164 Current Cancer Drug Targets, 2012, 12, 164-169
Transplacental Antioxidants Inhibit Lung Tumors in Mice Exposed to
Cigarette Smoke After Birth: A Novel Preventative Strategy?
R. Balansky1,2, G. Ganchev1, M. Iltcheva1, V.E. Steele3 and S. De Flora*,2
1National Center of Oncology, Sofia 1756, Bulgaria; 2Department of Health Sciences, University of Genoa, I-16132
Genoa, Italy; 3National Cancer Institute, Rockville, MD 20892, USA
Abstract: Birth is characterized by an intense oxidative stress resulting in nucleotide alterations and gene overexpression
in mouse lung. We showed that cigarette smoke (CS) is carcinogenic when exposure starts soon after birth and applied
this bioassay to evaluate the efficacy of chemopreventive agents. The present study evaluated whether administration of
the antioxidants N-acetyl-L-cysteine (NAC) and vitamin C or ascorbic acid (AsA) during pregnancy can protect strain H
Swiss mice exposed to CS after birth. Exposure to CS, for 4 months, of newborns from untreated mice resulted in
significant alterations at 8 months of life, including alveolar epithelial hyperplasia, emphysema, blood vessel proliferation,
microadenomas, adenomas, and malignant tumors in lung, liver parenchymal degeneration and urinary bladder epithelium
hyperplasia. Treatment throughout pregnancy with either NAC, a scavenger of reactive oxygen species, or AsA, an
electron donor, did not affect fertility, parity, and body weight of newborns. Prenatal antioxidants significantly inhibited
most lesions in adult mice exposed to CS since birth. For instance, the incidence of emphysema was reduced from 27.5%
in CS-exposed mice that were untreated during pregnancy to 7.1% and 14.0% in those treated prenatally with NAC and
AsA, respectively. Lung adenomas were reduced from 34.8% to 16.7% and 9.3%, respectively. Malignant lung tumors
were reduced from 13.0% to 4.7% by prenatal AsA. Liver parenchymal degeneration was reduced from 58.0% to 14.3%
by prenatal NAC. These data mechanistically support a “transplacental chemoprevention” strategy, aimed at protecting the
newborn from oxidative stress and the adult from CS-related diseases appearing later in life.
Keywords: Antioxidants, ascorbic acid, cigarette smoke, emphysema, Lung cancer, N-acetyl-L-cysteine, pregnancy.
Oxidants behave as double edge swords in the organism.
In fact, they play a physiological role by regulating the
expression of genes involved in cell signaling, growth, and
differentiation [1, 2] but, at the same time, they can damage
nucleic acids, proteins and lipids. Oxidatively generated
damage is involved in the pathogenesis of a variety of
diseases . The prenatal and perinatal periods are
particularly at risk for the induction of oxidative damage in
the fetus and in the newborn. In fact, most reactive oxygen
species (ROS) are membrane-permeable and can cross the
placenta to reach the fetus , a mechanism that is
antagonized by antioxidants. Protection of the fetus against
oxidants is provided both by maturation of antioxidant
enzymes during late gestation  and by the transplacental
passage of non-enzymic antioxidants such as tocopherol,
ascorbate, ?-carotene, and ubiquinone [6, 7]. However,
antioxidants do not cross the human placenta until the last
trimester of gestation, as also shown by the limited
antioxidant capacity of premature infants and by a lower
total plasma antioxidant status of premature babies as
compared with term babies .
A further, critical event is represented by the perinatal
period and in particular by birth, when there is a sudden
transition from the maternal-mediated respiration of the fetus
to the autonomous pulmonary respiration of the newborn.
This event is responsible for a hyperoxic challenge 
involving a shift from an intrauterine hypoxic environment,
*Address correspondence to this author at the Department of Health
Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy; Tel:
+39-010-3538500; Fax: +39-010-3538504; E-mail: firstname.lastname@example.org
with a pO2 of 20-25 mm Hg, to an extrauterine hyperoxic
environment of 100 mm Hg . As a consequence, in the
absence of any treatment of pregnant mice, nucleotide
alterations “physiologically” occur in the lung at birth, as
documented by a doubling of 8-hydroxy-2’-deoxyguanosine
and a 5-fold increase of bulky DNA adducts in the lung of
newborns as compared with the lung of fetuses collected few
hours before . Luckily, the birth-related DNA damage in
the lung tends to be attenuated by upregulation of genes
involved in adaptive functions, such as glutathione
metabolism, cellular stress, and damage to DNA and proteins
[11, 12]. The neonatal lung is able to increase the levels of
protective antioxidant enzymes during exposure to oxygen
, and the fact that almost all neonates exhibit a
“physiological” jaundice may be related to triggering of the
synthesis of the antioxidant bilirubin after birth .
According to the “Barker hypothesis”, early life exposure to
toxic agents is an important risk factor for the development
of late-life diseases , and perinatal exposure to
carcinogens is suspected to contribute to both childhood
cancers and cancers appearing later in life .
ROS generation is one of the main mechanisms of action
of cigarette smoke (CS) , which may further increase the
burden of oxidative stress in the lung at birth. These
premises prompted us to evaluate the ability of CS to induce
tumors in neonatal mice. We demonstrated that neonatal
mice are more susceptible than adult mice to molecular,
biochemical and cytogenetical alterations induced by CS
 and that CS becomes a potent carcinogen in mice when
exposure starts soon after birth and continues early in life
. We used the CS bioassay in neonatal mice in order to
assess the efficacy of chemopreventive agents, administered
under experimental conditions mimicking interventions
1873-5576/12 $58.00+.00 © 2012 Bentham Science Publishers
Transplacental Antioxidants Inhibit Lung Tumors Current Cancer Drug Targets, 2012, Vol. 12, No. 2 165
either in current smokers or in ex-smokers . In addition,
prompted by the previous finding that administration of N-
acetyl-L-cysteine (NAC) during pregnancy prevents birth-
related nucleotide alterations , we evaluated the ability of
this thiol to prevent CS-related lung tumors when
administered prenatally. Indeed, administration of NAC
during pregnancy was successful to inhibit lung tumors,
pulmonary emphysema, and hyperplasia of the urinary
bladder epithelium induced by CS soon after birth .
NAC, which has been in the clinical practice for 50 years as
a drug and also as a dietary supplement, works through
multiple mechanisms, the most important of which is its
ability to scavenge ROS either in the extracellular
environment or as precursor of intracellular L-cysteine and
reduced glutathione (GSH) .
The goal of the present study was to confirm, in a
separate experiment, the unusual and provocative finding
that prenatal NAC is able to inhibit lung tumors and other
histopathological alterations in mice exposed to CS soon
after birth and early in life. Moreover, in order to evaluate
whether this peculiar strategy
chemoprevention may be shared by other antioxidants, we
also tested ascorbic acid (AsA) or vitamin C, a major
antioxidant present in cells and plasma . Like NAC ,
AsA possesses pleiotropic properties, the most important of
which is its reducing ability as an electron donor .
The results of the present study provide evidence that
both antioxidants, given to pregnant mice, are able to
significantly attenuate a variety of histopathological lesions,
also including lung tumors induced by exposure to CS in
newborns and early in life.
MATERIALS AND METHODS
A total of 18 pregnant Swiss H mice, aged 9-11 weeks
and weighing 26-29 g, were used. Ten mice were left
untreated during pregnancy, while 8 mice were treated with
either NAC or AsA throughout pregnancy. After delivery, 34
newborns from untreated mice were kept for 8 months in
filtered air (sham-exposed mice). All remaining newborns,
including 69 newborns from untreated dams, 42 newborns
from NAC-treated dams, and 43 newborns from AsA-treated
dams, were exposed to CS starting within 12 h after birth.
The mice were kept in Makrolon cages on sawdust
bedding and maintained on rodent chow (Kostinbrod, Sofia,
Bulgaria) and tap water ad libitum. The animal room had a
temperature of 23 ± 2°C, a relative humidity of 55%, and a
12 h day-night cicle. Housing, breeding and treatment of
mice at the Animal Laboratory of the National Center of
Oncology (Sofia, Bulgaria) were in accordance with national
and institutional guidelines.
Treatment with Antioxidants During Pregnancy
NAC was used in the form of a pharmacological
preparation (Fluimucil, Zambon, Vicenza, Italy) and AsA
was obtained from Sigma Chemical Co. (St. Louis, MO,
USA). Both agents were added to the drinking water at a
concentration accounting for a calculated daily intake of 1
g/kg body weight. The solutions of the two antioxidants were
used as the only source of drinking water throughout
Exposure of Neonatal Mice to CS
A total of 154 neonatal mice, either from untreated dams
or dams treated with antioxidants, were exposed to CS,
starting at birth and continuing daily for 4 months.
Mainstream CS was generated by burning filter-tipped
commercial cigarettes (Melnik King Size, Bulgartabac)
having a declared content of 9 mg tar and 0.8 mg nicotine
and delivering 10 mg CO each in the mainstream CS. A
whole-body exposure of mice to CS was achieved by
drawing 15 consecutive puffs, each of 60 ml and lasting 6 s,
which were delivered into 22.5-l exposure chambers by
means of a syringe. Each daily session of treatment with CS
involved 6 consecutive exposures, lasting 10 min each, with
1 min intervals during which a total air change was made.
The average concentration of total particulate matter in the
exposure chambers was 647 mg/m3.
Evaluation of Histopathological Alterations
The mice exposed to CS for 4 months were kept in
filtered air for an additional 4 months. The mice that became
sick or moribund before the end of the experiment were
separated from the other mice of the same cage in order to
avoid the risk of communication of possible infections and to
avoid distress and cannibalism in case of death. Eight
months after birth, all surviving sham-exposed mice and CS-
exposed mice were deeply anesthesized with diethyl ether,
killed by cervical dislocation and subjected to complete
necropsy. Lungs, liver, stomach, kidney, urinary bladder,
and all organs with suspected macroscopical lesions of both
prematurely dead mice and mice killed after 8 months were
fixed in 10% formalin, cut into standardized sections, stained
with hematoxylin and eosin,
histopathological analysis. A total of 10 lung sections were
obtained by cutting the left lung into 3 pieces, the accessory,
cranial and middle lobes of the right lung into 2 pieces each,
and by collecting the right lung caudal lobe. Three sections
were analyzed per each kidney, and 4 sections per each liver.
and subjected to
Individual comparisons between groups regarding
survival of mice and incidence data were made by ?2
analysis. Body weights and tumor multiplicity data were
expressed as means ± SE within the mice composing each
experimental group. Individual comparisons either between
CS-exposed mice and sham-exposed mice of the same
gender or between CS-exposed mice born from dams treated
with antioxidants and those born from untreated dams of the
same gender were made by Student’s t test for unpaired data.
Parity, Body Weights, and Survival of Mice
Neither parity nor body weight at birth were affected by
treatment with antioxidants during pregnancy. In fact, the
166 Current Cancer Drug Targets, 2012, Vol. 12, No. 2 Balansky et al.
number of newborns/dam (mean ± SE) was 10.3 ± 0.60 in
newborns from untreated dams, 10.5 ± 1.00 in newborns
from NAC-treated dams, and 10.8 ± 1.90 in newborns from
AsA-treated dams. The body weights of newborns were 1.40
± 0.01 g, 1.36 ± 0.02 g, and 1.45 ± 0.03, respectively.
As shown in Table 1, irrespective of treatment with
antioxidants in pregnancy, the body weights were
significantly reduced after 2 and 4 months of exposure to
CS, as compared with sham-exposed mice. After 8 months,
i.e., 4 months after discontinuation of exposure to CS, there
was a recovery of body weight in all females. In males, the
recovery was significant only in mice whose dams had
treated with either NAC or AsA.
Survival after 8 months was 91.2% in sham-exposed
mice, 89.9% in CS-exposed mice born from untreated mice,
88.1% in CS-exposed mice born from NAC-treated mice,
and 81.3% in CS-exposed mice born from AsA-treated mice.
None of these differences was statistically significant.
Histopathological Alterations in CS-Exposed Mice
Table 2 shows the incidence of histopathological
alterations in mice of both genders, as related to treatment
with either NAC or AsA during pregnancy and subsequent
exposure to CS, starting immediately after birth.
CS produced a variety of alterations in different organs.
In the lung, there were nonsignificant increases in the
incidence of bronchial epithelial
hemangiomas and significant increases in the incidence of
alveolar epithelial hyperplasias, emphysema, blood vessel
proliferation, microadenomas, adenomas, and malignant
tumors. The histopathological diagnosis of emphysema was
based on the rupture of alveolar walls, either focal or diffuse,
with formation of spaces including at least 5 alveoli.
Microadenomas were classified
detectable lesions that are larger than hyperplastic foci but
smaller than adenomas . The only significant intergender
difference in response to CS was a higher incidence of both
Table 1. Body weights of mice (g) as related to treatment with either NAC or AsA during pregnancy and subsequent exposure to
CS after birth.
(Months) Gender Sham CS NAC ? CS AsA ? CS
2 M 27.5 ± 0.75 23.4 ± 0.48b
23.1 ± 0.54b
21.8 ± 0.60b
F 25.8 ± 0.64 19.3 ± 0.62b
20.9 ± 0.50b
20.3 ± 1.35a
4 M 38.0 ± 0.73 31.2 ± 0.67b
30.7 ± 0.62b
29.7 ± 0.53b
F 32.5 ± 1.16 26.5 ± 0.90b
27.7 ± 0.70a
24.7 ± 0.79b
8 M 36.6 ± 1.18 30.4 ± 1.07b
33.9 ± 1.08c
33.6 ± 1.10c
F 31.5 ± 1.38 30.1 ± 1.33 28.4 ± 1.02 29.6 ± 1.35
The results are means ± SE within each experimental group. Statistical analysis: aP < 0.01 and bP < 0.001, as compared with Sham; cP < 0.05, as compared with CS.
Table 2. Incidence of histopathological alterations in mice, as related to treatment with either NAC or AsA during pregnancy and
to subsequent exposure to CS after birth.
Organ Sham CS NAC ? CS AsA ? CS
Histopathology M (23) F (11) M + F (34)M (41) F (28) M + F (69) M (26) F (16) M + F (42)M (22) F (21) M + F (43)
Bronchial ep. hyperplasia1 (4.3%) 0 1 (2.9%)7 (17.1%)2 (7.1%) 9 (13.0%) 7 (26.9%)a
3 (18.8%) 10(23.8%)a
1 (4.6%)2 (9.5%) 3 (7.0%)
Alveolar ep. hyperplasia 1 (4.3%) 0 1 (2.9%)19 (46.3%)c
4 (25.0%) 16(38.1%)c11(50.0%)4 (19.0%) 15(34.9%)c
Emphysema 1 (4.3%) 0 1 (2.9%)4 (9.8%) 15 (53.6%)b19 (27.5%)b
4 (18.2%)2 (9.5%)e
Blood vessel proliferation1 (4.3%) 0 1 (2.9%)13 (31.7%)b
4 (14.3%)16 (23.2%)b
3 (18.8%)5 (11.9%)1 (4.6%)d
1 (4.8%)2 (4.7%)d
Hemangiomas 1 (4.3%) 0 1 (2.9%)4 (9.8%) 0 4 (5.8%) 1 (3.8%)0 1 (2.4%)1 (4.6%)1 (4.8%)2 (4.7%)
Microadenomas 0 0 0 16 (39.0%)c13 (46.4%)b29 (42.0%)c14(53.8%)c9(56.3%)b
23(54.2%)c7 (31.8%)b4 (19.0%)d
Adenomas 0 0 0 12 (29.3%)b12 (42.9%)b24 (34.8%)c5 (19.2%)a2 (12.5%)d7(16.7%)a,d
2 (9.1%) 2 (9.5%)e
Malignant tumors 0 0 0 2 (4.9%) 7 (25.0%)9 (13.0%)a
2 (12.5%)6 (14.3%)a
1 (4.6%)1 (4.8%)2 (4.7%)
Papillary ep. hyperplasia 0 0 0 5 (12.2%) 6 (21.4%)11 (15.9%)a4 (15.4%)a
2 (12.5%)6 (14.3%)a
Parenchymal degeneration1 (4.3%) 0 1 (2.9%) 26 (63.4%)c14 (50.0%)b40 (58.0%)c3 (11.5%)f3 (18.8%)d
6 (14.3%)f8(36.4%)b,d6 (28.6%)a14 (32.6%)b,e
Statistical analysis: aP < 0.05, bP < 0.01, and cP < 0.001, as compared with Sham; dP < 0.05, eP < 0.01, and fP < 0.001, as compared with CS.
Transplacental Antioxidants Inhibit Lung Tumors Current Cancer Drug Targets, 2012, Vol. 12, No. 2 167
emphysema (P < 0.001) and malignant tumors (P < 0.05) in
females. The histopathological analysis of malignant tumors
showed that one male had an indifferentiated bronchiolo-
alveolar carcinoma, while another one had multiple
malignant tumors, including 2 foci of spinocellular
carcinoma in the bronchi, 15 foci of spinocellular carcinoma
in situ, and 10 foci of bronchioloalveolar carcinoma in
different lobes. Of the 7 CS-exposed female mice affected by
malignant lung tumors, 3 had bronchioloalveolar carcino-
mas, 3 had foci of both bronchioloalveolar and spinocellular
carcinomas, and 1 had a spinocellular carcinoma and 5 foci
of spinocellular carcinoma in situ. Fig. (1) shows at a glance
the remarkable CS-related increases in both incidence and
multiplicity of microadenomas, adenomas and malignant
tumors in mouse lung (P < 0.001 for all lesions, as compared
with sham-exposed mice) and shows examples of
microscopical appearance of microadenoma (A), adenoma
(B), and bronchioloalveolar carcinoma (C). Examples of
other CS-related histopathological alterations are available in
our previous papers [18, 24].
In addition, as shown in Table 2, CS induced
histopathological alterations also in extrarespiratory organs.
The increase in the incidence of papillary hyperplasias of the
urinary bladder epithelium was borderline to statistical
significance in both genders (P < 0.1) and became
statistically significant when the two genders were
combined. The severe hepatotoxicity of CS was documented
by a significant and extensive induction of liver parenchymal
degeneration in mice of both genders. In fact, more than half
of CS-exposed mice were affected by this lesion, which had
a very low incidence in sham-exposed mice.
Protection of CS-Induced Histopathological Alterations
by Prenatal AsA and NAC
The transplacental administration of either NAC or AsA
resulted in a number of protective effects towards the
histopathological alterations induced by CS after birth. As
shown in Table 2, NAC totally abolished the occurrence of
emphysema that, in the absence of chemopreventive agents,
affected more than 50% of female mice exposed to CS. A
significant decrease of CS-related emphysema was also
observed in female mice born from dams treated with AsA
during pregnancy. Both antioxidants attenuated the incidence
of CS-induced blood vessel proliferation in the lung. This
protective effect was statistically significant in male mice
pre-treated with either NAC or AsA. AsA significantly
attenuated induction by CS of microadenomas in females.
Both antioxidants significantly inhibited formation of
adenomas in CS-exposed mice, especially in females, in
terms both of incidence (Table 2 and Fig. (1)) and
multiplicity (Fig. (1)). In addition, AsA decreased the
incidence of malignant tumors, but this effect was borderline
to statistical significance (P = 0.06) in female mice only.
Both AsA and NAC decreased the multiplicity of malignant
tumors (Fig. (1)), but this effect was not statistically
significant. The incidences
hyperplasias and of malignant tumors were significantly
higher in CS-exposed males treated prenatally with NAC as
compared with sham-exposed mice, but there was no
significant difference as compared with CS-exposed mice
receiving no treatment during pregnancy.
of bronchial epithelial
Prenatal AsA suppressed the CS-induced papillary
hyperplasias of urinary bladder epithelium. In addition, both
antioxidants significantly and convincingly reduced CS
hepatotoxicity in both genders.
The results of the present study provide evidence that
administration of antioxidants
attenuates the yield of
histopathological alterations induced by CS in mice soon
after birth and early in life.
lung tumors and other
Fig. (1). Incidence and multiplicity of lung microadenomas (A), adenomas (B) and malignant tumors (C) in combined gender mice, as
related to treatment with either NAC or AsA during pregnancy and to exposure to CS, starting immediately after birth. aP < 0.05 and bP <
0.01, as compared with CS-exposed mice born from untreated dams.
01234567890 10203040 5060
Incidence (%) Multiplicity (mean ± SE)
168 Current Cancer Drug Targets, 2012, Vol. 12, No. 2 Balansky et al.
Exposure to CS of neonatal mice born from untreated
dams, for 4 consecutive months, resulted, 4 months later, in a
significant induction of a variety of lesions, including
alveolar epithelial hyperplasia, emphysema, blood vessel
proliferation, microadenomas, adenomas, and malignant
tumors in the lung, papillary hyperplasia of the urinary
bladder epithelium, and severe signs of toxicity in the liver.
These findings are in line with our previous studies using
this animal model for CS [18-20, 24]. Females were more
susceptible than males to the induction by CS of emphysema
and malignant tumors. We refer to a previous paper  for
a thorough discussion of these intergender differences. The
high susceptibility of neonatal mice to the carcinogenicity of
CS is amenable to the previously discussed oxidative stress
and nucleotide alterations at birth and is related to other
mechanisms as well. For instance, there are an increased
proliferation of type II alveolar cells during the first two
weeks of life , when alveolarization takes place , and
alterations of xenobiotic metabolism  and DNA repair
mechanisms [28, 29].
Both NAC and AsA, administered during pregnancy,
exerted significant protective effects towards most CS-
related alteration in the lung, urinary bladder, and liver. The
results obtained with NAC confirm the conclusions of a
previous study using similar experimental conditions .
The only exception is that, in the previous study , NAC
inhibited microadenomas and malignant lung tumors, while
in the present study no significant effect was observed
relatively to these end-points. AsA, which mainly works as
an electron donor , was at least as effective as NAC,
which mainly works as a ROS scavenger . These two
agents were used at high doses, which is a drawback of all
animal studies. Nevertheless, the doses used were nontoxic
in pregnancy, as shown by the fact that fertility, parity, and
body weight at birth were not affected by treatment of dams
with antioxidants. On the contrary, the beneficial effects
continued after birth, not only in the protection against CS-
related toxic, preneoplastic and neoplastic lesions but also by
allowing a better recovery of body weight loss after
discontinuation of exposure to CS.
Although the transplacental passage of NAC has been
questioned, at least in an ovine model , prenatal NAC
has been shown to protect the fetus and the offspring from
several adverse effects in both humans  and experimental
animals [11, 32-34]. There is sound clinical experience that
NAC is safe in pregnancy, as shown by the extensive use of
this thiol, both orally and intravenously, as an antidote in
cases of acetaminophen overdose during pregnancy . In
untreated mice, NAC prevented birth-related nucleotide
modifications , and administration of NAC during
pregnancy prevented oxidative stress associated with birth in
neonatal rats . In mice exposed transplacentally to
environmental CS, prenatal NAC protected the fetus liver
against formation of DNA adducts, oxidative DNA damage,
gene overexpression, and cytogenetical damage . In
Ku86-deficient mice, prenatal NAC inhibited developmental
cell death in the liver . The convenience of
supplementing AsA in pregnancy is rather controversial ,
but there are several lines of evidence that antioxidant
strategies are of primary importance to protect the newborn
from oxidative stress [36-40].
Our data provide strong mechanistic support to the
strategy of using antioxidants in pregnancy with the goal of
preventing lung tumors, emphysema and other alterations
appearing later in life in subjects exposed to CS or other
carcinogens early in life. Since there are still uncertainties
regarding the safety of antioxidant supplements in
pregnancy, an antioxidant protection could be achieved by
assuming diets that are known to be rich in antioxidants.
Furthermore, use of antioxidant supplements could be
advisable especially in developing countries where the
seasonal availability of fruit and vegetables could result in
adverse clinical outcomes of pregnancy  and in long-
term adverse effects in the progeny.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
This study was supported by the Bulgarian Ministry of
Education, Youth and Science (National Science Fund) and
by U.S. National Cancer Institute contract N01-CN-53301.
AsA = ascorbic acid
CS = cigarette smoke
GSH = reduced glutathione
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Received: April 15, 2011 Revised: July 12, 2011 Accepted: October 13, 2011