Mutation Research 644 (2008) 38–42
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Mutation Research/Fundamental and Molecular
Mechanisms of Mutagenesis
journal homepage: www.elsevier.com/locate/molmut
Community address: www.elsevier.com/locate/mutres
Exposure of mice to cigarette smoke and/or light causes
DNA alterations in heart and aorta
Alberto Izzottia, Francesco D’Agostinia, Roumen Balanskya,b, Paolo Deganc,
Tanya M. Pennisia, Vernon E. Steeled, Silvio De Floraa,∗
aDepartment of Health Sciences, University of Genoa, Genoa, Italy
bNational Center of Oncology, Sofia 1756, Bulgaria
cCancer Research Institute (IST), Genoa, Italy
dNational Cancer Institute, Rockville, MD, USA
a r t i c l ei n f o
Received 12 March 2008
Received in revised form 12 June 2008
Accepted 24 June 2008
Available online 3 July 2008
Environmental cigarette smoke
Oxidatively damaged DNA
a b s t r a c t
Cigarette smoke (CS) is a major risk factor for cardiovascular diseases, cancer, and other chronic degen-
erative diseases. UV-containing light is the most ubiquitous DNA-damaging agent existing in nature, but
its possible role in cardiovascular diseases had never been suspected before, although it is known that
mortality for cardiovascular diseases is increased during periods with high temperature and solar irradi-
ation. We evaluated whether exposure of Swiss CD-1 mice to environmental CS (ECS) and UV-C-covered
halogen quartz lamps, either individually or in combination, can cause DNA damage in heart and aorta
cells. Nucleotide alterations were evaluated by32P postlabeling methods and by HPLC-electrochemical
2?-deoxyguanosine (8-oxodGuo) and of bulky DNA adducts in both heart and aorta. Surprisingly, even
exposure to a light that simulated solar irradiation induced oxidatively generated damage in both tissues.
The genotoxic effects of UV light in internal organs is tentatively amenable to formation of unidenti-
fied long-lived mutagenic products in the skin of irradiated mice. Nucleotide alterations were even more
pronounced when the mice were exposed to smoke and/or light during the first 5weeks of life rather
than during adulthood for an equivalent period of time. Although the pathogenetic meaning is uncertain,
DNA damage in heart and aorta may tentatively be related to cardiomyopathies and to the atherogenesis
© 2008 Elsevier B.V. All rights reserved.
Cigarette smoke (CS) is one of the major risk factors for
atherosclerosis, which accounts for nearly 50% of deaths in west-
ern societies . Environmental CS (ECS), which is a form of indoor
air pollution resulting from the mixture of sidestream CS (SCS) and
that portion of mainstream CS (MCS) which is released into ambi-
ent air by actively smoking individuals, contains many free radicals
and redox-active compounds . The involuntary exposure to ECS
in never smokers is associated with a significant excess of coronary
of antioxidant enzymes in these tissues , while in the long-term
ECS created a state of permanent inflammation and an imbalance
in the profile of lipid peroxidation products .
∗Corresponding author. Tel.: +39 010 3538500; fax: +39 010 3538504.
E-mail address: firstname.lastname@example.org (S. De Flora).
In spite of its essential role in life, solar irradiation is the most
ubiquitous DNA-damaging agent existing in nature. Especially in
degenerative diseases of the skin, also including the 10% of all
human cancers . We previously showed that certain widespread
artificial light sources, and above all traditional halogen quartz
bulbs, emit UV-A, UV-B, and even UV-C wavelengths . Their light
mice to either ECS or UV-C-covered halogen quartz bulbs, simulat-
ing solar irradiation, produced extensive biochemical, molecular,
and cytogenetic alterations not only in the expected target organs
but even at a systemic level [11,12]. However, a possible involve-
ment of exposure to UV-containing light in cardiovascular diseases
has never been investigated.
The goal of the present study was to evaluate whether exposure
of Swiss CD-1 albino mice to ECS and UV-C-covered halogen light,
either individually or in combination, can cause DNA damage in
0027-5107/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
A. Izzotti et al. / Mutation Research 644 (2008) 38–42
deoxyguanosine (8-oxodGuo) were measured. Bulky DNA adducts
are premutagenic lesions that have been involved in the initiation
of cancer and other chronic degenerative diseases , while 8-
the nucleoside 2?-deoxyguanosine by reactive oxygen species .
A further goal of the present study was to investigate the influence
paring adult mice with mice exposed to ECS and/or light starting
at birth until weaning. In fact, the analysis of respiratory tissues
from the same animals has shown that both ECS and light induce a
greater oxidative DNA damage early in life than in adults .
2. Materials and methods
2.1. Treatment of mice
Twenty-five pregnant Swiss CD-1 albino mice (Harlan, Italy, San Pietro al Nati-
mice and their dams were divided into four groups, including (a) sham-exposed
mice, kept in filtered air; (b) ECS-exposed mice; (c) light-exposed mice; and (d)
mice exposed to both ECS and light. All treatments were in accordance with NIH
and our national and institutional guidelines.
2.2. Exposure to ECS and/or light
(model TE-10, Teague Enterprises, Davis, CA), adjusted to produce a combination
of SCS (89%) and MCS (11%), which, on an average, yielded a total of suspended
particulate of 63.3mg/m3. Exposure was daily, 6h/day, divided into two rounds
with a 3-h interval. An illumination system simulating sunlight was obtained by
using halogen quartz bulbs (Leuci, File, Lecco, Italy), incorporated into dichroic spot
Division, Mainz, Germany) . The mice were exposed daily, 6h/day, at a distance
of approximately 50cm, yielding an illuminance level of 10,000lx. A daily exposure
of mice to both ECS and light was achieved according to the following schedule: 3h
ECS+3h light+3h ECS+3h light.
2.3. Collection of heart and aorta, and extraction of nucleic acids
Approximately 5weeks after birth, 10 post-weanling mice (5 males and 5
females)/group and all available dams (5–7/group) were deeply anesthetized with
diethyl ether and killed by cervical dislocation. All remaining mice were kept alive
for a carcinogenesis study, which is now in progress. In addition to biological fluids
and other organs, which have been used for a separate study evaluating alterations
in the respiratory tract , the heart and thoracic aorta were removed, washed in
phosphate buffered saline, pH 7.4, and stored at −80◦C.
Nucleic acids were extracted from 50 to 100mg tissue, in the presence of an
antioxidant (dithiothreitol), by means of commercially available kits using phenol-
free reagents (Genelute DNA Miniprep kit, Sigma, St. Louis, MO, and Purelink RNA
Purification kit, Invitrogen, Carlsbad, CA).
2.4. Evaluation of bulky DNA adducts
Bulky lipophilic DNA adducts were detected after butanol extraction and32P
ing the ratio between cpm detected in DNA adducts and cpm in normal nucleotides.
All samples were analyzed in two separate experiments. The results are expressed
as medians and 95% confidence intervals (CI) of the data obtained within the mice
composing each experimental group.
2.5. Evaluation of oxidatively damaged DNA
8-OxodGuo was evaluated both by HPLC-electrochemical detection (ECD) and
treated with nuclease P1 (Boehringer Mannheim, Germany), pH 4.0, and alkaline
then filtered through 0.22?m cellulose acetate filter units (Millipore Corp., Bedford,
MA), and 5–10?l aliquots were dispensed in autosampling vials. Separation of 8-
oxodGuo and normal 2?-deoxyribonucleosides was performed in 75mm×4.6mm
LC-18-DB columns equipped with a LC-18-DB guard column cartridge (Supelco Co.,
sium phosphate, pH 5.5 (90%), and methanol (10%). Electrochemical analysis was
carried out by using a Coulochem II detector (Esa Inc., Chelmsford, MA). Identity
of the 8-oxodGuo peak was confirmed by co-elution with a standard reference
(Sigma–Aldrich). 8-OxodGuo was quantified by integrating the area of the peak
eluted from the electrochemical detector. The levels of 8-oxodGuo were related to
the amount of 2?-deoxyguanosine (dGuo), detected by UV absorbance at 254nm in
the same samples. The amount of DNA was determined by a calibration curve vs.
known amounts of calf thymus DNA. The results are expressed as medians (95% CI)
of three replicate analyses on pooled heart or aorta sample from each experimental
The32P postlabeling reaction for 8-oxodGuo  was performed, following tri-
fluoroacetic acid enrichment, by polynucleotide kinase and [?-32P]ATP (16?Ci/?l,
specific activity 325Ci/mmol) (ICN, Irvine, CA).32P-labeled 8-oxodGuo molecules
were purified by monodirectional thin layer chromatography (TLC) in formic acid
and detected by32P imaging (InstantImager, Packard, Meriden, CT). 8-OxodGuo lev-
els were quantified by calculating the ratio between the radioactive level (cpm)
detected in the 8-oxodGuo-related spot and cpm detected in normal nucleotides.
A DNA-free sample was used as a negative control. The following reference stan-
dards were used for 8-oxodGuo identification: (a) untreated calf thymus DNA; (b)
calf thymus DNA incubated at 37◦C for 30min with 50-mM hydrogen peroxide; (c)
untreated Salmonella typhimurium DNA; and (d) a standard of authentic 8-oxodGuo
(National Cancer Institute Chemical Carcinogen Reference Standard Repository,
Mid-West Research Institute, Kansas City, MO), used in order to optimize, under
fluorescence detection, the trifluoroacetic acid enrichment procedure and TLC con-
ditions. All samples were analyzed in two separate experiments. The results are
expressed as medians (95% CI) of five replicate analyses on pooled heart or aorta
samples from each experimental group.
2.6. Analysis of OGG1 and GTPase expression
The transcriptional activities of 8-oxoguanine DNA glycosylase 1 (OGG1) and 8-
oxo-7,8-dihydroguanosine triphosphatase (GTPase) genes were evaluated by mRNA
reverse transcription reaction followed by qPCR . Each sample was tested in
2.7. Statistical analysis
The statistical significance of the differences recorded between the analyzed
experimental groups was evaluated by nonparametric Mann–Whitney U-test. The
correlation between the data obtained with different methodologies was evalu-
ated by simple regression analysis using the StatView software (Abacus Concept,
3.1. Bulky DNA adducts
samples from both sham-exposed and light-exposed mice. In con-
trast, an evident diagonal radioactive zone (DRZ) and an individual
spot in the upper end of the DRZ were detected in both heart and
aorta samples from ECS-exposed mice, irrespective of co-exposure
to light (chromatograms not shown). As reported in Table 1, the
baseline levels of bulky DNA adducts detected in sham-exposed
mice were significantly higher in both heart (1.5-fold) and aorta
(2.6-fold) of adult females than in the corresponding organs of
modify DNA adduct levels in either heart or aorta, irrespective of
the age of mice. In contrast, the levels of adducts to heart DNA were
significantly higher in mice exposed to ECS from birth to wean-
ing, both males and females, than in their dams exposed under
identical conditions. Similarly, following exposure to both ECS and
light, the increase in DNA adduct levels in heart was higher in post-
weanling mice, both males and females, than in their dams. Also in
aorta, the increase in DNA adduct levels stimulated by either ECS
or ECS+light was more pronounced in post-weanling mice, both
males and females, than in their dams.
3.2. Oxidatively damaged DNA
The results relative to the assessment of 8-oxodGuo in mouse
heart and aorta by HPLC-ECD and32P postlabeling are summarized
in Tables 2 and 3, respectively.
HPLC-ECD analyses showed that the baseline 8-oxodGuo lev-
els were not significantly affected either by age, gender, or organ.
Exposure of mice to light alone slightly increased oxidatively dam-
A. Izzotti et al. / Mutation Research 644 (2008) 38–42
Levels of bulky DNA adducts/108nucleotides, as detected by32P postlabeling, in the heart and aorta of mice exposed either to ECS and/or UV-containing light
OrganPost-weanling mice Dams
2.3 (1.1–3.5) [×1.2]
1.9 (1.1–2.8) [×1.3]
2.2 (1.2–3.2) [×1.0]
1.4 (0.9–1.9) [×1.4]
1.1 (0.9–1.4) [×1.3]
2.0 (1.0–3.0) [×0.8]
Treatments lasted either since birth until weanling or, in adult mice, for an equivalent period of time. The results are expressed as medians (95% CI) for 5–7
mice/treatment/gender. The values in square brackets indicate the fold increase over the corresponding sham. Statistical analysis:aP≤0.01, significantly higher as com-
pared with the corresponding post-weanling females;bP≤0.05, significantly lower as compared with the corresponding post-weanling females;cP<0.001, significantly
increased as compared with the corresponding sham.
Levels of 8-oxodGuo, as detected by HPLC-ECD (8-oxodGuo/106dGuo), in the heart and aorta of mice exposed either to ECS and/or UV-containing light
0.8 (0.3–1.2) [×1.0]
0.9 (0.5–1.3) [×2.5]
1.0 (0.7–1.2) [×1.5]
1.1 (0.7–1.6) [×2.2]
0.8 (0.4–1.3) [×1.6]
Treatments lasted either since birth until weanling or, in adult mice, for an equivalent period of time. The results are expressed as medians (95% CI) of three replicate analyses
on pooled samples from each experimental group. The values in square brackets indicate the fold increase over the corresponding sham. NA, not available. Statistical analysis:
aP≤0.05, significantly increased as compared with the corresponding sham.
aged DNA in both heart and aorta of post-weanling mice, but only
to ECS alone significantly increased 8-oxodGuo levels in the heart
of male and female post-weanling mice and of their dams. In the
aorta, the effect of ECS was detectable in post-weanling mice but
not in their dams. Similar alterations occurred in mice exposed to
both ECS and light. In addition, a significant increase of oxidatively
generated DNA damage was also observed in the aorta of dams.
Similar trends were observed when heart and aorta sam-
ples were analyzed by32P postlabeling (Table 3). However, the
effects of both ECS and light became even more evident by using
this technique. In particular, the baseline 8-oxodGuo levels were
higher in the heart than in the aorta of post-weanling males (1.6-
fold, P<0.01) and females (1.2-fold, not significant) and of adult
mice (1.9-fold, P<0.001). The baseline levels observed in sham-
(1.3-fold, not significant) of adult females than in post-weanling
stimulation of oxidatively damaged DNA in the heart and aorta of
post-weanling mice, both males and females. In adult mice, expo-
but had no effect in heart. Even more potent was the stimulation of
Levels of 8-oxodGuo, as detected by32P postlabeling (8-oxodGuo/105nucleotides), in the heart and aorta of mice exposed either to ECS and/or UV-containing light
3.5 (1.7–5.4) [×1.1]
higher as compared with the corresponding post-weanling females;bP<0.05;cP<0.001 significantly higher as compared with the corresponding sham.
A. Izzotti et al. / Mutation Research 644 (2008) 38–42
oxidatively generated DNA damage in mice exposed to either ECS
or ECS+light. The ECS-related increases of 8-oxodGuo levels were
more evident in aorta than in heart and much more pronounced in
post-weanling mice than in adult mice.
The average 8-oxodGuo data detected in the analyzed experi-
mental groups by32P postlabeling (x) were significantly correlated
(r=0.804; P<0.0001) with those detected by HPLC-ECD (y). The
equation of the regression line was y=0.324+0.148x.
3.3. OGG1 and GTPase transcriptional activities
The observed differences in 8-oxodGuo levels were not
these DNA repair activities were not significantly affected either
by organ (heart vs. aorta), gender (male vs. female post-weanling
mice), age (post-weanling vs. adult females), or treatment (ECS
OGG1 and GTPase were around 1 (data not shown).
The results obtained in the present study provide evidence that
exposure of mice to ECS resulted in a considerable stimulation of
8-oxodGuo and bulky DNA adduct formation in both aorta and
heart. In addition, the simple exposure to light containing UV-A
and UV-B significantly increased 8-oxodGuo levels in both tissues.
This conclusion was drawn by using two different methodologies.
HPLC-ECD requires relatively high amounts of DNA but is quantita-
tive and specific . In contrast,32P postlabeling uses much lower
of quantitative phosphorylation yield and artefactual 8-oxodGuo
formation from dGuo as a consequence of ?-radiation emitted by
32P. Although the results generated by these techniques cannot be
directly compared since they necessarily use different units, it is
noteworthy that the two sets of 8-oxodGuo data were highly cor-
related. Differences related to age, gender, organ, and exposure to
ECS and/or light that were statistically significant by both methods
were more pronounced at32P postlabeling. Moreover, some dif-
ferences that did not reach the statistical significance threshold at
HPLC-ECD became significant when the samples were analyzed by
of samples tested with the former technique, which requires larger
amounts of DNA.
els of bulky DNA adducts in rodent heart [16,19,20] and aorta ,
and to induce the formation of 8-oxodGuo in heart . Removal of
DNA adducts after discontinuation of exposure to ECS was slower
in heart than in other rat organs . ECS-related DNA adduct lev-
els in the heart were higher in p53 mutant mice than in wildtype
mice . Low levels of 8-oxodGuo were also observed in the heart
after short exposures of mice to SCS . Co-exposure to MCS and
ethanol further increased DNA adduct levels in the heart of rats
 and enhanced lipid peroxidation in the heart of mice .
Cigarette smoking was associated with an increase of DNA adduct
levels also in the human heart [24–26] and aorta [27–29] as well as
with oxidatively generated DNA damage in the aorta of atheroscle-
rotic patients , which was significantly related to an impaired
survival as compared with patients with lower 8-oxodGuo levels
On the other hand, the possibility that sunlight simulating irra-
diation may cause oxidatively generated DNA damage in mouse
heart and aorta is a totally new finding. Although the epidemio-
logical data are not unequivocal, there is some suspicion that solar
irradiation may be involved in the genesis of human hematolym-
phopoietic malignancies, such as non-Hodgkin lymphoma .
These effects have usually been ascribed to the immunosuppres-
sive activity of UV light , but our studies in hairless mice [11,12]
and Swiss mice, both in the respiratory tract  and in the cardio-
vascular system (this study), unequivocally demonstrate that the
light is able to induce a systemic genotoxic damage. The results
clearly indicate a potential oxidative stress-related effect, which
might either relate to oxidative stress-induced intracellular signal-
ing and altered gene expression or to oxidative damage to either
mitochondrial or genomic DNA. Formation of 8-oxodGuo not only
in the skin but also in internal organs of UV-exposed hairless mice,
including brain, liver and spleen, has also been reported by another
laboratory . Presumably, DNA damage in internal organs is due
can induce nucleotide alterations at a distance from the skin.
A further mechanism by which light-induced oxidative stress
may cause pathological consequences in the aorta is an increased
instability of existing atherosclerotic plaques. In fact, oxidative
damage of leukocyte membranes results into the release of inflam-
matory and trombophilic factors that contribute to worsening the
health. In fact, epidemiological studies have suggested that cardio-
high temperature and solar irradiation. These extramortality peaks
have been associated with a light-related formation of oxidizing
pollutants, such as ozone .
The differences in oxidatively generated DNA damage among
the various experimental groups were not due to variations in the
expression of either OGG1 or GTPase. In mammalian cells, OGG1
is the main DNA glycosylase removing 8-oxodGuo, while GTPase
hydrolyzes oxidized purine nucleoside triphosphates, thus avoid-
these data are in agreement with the findings of in vitro studies,
showing that OGG1−/−cells do not exhibit an increased frequency
of mutations compared to OGG1+/+cells after exposure to either
UV-A or UV-B radiation .
An additional new finding of the present study was that the
baseline levels of 8-oxodGuo and bulky DNA adducts in both heart
and aorta were affected by age, being significantly higher in adult
mice than in post-weanling mice. On the other hand, the response
to either ECS or light in both tissues was much more pronounced
when exposures occurred during the first 5weeks of life. Similar
findings were obtained in the respiratory tract of the same mice
. Experimental and epidemiological data as well as mechanis-
vulnerable to carcinogens than adults [15,35–38]. However, we
cannot speculate whether the quantitative age-related differences
observed in light-exposed mice are due to an enhanced suscepti-
bility or just to the fact that mice are hairless at birth and need
2–3weeks to form a well developed hairy screen.
The presence of promutagenic lesions in the lung and other tar-
get organs is recognized to be predictive of a possible evolution
towards cancer . Several lines of evidence suggest that oxida-
tively generated DNA damage and formation of bulky DNA adducts
in arteries are related to the atherosclerotic process [13,21,26–30],
thereby supporting the mutational origin theory of atheroscle-
rotic plaques, which in any case is not in contrast with the
well-known response-to-injury theory . The situation is dif-
ferent in the heart, because the lack of proliferation of cardiac
myocytes is incompatible with a neoplastic evolution, at least
in adults. Nucleotide alterations in these cells have tentatively
been associated with nonproliferative degenerative diseases, such
as cardiomyopathies . In humans, smoking is related to both
cardiomyopathy and transmural myocardial infarction  and,
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A. Izzotti et al. / Mutation Research 644 (2008) 38–42
in experimental models, cigarette smoking causes considerable
metabolic and morphological alterations in the heart, which are
known as “smoke cardiomyopathy” [41,42].
The relevance of animal data to the human situation is always
uncertain, especially due to the circumstance that experimental
animals need to be exposed to high doses for relatively short
periods of time. The present study does not make an exception.
Nevertheless, the results reported herein show that not only ECS
but even a halogen light irradiation containing UV-A and UV-B,
which simulates sunlight, can potentially cause molecular alter-
ations in the heart and aorta of mice, especially when the animals
are exposed early in life. These findings deserve attention in the
framework of cardiovascular disease prevention.
Conflicts of interest
This work was supported by the US National Cancer Institute
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