High susceptibility of neonatal mice to molecular, biochemical and
cytogenetic alterations induced by environmental
cigarette smoke and light
Silvio De Floraa,*, Francesco D’Agostinia, Roumen Balanskya,b, Anna Camoiranoa,
Cristina Cartigliaa, Mariagrazia Longobardia, Giorgia Travainia, Vernon E. Steelec,
Carlo Pesced, Alberto Izzottia
aDepartment of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy
bNational Centre of Oncology, Sofia 1756, Bulgaria
cNational Cancer Institute, Rockville, MD 20892, USA
dDepartment of Biophysical, Medical and Odontostomatological Sciences and Technologies, University of Genoa, I-16132 Genoa, Italy
Received 31 July 2007; received in revised form 25 October 2007; accepted 1 November 2007
Available online 17 November 2007
Our recent studies have shown that both cigarette smoke and UV-containing light, which are the most widespread and ubiquitous mutagens and
of mice to mainstream cigarette smoke, starting at birth, caused an early and potent carcinogenic response in the lung and other organs. Our further
experiments showed that exposure of mice to environmental cigarette smoke, during the first 5 weeks of life, resulted in a variety of significant
increase of bulky DNA adduct levels, induction of oxidative DNA damage, and overexpression of OGG1 gene in lung, stimulation of apoptosis,
in the respiratory tract. Moreover, exposure of mice to UV-containing light, mimicking solar irradiation, significantly enhanced oxidative DNA
expression in lung was particularly high at birth and decreased in post-weanling mice. Oxidative DNA damage and other investigated end-points
the neonatal period and early stages of life are critical in affecting susceptibility to carcinogens.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Environmental cigarette smoke; Mainstream cigarette smoke; UV-containing light; Carcinogenicity; Intermediate biomarkers; Genotoxicity; Neonatal
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Susceptibility to carcinogens at birth and early in life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Carcinogenicity of mainstream cigarette smoke in neonatal mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Available online at www.sciencedirect.com
Mutation Research 659 (2008) 137–146
Abbreviations: B2m, beta-2 microglobulin; CA, chromosomal aberrations; CS, cigarette smoke; ECS, environmental cigarette smoke; MCS, mainstream
cigarette smoke; MDA, malondialdehyde; MN, micronuclei; NCE, normochromatic erythrocytes; OGG1, 8-oxoguanosine-glycosylase 1; 8-oxo-dG, 8-oxo-20-
deoxyguanosine; PAM, pulmonary alveolar macrophages; PCE, polychromatic erythrocytes; PCNA, proliferating cell nuclear antigen; qPCR, quantitative real-time
polymerase chain reaction; Sca-1, stem cell antigen 1; TBARS, thiobarbituric acid reactive substances; TLC, thin layer chromatography; TUNEL, TdT-mediated
dUTP nick end labeling.
* Corresponding author. Tel.: +39 010 3538500; fax: +39 010 3538504.
E-mail address: firstname.lastname@example.org (S. De Flora).
1383-5742/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
4. Alterations of intermediate biomarkers in neonatal mice exposed to environmental cigarette smoke and/or light. . . . . . . . . . . .
4.1.General design of the studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Body weights. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Bulky DNA adducts, 8-oxo-dG, and TBARS in lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.Expression of OGG1 in lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.Apoptosis and Fhit protein in pulmonary alveolar macrophages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6.PCNA, apoptosis, P53 and Fhit in the bronchial epithelium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7. Cytogenetic damage in peripheral blood and bone marrow erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.8.Histopathological analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Systemic genotoxicity of ECS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Systemic genotoxicity of UV-containing light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Increased susceptibility of mice exposed early in life. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tobacco smoking is the most important cause of death for
cancer and other chronic degenerative diseases worldwide.
existing in nature, and UV-emitting artificial light sources have
been shown to be genotoxic  and carcinogenic [2,3].
Collectively, tobacco smoking and sunlight have been
estimated to account for 40% of all human cancers .
Recently, we provided evidence that exposure of SKH-1
hairless mice not only to environmental cigarette smoke (ECS)
but even to the light emitted by UV-C-covered halogen quartz
bulbs, simulating sunlight, produces extensive alterations in the
expected target organs as well as at a systemic level . In fact,
the light caused formation of bulky DNA adducts in both lung
and bone marrow and induced cytogenetic damage in bone
marrow and peripheral blood erythrocytes. We speculate that
the alterations causedbylightatasystemiclevelare likelytobe
mediated by formation of long-lived genotoxic derivatives,
such as malondialdehyde (MDA) and other lipid peroxidation
products, in the skin of light-exposed mice . In addition,
there was a synergism between light and ECS in the respiratory
tract, as shown by increased levels of thiobarbituric acid
reactive substances (TBARS) and bulky DNA adducts in lung
and by an increased frequency of micronucleated (MN)
pulmonary alveolar macrophages (PAM), as compared with
mice exposed to ECS only . These results were further
supported by multigene expression analyses of mouse skin and
lung, showing that exposure to the light results in over-
expression of genes in lung, such as catalase and glutathione S-
transferase-Pi, and tends to upregulate ECS-induced gene
expression in lung .
2. Susceptibility to carcinogens at birth and early in life
Susceptibility to carcinogens may be enhanced during
certain stages of life, and there is adequate reason to suspect
that perinatal exposures contribute to both childhood cancers
25 years (1980–2004) provided evidence that chromosomal
aberrations (CA) and, to a lesser extent, MN levels are
increased in children exposed to environmental contaminants
. Specifically, increased frequencies of CA and increased
levels of DNA adducts and hemoglobin adducts were
associated with prenatal and postnatal exposure of children
to CS . Epidemiologic work has suggested that exposure to
tobacco carcinogens at an early age may be an independent risk
factor for lung cancer, and adolescence constitutes a critical
period during which tobacco carcinogens can induce fields of
damaging effects of continuing smoking .
Our previous studies demonstrated that birth itself, without
any treatment, causes strong nucleotide alterations in mouse
lung,asshownby a5-foldincreaseofbulkyDNAadducts and a
2-fold increase of oxidative damage, and by overexpression of
33 out of 746 genes analyzed by cDNA array, including genes
involved in adaptivefunctions, such as glutathione metabolism,
cellular stress, and response to DNA and protein damage .
Affymetrix analyses confirmed that a largenumber of genes are
differently expressed in mouse fetal lung as compared with
postnatal lung .
3. Carcinogenicity of mainstream cigarette smoke in
These premises prompted us to implement a series of studies
aimed at evaluating whether exposure of mice, early in their
life, to cigarette smoke (CS) and/or light may enhance their
susceptibility to alterations of intermediate biomarkers and
induction of tumors. The earliest study  provided evidence
that exposure of mice to mainstream cigarette smoke (MCS),
starting within 12 h after birth and continuing for 4 months,
resulted in a potent carcinogenic response, characterized by (a)
a short latency time, (b) a high yield of preneoplastic lesions
and benign tumors in lung, (c) occurrence of pulmonary
malignancies, and (d) occurrence of both primary lesions and
metastases outside the respiratory tract. Similar conclusions are
being confirmed by the preliminary results of a further study
with MCS (R. Balansky et al., study in progress). These
findings contradict the widespread view that CS is a weak
carcinogen in experimental animals. A new model is thus
available for evaluating the mechanisms involved in CS
S. De Flora et al./Mutation Research 659 (2008) 137–146138
carcinogenicity and for assessing efficacy and safety of cancer
chemopreventive agents. We refer to the original paper  for
4. Alterations of intermediate biomarkers in neonatal
mice exposed to environmental cigarette smoke and/or
We implemented studies in mice exposed to ECS and UV-
containing light, either individually or in combination. Like in
the above described study with MCS, exposures started within
12 h after birth. Subgroups of variously treated mice were
sacrificed after weanling, approximately 5 weeks after birth,
together with the dams that had been exposed under identical
conditions. A variety of intermediate biomarkers were
comparatively evaluated in male and female post-weanling
mice and their dams.
4.1. General design of the studies
A total of 26 pregnant Swiss CD-1 albino mice were
purchased from Harlan Italy (San Pietro al Natisone, Udine,
sawdust bedding and maintained on standard rodent chow
(MIL, Morini, S. Polo d’Enza, Italy) and tap water ad libitum.
The cages were kept in a cabinet where filtered air was
circulated. The temperature of the animal room was 23 ? 2 8C,
with a relative humidity of 55%, ventilation accounting for 15
air renewal cycles per hour, and a 12-h day/night cycle. The
housing and treatments of mice were in accordance with our
national and institutional guidelines, and were approved by the
Italian Ministry of Health.
Each dam generated an average of 10 pups/litter, for a total
of 260 new generation mice. The neonatal mice belonging to
one litter were sacrificed immediately after birth in order to
evaluate OGG1 expression in lung. The remaining neonatal
mice and the dams were divided into 4 experimental groups,
exposedmice; (C)light-exposedmice; and (D) mice exposedto
both ECS and light. All treatments started within the first 12 h
of life. Each dam was kept with its litter during treatments.
ECS was generated by burning Kentucky 2R4F reference
cigarettes (Tobacco Research Institute, University of Kentucky,
Lexington, KY, USA), having a declared content of 9.2 mg tar
and 0.8 mg nicotine each, with a 23 mm butt remaining after
smoking. Awhole-body exposure of mice to ECS was achieved
by using a smoking machine (model TE-10, Teague
Enterprises, Davis, CA, USA). The machine was adjusted to
produce a combination of sidestream smoke (89%) and
mainstream smoke (11%), mimicking a high-dose exposure
total suspended particulate of 63.3 mg/m3in the exposure
chambers. Exposure was daily, 6 h/day divided into two rounds
with a 3-h interval.
Exposure to light was obtained by using halogen quartz
bulbs, incorporated into dichroic spot light lamps (12 V, 50 W),
which were supplied by Leuci (File, Lecco, Italy). The lamps
were equipped with filters cutting UV-C light (WG 280, Schott
Optics Division, Mainz, Germany). The distance from the back
of the mice was approximately 50 cm and yielded an
illuminance level of 10,000 lx. Exposure was daily, 6 h/day.
by applying the following schedule: 3 h ECS, 3 h light, 3 h
ECS, and 3 h light. Light and ECS were not simultaneously
applied in order to avoid the risk of alterations of ECS
components by light .
When the infant mice became post-weanling, approxi-
mately 5 weeks after birth, they were weighed and housed
separately according to gender. All dams (5–7/group) and
subgroups of 10 post-weanling mice (5 males and 5 females/
group) were deeply anesthesized with diethyl ether and killed
by cervical dislocation.All remainingmicewere kept alivefor
a carcinogenesis study, which is now in progress. Bronch-
oalveolar lavage was immediately performed by lavaging the
lungs with three 2-ml aliquots of cold (4 8C) 0.15 M NaCl
infused via a cannula inserted in the trachea. The cells were
washed twice with RPM1 1640 and then spun in a
cytocentrifuge and fixed with methanol. Peripheral blood
was collected by the lateral tail vein and smeared on duplicate
slides. The left femur was collected, and bone marrow was
smeared on duplicate slides. The right lung of each mousewas
stored at ?80 8C for molecular analyses, whereas the left lung
was fixed in formalin.
As detailed in next sessions, we implemented a series of
studies in order to comparatively evaluate the following end-
points in male and female post-weanling mice and in their
dams, either unexposed or exposed to ECS and/or light:
- body weights;
- apoptosis and loss of Fhit oncoprotein in PAM;
- proliferating cell nuclear antigen (PCNA), apoptosis, inacti-
vated or mutated P53, and loss of Fhit in the bronchial/
- bulky DNA adducts, 8-oxo-20-deoxyguanosine (8-oxo-dG),
TBARS, 8-oxoguanosine-glycosylase 1 (OGG1) expression,
and histopathological alterations in whole lung;
- cytogenetic damage in bone marrow and peripheral blood
4.2. Body weights
After weanling, when the mice were divided into males and
females,thebodyweights(means ? S.E.)oftheuntreatedmice
(Group A, Sham), ECS-exposed mice (Group B), light-exposed
mice (Group C), and mice exposed to both ECS and light
(Group D) were 22.2 ? 0.63, 18.1 ? 0.40, 23.5 ? 0.76, and
17.0 ? 0.30 g
21.1 ? 0.64, and 15.3 ? 0.30 g in females, respectively. The
body weight loss observed in mice exposed to either ECS or
ECS + light, ranging between 17.8 and 23.4% as compared
with the corresponding Sham,was significant in bothmales and
females (P < 0.001 in all cases, as assessed by Student’s t-test).
We observed a similar weight loss in adult mice exposed to
cigarette smoke, either MCS  or ECS .
inmales, and19.6 ? 0.43,16.1 ? 0.34,
S. De Flora et al./Mutation Research 659 (2008) 137–146139
4.3. Bulky DNA adducts, 8-oxo-dG, and TBARS in lung
This study involved the analysis of bulky DNA adducts and
8-oxo-dG and the measurement of MDA and other TBARS in
the lung of 64 mice, including 40 post-weanling mice and 24
dams. All end-points were evaluated in two separate experi-
ments, whose means were used in order to generate the data
summarized in Table 1, which are means ? S.E. of the results
obtained within each experimental group.
Bulky lipophilic DNA adducts were detected, after DNA
purification, butanol enrichment and
previously described [14–16]. A diagonal radioactive zone
was evident in TLC sheets of all mice exposed either to ECS or
ECS + light (not shown). As reported in Table 1, the increases
in bulky DNA adduct levels in the lung of male (5.8-fold) and
female (5.1-fold) ECS-exposed post-weanling mice (Group B),
as compared with Sham (Group A), were similar to those
observed in dams (4.8-fold). Interestingly, exposure to the light
alone (Group C) caused the formation of a diffuse radioactive
signal, without any well-defined spot, resulting in a slight but
significant increaseoflipophilicDNAadducts inpost-weanling
females(1.8-fold).Moreover,although the differenceswere not
statistically significant, ECS-related DNA adducts were further
increased in both male (6.3-fold) and female (6.2-fold) post-
weanling mice exposed to ECS + light.
8-Oxo-dG was measured by using a
procedure, as described previously . A standard of
authentic 8-oxo-dG (National Cancer Institute Chemical
32P postlabeling, as
Research Institute, Kansas City, MO, USA) was used in order
to localize the position of this oxidatively modified base in
TLCs. In addition, an 8-oxo-dG positive reference sample was
obtained by incubating at 37 8C calf thymus DNA with 1 mM
CuSO4plus 50 mM hydrogen peroxide, as reported previously
. The results (Table 1) can be summarized as follows: (a) 8-
oxo-dG levels were significantly higher in untreated (Sham)
adult female mice, i.e., the dams, than in post-weanling female
litters (Group A); (b) exposure to ECS alone (Group B) resulted
in a considerable increase of 8-oxo-dG levels in both male (8.7-
fold) and female (10.3-fold) post-weanling mice, whereas the
increase was still significant but less pronounced in dams (2.2-
fold). Therefore, in spite of the lower baseline levels, 8-oxo-dG
levels in ECS-exposed post-weanling mice, both males and
females,became significantlyhigherthan indams;(c) exposure
to the light alone (Group C) resulted in a statistically significant
increase of 8-oxo-dG levels in the lung of all mice. Again, this
increase was more evident in male (2.8-fold) and female (4.1-
fold) post-weanling mice than in dams (1.5-fold); (d) 8-oxo-dG
levels were also increased in post-weanling mice exposed to
ECS + light (Group D), both males (8.6-fold) and females
(13.3-fold). The difference between ECS + light- and ECS-
exposed post-weanling females was statistically significant.
Conversely, in dams exposed to ECS + light the increase of 8-
oxo-dG levels (2.6-fold) was similar to that observed in dams
exposed to ECS alone (2.2-fold).
MDA and other TBARS were measured in lung samples by
using the thiobarbituric acid method . As shown in Table 1,
two findings are noteworthy: (a) the baseline TBARS levels
were significantly higher in the lung of untreated post-weanling
females than in the corresponding dams (Group A); (b)
exposure to either ECS alone (Group B) or ECS + light (Group
D) resulted in a moderate but significant increase of TBARS in
the lung of all mice.
4.4. Expression of OGG1 in lung
Analysis of OGG1 gene transcriptional activity was
performed by mRNA retrotranscription reaction followed by
qPCR. Primers, qPCR molecular beacons (TIB MolBiol,
Genoa, Italy), and PCR reaction conditions were designed
using the Beacon Designer 6.0 software (Premier Biosoft
International, Palo Alto, CA, USA). The results obtained with
housekeeping gene beta-2 microglobulin (B2m).
Table 2 summarizes the results relative to evaluation of
OGG1 expression in the lung of 74 mice, including 10 mixed
gender newborns, 40 male and female post-weanling mice, and
24 dams. The baseline OGG1 expression was significantly
higher in newborn mice as compared with dams and, evenmore
sharply, as compared with post-weanling mice. The baseline
values recorded in post-weanling mice were significantly lower
than those recorded in dams. Exposure to either ECS alone
(Group B) or ECS + light (Group D) resulted in a significant
upregulation of OGG1 in lung, but only in dams. No significant
effect was produced by light alone (Group C).
Bulky DNA adducts, oxidative DNA damage, and TBARS in the lung of post-
weanling mice and their dams
(Group) treatment Post-weanling miceDams
Bulky DNA adducts/108nucleotides
(D) ECS + light
1.4 ? 0.13
7.8 ? 0.54c
1.9 ? 0.24
8.5 ? 0.80c
1.3 ? 0.18
6.6 ? 0.68c
2.3 ? 0.15a
8.1 ? 0.69c
1.3 ? 0.08
6.2 ? 0.51c
1.5 ? 0.23
6.6 ? 0.59c
(D) ECS + light
1.6 ? 0.19e
13.9 ? 0.54c,i
4.5 ? 0.29c
13.8 ? 1.04c,i
1.1 ? 0.12f
11.3 ? 1.07c,h
4.5 ? 0.39c
14.6 ? 0.70c,d,i
2.9 ? 0.38
6.4 ? 0.49c
4.3 ? 0.36a
7.4 ? 0.47c
TBARS (nmol/g tissue)
(D) ECS + light
167.79 ? 18.83g
241.82 ? 21.04a,h
172.76 ? 10.64h
285.46 ? 10.48c,i
170.02 ? 7.67h
261.15 ? 12.91c,i
190.98 ? 13.30h
297.23 ? 18.08c,i
111.14 ? 11.37
153.73 ? 13.98b
109.15 ? 12.80
162.96 ? 8.02b
Values are expressed as means ? S.E. for 5–7 mice treatment/gender. The
significance of differences between experimental groups was evaluated by
aP < 0.05,bP < 0.01,andcP < 0.001,significantlyincreasedascomparedwith
the corresponding Sham.
dP < 0.05, significantly increased as compared with the corresponding ECS.
eP < 0.01 andfP < 0.001, significantly lower than the baselinevalues recorded
gP < 0.05,hP < 0.01, andiP < 0.001, significantly higher than the correspond-
S. De Flora et al./Mutation Research 659 (2008) 137–146140
4.5. Apoptosis and Fhit protein in pulmonary alveolar
Apoptosis in PAM samples and bronchial/bronchiolar
epithelium was evaluated by TUNEL (TdT-mediated dUTP
nick end labeling) method using the Tacs XL Blue Label In Situ
Apoptosis Detection Kit (Trevigen, Gaithersburg, MD, USA),
following the manufacturer’s instructions. The slides were
scored at a magnification of ?400 and 1000 cells/mouse were
examined. Table 3 summarizes the data relativeto evaluation of
apoptosis in the PAM collectedby bronchoalveolar lavage from
40 post-weanling mice. Exposure to ECS, either alone (Group
B) or combined with light (Group D), resulted in a considerable
increase in the proportion of apoptotic PAM, ranging between
4.7-fold and 5.3-fold.
Fhit protein was evaluated by using a rabbit anti-Fhit
polyclonalantibody,kindly supplied byDr.Kay Huebner (Ohio
State University Comprehensive Cancer Center, Columbus,
OH, USA), at a final dilution of 1:2000. Formalin-fixed,
paraffin-embedded sections were routinely processed by using
the HistoMouse-SP kit (Zymed Laboratories, San Francisco,
CA, USA), according to the manufacturer’s instructions. The
slides were scored at a magnification of ?400, and 1000 cells
showing an evident loss of Fhit out of those examined, as
previously described . As shown in Table 3, the large
majority of the scored PAM was positive for Fhit in Sham-
exposed mice. Exposure to ECS, either alone (Group B) or
combined with light (Group D), resulted in a significant loss of
Fhit-positive PAM in both genders.
4.6. PCNA, apoptosis, P53 and Fhit in the bronchial
Table 4 summarizes the data relative to the immunohisto-
chemical evaluation of proliferation, apoptosis, mutated or
inactivated P53, and loss of Fhit protein in bronchial epithelial
cells from 64 mice, including 40 post-weanling mice and 24
For the analysis of PCNA, 5 mm sections were cut and
placed onto slides treated with poly-L-lysine (Poly-PrepTM
OGG1expression(qPCRrelativeexpressionnormalizedfor B2m) inthe lungof
newborn mice, post-weanling mice and their dams
(Group) treatment NewbornsOGG1 expression
(D) ECS + light
2.3 ? 0.38a
0.7 ? 0.05c
0.4 ? 0.11c
0.7 ? 0.14b
0.5 ? 0.09c
0.5 ? 0.04c
0.5 ? 0.12c
0.7 ? 0.16b
0.7 ? 0.23c
1.1 ? 0.13
1.7 ? 0.05e
1.1 ? 0.10
1.6 ? 0.18d
Values are expressed as means ? S.E. for 5–10 mice/treatment/gender. NA, not
available. The significance of differences between experimental groups was
evaluated by Student’s t-test.
aP < 0.001, significantly higher as compared with both post-weanling mice and
bP < 0.05 andcP < 0.001, significantly lower as compared with dams.
dP < 0.05 andeP < 0.001, significantly increased as compared with the corre-
Frequency of apoptotic and Fhit-positive pulmonary alveolar macrophages
(PAM) collected from post-weanling mice
(Group) treatmentMales FemalesMales + females
Apoptotic cells (%)
(D) ECS + light
1.8 ? 0.36
8.8 ? 0.69a
1.4 ? 0.35
8.4 ? 1.04a
1.6 ? 0.20
7.5 ? 0.61a
1.4 ? 0.25
8.4 ? 1.07a
1.7 ? 0.20
8.2 ? 0.49a
1.4 ? 0.20
8.4 ? 0.70a
Fhit-positive cells (%)
(D) ECS + light
87.8 ? 2.94
61.9 ? 3.55c
88.9 ? 2.88
60.1 ? 4.45c
87.4 ? 3.12
61.4 ? 6.32b
88.3 ? 2.80
71.8 ? 2.88b
87.6 ? 2.02
61.6 ? 3.42c
88.6 ? 1.90
66.0 ? 3.17c
Values are expressed as means ? S.E. for 5 mice/treatment/gender. The sig-
nificance of differences between experimental groups was evaluated by Stu-
aP < 0.001, significantly increased as compared with the corresponding Sham.
bP < 0.01 and
cP < 0.001, significantly decreased as compared with the
Frequency of cells positive for either PCNA, apoptosis, inactivated or mutated
P53, or loss of Fhit in the bronchial epithelium of post-weanling mice and their
(Group) treatmentPost-weanling miceDams
(D) ECS + light
0.24 ? 0.12
1.12 ? 0.14b
0.24 ? 0.08
1.20 ? 0.21b
0.20 ? 0.09
1.24 ? 0.17c
0.12 ? 0.08
1.12 ? 0.26b
0.11 ? 0.06
0.83 ? 0.15c
0.16 ? 0.08
0.80 ? 0.18b
(D) ECS + light
0.20 ? 0.06
1.20 ? 0.09c
0.08 ? 0.05
1.76 ? 0.08c,d,e
0.24 ? 0.10
1.60 ? 0.09c
0.32 ? 0.14
1.88 ? 0.19c,e
0.17 ? 0.05
1.17 ? 0.24b
0.16 ? 0.08
1.28 ? 0.15c
Inactivated or mutated P53 (%)
(D) ECS + light
0.08 ? 0.05
0.08 ? 0.08
0.04 ? 0.04
0.12 ? 0.08
0.04 ? 0.04
0.08 ? 0.05
0.04 ? 0.04
0.16 ? 0.09
0.06 ? 0.04
0.09 ? 0.06
0.04 ? 0.04
0.12 ? 0.08
Loss of Fhit (%)
(D) ECS + light
Values for PCNA, apoptosis, and inactivated or mutated P53 are expressed as
means ? S.E. for 5–7 mice/treatment/gender. The significance of differences
between experimental groups was evaluated by Student’s t-test. Values for Fhit
are expressed as fraction of mice showing a loss of Fhit as related to the number
was evaluated by x2analysis.
aP ? 0.05,bP < 0.01,andcP < 0.001,significantlyincreasedascomparedwith
the corresponding Sham.
dP < 0.01, significantly increased as compared with the corresponding ECS.
eP < 0.05, significantly higher than the corresponding dams.
S. De Flora et al./Mutation Research 659 (2008) 137–146141
Slides, Sigma Diagnostics, St Louis, MO, USA). PCNA was
detected by using the NCL-PCNA kit (Novocastra Labora-
tories, Newcastle upon Tyne, UK), following the manufacurer’s
instructions. This detection kit is based on an anti-PCNA
monoclonal antibody (clone PC10) and employs avidin-
biotinylated horseradish peroxidase complex technology
(ABC technique). The slides were scored at a magnification
of ?400 and 1000 cells/mousewere examined. As summarized
in Table 4, the baseline proportion of PCNA-positive cells was
twice in post-weanling mice as compared with dams, but this
difference was not statistically significant. Exposure to ECS
(Group B) resulted in a significant stimulation of both
proliferation and apoptosis in post-weanling mice and dams.
The combined exposure to ECS + light (Group D) did not
further affect the proportion of PCNA-positive cells, whereas a
further significant increase of apoptotic cells was observed in
post-weanling mice, both males and females, but not in dams.
P53 oncoprotein was detected in sections of lung from each
mouse by using CM5 polyclonal rabbit antibody (NCL-P53
CM5p, Novocastra Laboratories), which detects both over-
expression and mutation of P53 gene. Formalin-fixed, paraffin-
embedded sections(5 mm)werepretreatedwith 0.01 Msodium
citrate buffer (pH 6.0) in a microwave oven at a high
temperature for 10 min. The sections were routinely processed
using the Vectastain ABC kit (Vector Laboratories, Burlin-
game, CA, USA), following the manufacturer’s instructions. A
slide of the same tissue, incubated with normal serum without
CM5 antibody, was used as negative control. Slides of mouse
3T3 cells were used as positivecontrols. The slides were scored
at a magnification of ?400 and 1000 cells/mouse were
examined. Any nucleus showing a partial or total brownish
staining was defined as positive. Table 4 shows that the
proportion of bronchial epithelial cells harboring mutated or
inactivated P53, as detected by the monoclonal antibody used,
was very low, irrespective of the age and gender of mice and of
Since it is difficult to calculate the percentage of cells
positive for Fhit protein, which is localized in the cytoplasm of
expressed in terms of mice showing an evident loss of Fhit
out of those examined. All samples from Sham-exposed mice
(Group A) and light-exposed mice (Group C) exhibited a
diffuse positivity for Fhit in virtually all cells of the bronchial/
bronchiolar epithelium, while all ECS-exposed (Group B)
and ECS + light-exposed mice (Group D) had a significant loss
4.7. Cytogenetic damage in peripheral blood and bone
The cytogenetic damage was evaluated in bone marrow
polychromatic erythrocytes (PCE) and in peripheral blood
normochromatic erythrocytes (NCE). Briefly, bone marrow
smears from 5 mice per group were air-dried and stained with
for the presence of micronucleated (MN) erythrocytes. The
indicator of toxicity to bone marrow cells. Peripheral blood
smears from 10 mice per group were air-dried and stained with
May–Gru ¨nwald–Giemsa, and 50,000 NCE per mouse were
scored for the presence of MN erythrocytes.
Table 5 summarizes the results of cytogenetic analyses
performed in bone marrow and peripheral blood samples
collected from 64 mice, including 40 post-weanling mice and
significant increase in the frequency of both MN PCE in bone
marrow and MN NCE in peripheral blood, irrespective of
gender and age of mice. This effect was less evident in mice
exposed to ECS + light (Group D), such increase being
statistically significant in post-weanling females only. The
PCE/NCEratio inbone marrowwas not affected by gender,age
4.8. Histopathological analyses
Sections of lung from 64 mice were fixed, stained by
hematoxylin and eosin, and examined microscopically for the
detection of early histopathological alterations. The histo-
pathological analysis showed, in the bronchial and bronchiolar
epithelium of dams exposed either to ECS (Group B) or
ECS + light (Group D), vacuolar degeneration (Fig. 1A) and
morphological signs of apoptosis, such as pycnosis, condensed
chromatin, and nuclear fragmentation (Fig. 1B). These
alterations, which were totally absent in Sham-exposed mice
(Group A) and light-exposed mice (Group C), were detectable
of 7 ECS-exposed dams and 4 out of 5 ECS + light-exposed
dams showed signs of alveolar hemorrhage (Fig. 1C).
Frequency of micronucleated (MN) peripheral blood normochromatic erythro-
cytes (NCE) and bone marrow polychromatic erythrocytes (PCE) collected
from post-weanling mice and their dams, and PCE/NCE ratio in bone marrow
(Group) treatment Post-weanling miceDams
MN NCE (%)
(D) ECS + light
0.81 ? 0.097
1.09 ? 0.082a
0.64 ? 0.089
0.87 ? 0.082
0.75 ? 0.075
1.21 ? 0.070a
0.69 ? 0.079
0.93 ? 0.090
0.70 ? 0.159
1.14 ? 0.100a
0.57 ? 0.086
0.70 ? 0.159
MN PCE (%)
(D) ECS + light
1.77 ? 0.494
2.82 ? 0.542a
1.57 ? 0.413
2.53 ? 0.170
1.15 ? 0.123
3.20 ? 0.861a
1.40 ? 0.164
2.73 ? 0.413b
1.84 ? 0.415
3.89 ? 0.692a
2.80 ? 0.326
3.40 ? 0.734
(D) ECS + light
1.43 ? 0.181
1.19 ? 0.117
1.29 ? 0.156
1.89 ? 0.178
1.52 ? 0.279
1.60 ? 0.239
1.68 ? 0.152
1.85 ? 0.273
1.48 ? 0.202
1.86 ? 0.188
1.51 ? 0.188
1.60 ? 0.280
Values are expressed as means ? S.E. for 5–7 mice/treatment/gender. The
significance of differences between experimental groups was evaluated by
aP < 0.05, andbP < 0.01, significantly increased as compared with the corre-
S. De Flora et al./Mutation Research 659 (2008) 137–146 142
The same type of alterations were detected in all post-
weanling mice. In addition, a modest hyperplasia of the
bronchial and bronchiolar epithelium (Fig. 1D) was detected in
ECS and ECS + light-exposed post-weanling mice, both males
(6 out of 10) and females (7 out of 10). Alveolar hyperplasia
(Fig. 1E) was detected in the same mice.
5. Systemic genotoxicity of ECS
The results of the studies described in the previous sections
confirm that the whole-body exposure of mice to ECS affects a
broad variety of early biomarkers in different organs. In
particular, exposure to ECS of post-weanling mice and/or
dams, for 5 weeks, resulted in a number of significant
alterations, including cytogenetic damage in bone marrow and
peripheral blood erythrocytes, formation of lipid peroxidation
products in lung, a strong increase of bulky DNA adduct and 8-
oxo-dG levels in lung, accompanied by overexpression of the
OGG1 gene in lung, induction of apoptosis both in PAM and in
the bronchial epithelium, stimulation of proliferation, and loss
line with the conclusions of previous studies performed in our
laboratory, indicating that high-dose ECS produces biochem-
ical, cytogenetic and molecular alterations in various organs of
mice and rats [5,6,16,20–33]. Interestingly, in spite of the short
exposure period (5 weeks), histopathological alterations, such
as vacuolar degeneration, morphological signs of apoptosis and
a modest hyperplasia of the bronchial/bronchiolar epithelium,
and alveolar hyperplasia and hemorrhage, were readily
detectable in ECS-exposed mice.
Fig. 1. Histopathological alterations in the lung of mice exposed to ECS. (A) Vacuolar degeneration of the bronchial and bronchiolar epithelium. (B) Pycnosis,
condensed chromatin, and nuclear fragmentation. (C) Alveolar hemorrhage. (D) Hyperplasia of the bronchial and bronchiolar epithelium. (E) Alveolar hyperplasia.
Stained with hematoxylin-eosin. Original magnifications: ?200 (A, C–E) and ?1000 (B).
S. De Flora et al./Mutation Research 659 (2008) 137–146143
6. Systemic genotoxicity of UV-containing light
Exposure to the light alone produced several molecular
alterations, including significant increases in bulky DNA
adduct levels and 8-oxo-dG levels in lung. Moreover, some of
the alterations produced by exposure to ECS, such as the
increase of 8-oxo-dG levels and stimulation of apoptosis in
the bronchial/bronchiolar epithelium, were further enhanced by
the combined exposure to light. These results, generated in
Swiss mice, confirm the findings of our previous studies in
SKH-1 hairless mice [5,6] and provide further evidence for the
fact that UV-containing light, mimicking solar irradiation, has
the ability to produce systemic alterations and to synergizewith
CS in the respiratory tract.
7. Increased susceptibility of mice exposed early in life
Several investigated end-points exhibited differential pat-
terns in post-weanling mice and in dams. While the baseline
concentrations of TBARS in lung were significantly higher in
young mice than in adult mice, 8-oxo-dG levels in lung showed
an opposite trend. The ‘‘spontaneous’’ oxidative DNA damage
in the lung of post-weanling mice observed in the present study
was lower than that observed at birth in a previous study .
Also in humans, the sudden increase of oxygenation at birth
exposes the neonate to oxidative stress, documented by DNA
damage and alterations of the redox state in peripheral blood
mononuclear cells .
The baseline levels of bulky DNA adducts measured in the
lung of both post-weanling mice and dams were lower than
those recorded at birth, presumably due to the fact that several
adaptivegenes are upregulated at birth . The lower levels of
8-oxo-dG in post-weanling mice, as compared both with
newborns  and adult mice [this article], are likely to be
cells is the main DNA glycosylase removing this altered DNA
base. In fact, the baseline OGG1 expression was significantly
lower in post-weanling mice than in dams, and was particularly
high in newborns. Therefore, the intensity of OGG1 expression
can be interpreted as an adaptive mechanism triggered both by
the perinatal oxidative burst and by aging. Also in humans,
there is an age-related increase of OGG1 expression, as shown
by the finding that the mean urinary 8-oxo-dG excretion in
normal children increased with postnatal age .
The greater susceptibility of mice to the oxidative stress
induced early in life by ECS is documented by the significantly
higher concentrations of TBARS as well as by higher 8-oxo-dG
levels in lung, as compared with adult mice exposed under
identical conditions. It is noteworthy that, while the lung of
adult mice responded to ECS exposure by upregulating OGG1,
no variation in the expression of this gene occurred in mice
exposed to ECS during the first 5 weeks of life. Indeed, the
ECS-induced increases of 8-oxo-dG levels recorded in post-
weanling mice, especially in females, are the highest ones that
we have ever observed in studies in rodents, using the same
methodology [5,10,16,24,26,27]. This conclusion is consistent
with the finding that female mice were more susceptible than
male mice to the induction of lung tumors when exposure to
MCS started at birth . In addition, post-weanling mice
suffered from early ECS-related histopathological alterations,
such as bronchial and alveolar hyperplasias, which were not
detectable in dams.
Young mice were also more susceptible than adult mice to
alterations induced by light, either alone or in combination with
ECS. In fact, they had higher concentrations of TBARS, a
greater increase of 8-oxo-dG levels in lung as well as a higher
frequency of bronchial/bronchiolar epithelial cells undergoing
apoptosis as a consequence of the combined exposure to ECS
and light. We cannot speculate whether these age-related
differences are due to a greater susceptibility of young mice, as
observed in ECS-exposed mice, or simply to the fact that their
skin is less protected by hair. In fact, the mice are hairless at
birth and start forming a soft down in about 1 week, which
becomes a well-developed hairy screen after about 2–3 weeks.
In conclusion, these data provide sound evidence that MCS
behaves as a potent carcinogen when exposure of mice starts at
birth. Moreover, evidence is provided that not only ECS but
also UV-containing light produces alterations of early
biomarkers in different organs of mice. Young mice, exposed
to ECS- and light-induced genotoxic damage, as assessed by
the types of molecular alterations we measured. The general
conclusion that the neonatal period and the early stages of life
are critical in affecting susceptibility to carcinogens is
supported by a series of mechanistic considerations. First of
all, the sudden transition from the maternal-mediated respira-
tion of the fetus to the autonomous pulmonary respiration at
birth, even in the absence of any exogenous insult, results in a
strong induction of oxidative DNA damage and in a sharp
increase of bulky DNA adduct levels in lung . In parallel,
birth is accompanied by dysregulation of a number of genes in
mouse lung, which is mainly aimed at attenuating oxidative
stress and genotoxic damage [10,11]. Moreover, oxidative
DNA damage in lung is significantly higher in mice exposed
either to ECS and/or light during the first 5 weeks of life than in
their dams exposed under identical conditions (Section 4.3). It
is also known that neonatal organs, among which the lung,
display an increased proliferation rate. In particular, alveolar-
ization occurs during the first 12 days of postnatal life ,
while proliferation of type-II cells increases after birth and
declines after twoweeks . In our studies, the baseline levels
of PCNAin bronchial epithelial cells were approximately twice
in post-weanling mice as compared with their dams, but this
difference was not statistically significant (Section 4.6). It has
also been reported that, during the first week of life, there are
alterations of xenobiotic metabolism in mice, such as lower
levels of glutathione S-transferases . The conclusion that
certain DNA repair mechanisms are less efficient at birth
[39,40] is supported by our finding that OGG1 expression in
lung is lower in post-weanling mice than in their dams and, in
S. De Flora et al./Mutation Research 659 (2008) 137–146 144
4.4). Finally, we raise the tentative hypothesis that stem cells,
that we have shown to have an increased susceptibility to
genotoxic carcinogens , may be the preferential target for
carcinogens during the earliest stages of life. For instance, we
showed that the expression of Sca-1 (stem cell antigen 1) in
lung is significantly higher in newborn mice than in adult mice
and is inducible by ECS only when exposure starts at birth (our
unpublished data). Thus, on thewhole, the greater vulnerability
of mice to carcinogens when exposure starts at birth and
continues during early stages of life is accounted for by a
variety of composite mechanisms, some of which were
explored in our studies and reviewed in the present article.
This study was supported by the U.S. National Cancer
Institute contract N01-CN53301 and by the Bulgarian Ministry
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