apoptotic cells in the hair bulbs was
greatly enhanced either by heterozygosity for FHIT or admin-
istration of B[a]P. Alopecia areata is a nonscarring, inflamma-
tory form of patchy hair loss that is determined by a combination
of genetic factors and environmental stimuli (26). Among the
multiple genes involved in its pathogenesis, P53 has been shown
to be involved in the control of apoptosis in the hair follicle
during physiological regression (27). The results of our study,
highlighting an enhanced hair bulb cell apoptosis and an in-
creased alopecia in untreated FHIT-deficient mice, suggest that
FHIT plays a protective role in alopecia areata. Interestingly,
FHIT-deficient B6?129 F1 mice can also develop tumors of
sebaceous glands, similar to those observed in Muir–Torre
black C57BL?6J mice, and C57BL?6 and related strains are
Fig. 2.Appearance of alopecia areata in B6?129 F1mice and time course formation of alopecia related to FHIT status and treatment of mice.
www.pnas.org?cgi?doi?10.1073?pnas.0601412103Balansky et al.
susceptible to hair loss. For instance, alopecia-dependent apo-
ptosis was induced in C57BL?6 mice either treated with doxo-
rubicin (28), an anticancer drug that typically induces oxidative
DNA damage, or exposed whole-body to environmental ciga-
rette smoke (29). The involvement of environmental factors in
the induction of alopecia areata is further supported by the
present finding that B[a]P, a typical component of cigarette
smoke and other complex mixtures resulting from combustion
processes, induced hair loss in wild-type mice. Also in humans,
hair follicle cells have been shown to be able to convert B[a]P to
ultimate DNA-damaging and carcinogenic metabolites (30).
Irrespective of FHIT status, no lung tumors or glandular
stomach hyperplasia were observed in untreated B6?129 F1
mice, and the spontaneous incidence of forestomach tumors was
very low. These findings are in line with the conclusion that the
incidence of spontaneous lung tumors is approximately the same
in wild-type and FHIT-deficient B6?129 F1mice (6). In addition,
the baseline frequency of MN NCE, monitored at periodic
intervals in peripheral blood, was particularly low in this mouse
Multiple administrations of B[a]P by gavage induced, in both
A?J and B6?129 F1mice, an evident increase of MN NCE in
peripheral blood, which reflects the induction of cytogenetic
damage in bone marrow cells. Consistent with the fact that
treatment with B[a]P lasted 28 days and that the half-life of NCE
in mice is ?30 days (31), the levels of B[a]P-induced MN NCE
declined by the end of the monitoring period. As expected, B[a]P
induced a high multiplicity of lung tumors in all treated A?J
mice, whereas both incidence and multiplicity of lung tumors
were modest in B6?129 F1mice. Note that these mice are derived
from C57BL?6 mice, which are rather insensitive to the induc-
tion of lung tumors (32). In addition, B[a]P caused hyperplasia
of the glandular stomach in an appreciable proportion of A?J
mice, whereas these histopathological alterations were infre-
quent in B6?129 F1mice. Both mouse strains were conversely
sensitive to the induction of forestomach keratoses, hyperplasias,
and tumors by B[a]P. In addition, irrespective of FHIT status, the
oral administration of B[a]P to B6?129 F1mice caused hyper-
plasia of both bronchial epithelium and alveolar walls. Interest-
ingly, a B[a]P-related formation of preneoplastic lesions of the
uterus was only evident in FHIT?/?mice.
Thus, on the whole, the yield of both forestomach tumors and
lung tumors by B[a]P does not appear to be affected by heterozy-
gosity for FHIT. However, FHIT?/?mice were more sensitive than
wild-type mice to the induction by B[a]P of uterus and liver
pathological lesions, which suggests a possible protective role of
FHIT in these organs. It should be noted that FHIT inactivation is
an early event in carcinogenesis of the endometrium (33) and that
deficient mice (6). In previous studies, FHIT?/?mice had been
shown to be more susceptible than their wild-type counterparts to
the induction of forestomach tumors by N-nitrosomethylben-
zylamine (NMBA) (6), whereas there was no difference in the
induction of lung tumors by 4-methylnitrosamino-1,3-pyridyl-1-
butanone (NNK) in FHIT heterozygous mice (34).
The oral administration of the chemopreventive agents NAC
and budesonide inhibited hair bulb cell apoptosis and formation
of alopecia areata. Moreover, budesonide and NAC exerted a
variety of protective effects on preneoplastic and neoplastic
alterations induced by B[a]P. The protective effect of budes-
onide is consistent with the well known antiinflammatory prop-
erties of glucocorticoids (14). Among the variety of mechanisms
of NAC, this thiol compound has antiinflammatory properties
and inhibits triggering of apoptosis consequent to DNA damage
and redox imbalances (22). NAC has been shown to prevent
alopecia induced by either doxorubicin (28) or cigarette smoke
(29) in C57BL?6 mice or by 2-chloroethyl ethyl sulfide in guinea
pigs (35). Interestingly, NAC was able to inhibit the loss of Fhit
protein induced by cigarette smoke in the bronchial epithelium
of Sprague–Dawley rats, whereas other chemopreventive agents,
including oltipraz, 5,6-benzoflavone, phenethyl isothiocyanate,
and indole-3-carbinol, were ineffective (36). As an antioxidant,
nucleophile, and scavenger of free radicals, NAC is likely to
suppress the stimuli that alter the FHIT gene and cause the loss
of Fhit protein. In addition, because Cu(II) is a strong inhibitor
of the enzymatic activity of Fhit due to its reaction with Cys-39,
which bears the only thiol group in the Fhit monomer (37),
protection of Fhit by NAC may also be ascribed to the ability of
thiols to react with Cu(II) and other metals (38).
In conclusion, heterozygosity for FHIT does not appear to
confer an increased susceptibility of B6?129 F1mice to B[a]P
tumorigenicity in lung and stomach but renders the animals
and B[a]P-induced preneoplastic lesions of the uterus. Budes-
onide and NAC are able to inhibit B[a]P-induced forestomach
tumors and preneoplastic lesions in the respiratory tract. In
addition, they prevent both spontaneous and B[a]P-induced
alopecia areata, an inflammatory skin disease in which FHIT
appears to play an evident protective role.
Materials and Methods
Mice. Eighty female A?J mice weighing 18–19 g at the start of the
experiment were commercially available (Harlan Italy, S. Pietro al
Natisone, Italy). A total of 163 female B6?129 F1, 74 wild-type, and
Genoa. They weighed 24–27 g at the start of the experiment. The
mice were housed in Makrolon cages on sawdust bedding and were
maintained on standard mouse chow (MIL Morini, San Polo
d’Enza, Italy) and tap water ad libitum. The temperature of the
animal room was 23 ? 2°C, with a relative humidity of 55%,
light–dark cycle. The housing and treatment of animals were in
accordance with our national and institutional guidelines.
Chemicals. B[a]P and budesonide were purchased from Sigma,
and NAC was purchased from Zambon (Bresso, Italy) in the
form of a commercially available product (Fluimucil).
Treatment of Mice. After a 2-week acclimatization, A?J, B6?129
F1wild-type, and B6?129 F1FHIT?/?mice were divided into
four groups, each composed of 14–23 animals. The mice be-
longing to the first group (controls) were treated with corn oil
were treated with B[a]P dissolved in corn oil (0.1 ml) and given
by gavage in eight doses (1 mg per dose) twice per week for 4
weeks. The mice belonging to the third group (B[a]P plus
budesonide) were treated with B[a]P in the same way as mice in
the second group and received budesonide with the diet (2.4
mg?kg diet), which was prepared once per week. The mice
belonging to the fourth group (B[a]P plus NAC) were treated
with B[a]P in the same way as mice in the second group and
of 1 g?kg body weight. Treatment with the two chemopreventive
agents started 3 days before the first B[a]P administration and
continued until the end of the experiment. The mice were
inspected daily for their general appearance and weighed indi-
vidually at monthly intervals. All A?J mice were killed 7 months
after the first B[a]P administration, whereas all B6?129 F1mice
were killed 11 months after the first B[a]P administration to
allow for a better growth of lung tumors. The mice were killed
by cervical dislocation after deep anesthesia with diethyl ether.
Stomach, lungs, kidneys, and liver were collected from all mice.
A complete necropsy was performed, and tissues with macro-
scopically visible alterations were subjected to standard his-
Balansky et al.
May 16, 2006 ?
vol. 103 ?
no. 20 ?
Cytogenetic Monitoring. At periodic intervals (see Fig. 1), samples Download full-text
of blood were collected from the tail lateral vein of 10 mice per
group. Duplicates of smears of peripheral blood were stained
with May–Gru ¨nwald–Giemsa. An average of 50,000 NCE were
scored per mouse, accounting for a total of 42 million NCE
scored for the presence of MN. The results are expressed as MN
frequency per 1,000 NCE.
Forestomach Tumors and Glandular Stomach Hyperplasia. The fore-
stomach of each mouse was cut longitudinally, and forestomach
tumors were detected with the aid of a stereomicroscope. The
forestomach and glandular stomach of each mouse were cut into
10 sections each and subjected to standard microscopic analysis
to evaluate possible histopathological alterations.
after 48 h, the tumors were detected on the lung surface with the
aid of a stereomicroscope. The two main diameters of each
tumor were measured with a linear micrometer, and the mean
diameter was calculated. All tumors were subjected to standard
P53, Fhit, PCNA, and Apoptosis. Mutated or inactivated P53 protein
was detected in skin fragments and lung tumors by immunohis-
tochemistry using the CM-5 polyclonal antibody (NovoCastra,
Newcastle upon Tyne, U.K.). Positivity for Fhit in skin cells and
lung tumors was evaluated by immunohistochemistry using a
rabbit anti-Fhit polyclonal antibody, kindly supplied by Kay
Huebner (Ohio State University Comprehensive Cancer Center,
Columbus), at a final dilution of 1:2,000. Formalin-fixed, paraf-
fin-embedded sections were routinely processed by using the
HistoMouse-SP kit (Zymed Laboratories), according to the
manufacturer’s instructions. The slides were scored at a magni-
fication of ?400, and 1,000 cells per slide were examined.
PCNA was detected by immunohistochemistry using the
PCNA staining kit (Zymed Laboratories). This kit is based on an
anti-PCNA monoclonal antibody (clone PC10) and uses avidin?
biotinylated peroxidase complex (ABC) technology. The slides
were scored at a magnification of ?400, and 1,000 cells per slide
The frequency of apoptotic cells was evaluated by the TUNEL
method with two different commercially available kits. For skin
cells, we used the Dermatacs in situ apoptosis detection kit
the Tacs XL Blue Label in situ apoptosis detection kit (Trevi-
gen). Both kits were used according to the manufacturer’s
instructions. The slides were scored at a magnification of ?400,
and 1,000 cells per mouse were examined.
Statistical Analyses. Comparisons of treatment-related differ-
ences regarding frequency of alopecia, incidence of tumors and
preneoplastic lesions, and frequency of P53-positive, PCNA-
positive, and apoptotic cells were made by ?2analysis. Compar-
isons regarding quantitative data, expressed as means ? SE,
including body weight, tumor multiplicity, and size of tumors,
were made by Student’s t test for unpaired data. Differences with
P ? 0.05 were taken as statistically significant.
This work was supported by grants from the Italian Ministry of Health,
the National Institutes of Health National Cancer Institute, and the
Bulgarian Ministry of Education and Science.
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