50
B. Binkovµ et al.
classified according to its macroscopic appearance of
atherosclerotic changes by the following way: (1) fatty
dots and streaks; (2) fibrous plaques; (3) atheromatous
plaques; (4) ulcerothrombotic changes. The fragments
were cleaned of calcifications, fatty deposits and throm-
botic materials and washed with physiological saline to
remove debris and blood residues. The tunica media,
which is composed mainly of SMCs, was isolated and
immediately stored at 808C until DNA isolation (within
two weeks).
Blood samples were collected during autopsy in hepar-
inized vacutainers and processed within 2 h to separate
plasma for lipids, vitamins and cotinine analyses. Plasma
samples were stored at 808C until analysis.
The standardized autopsy protocol was used to assess
causes of death. Marker for body fatness (according to
measure of subcutaneous fat) and weight of the heart were
also ascertained. Total atherosclerotic changes in the body
(thoracic and abdominal aortas, renal and cerebral
arteries) were classified as follows: 1 ± rare; 2 ± few; 3 ±
frequent.
fore, there may be common similarities between
carcinogenesis and atherosclerosis, the diseases
generally grouped as ªmultifactorial diseasesº (San-
karanarayanan et al., 1999).
The recent use of biomarkers in molecular epide-
miological studies is aimed at elucidating what
represents the latency period between exposure to
risk factors and the onset of chronic degenerative
diseases including atherosclerosis. A well-estab-
lished biomarker is the formation of DNA adducts
from covalent reactions between electrophilic mo-
lecules and DNA. DNA adducts, if not repaired
correctly, represent an early event between exposure
to genotoxic compounds and progression of multi-
stage diseases (De Flora et al., 1996).
Several studies in rodents provided evidence for
preferential binding of carcinogenic compounds not
only in the lungs but also in the heart and other
tissues (Randerath et al., 1988; Gairola and Gupta,
1991; Izzotti et al., 1992; Binkova et al., 1994 and
1996). In a pilot study in patients, diverse bulky-
aromatic DNA adducts were detected in abdominal
aorta taken at surgery (Izzotti et al., 1995). After-
wards, a molecular epidemiology study was carried
out by analyzing the DNA isolated from the SMCs of
abdominal aorta taken at surgery from 85 patients
suffering from severe atherosclerotic lesions (De
Flora et al., 1997). DNA adducts were detected in all
these samples using 32P-postlabeling technique. Sig-
nificant correlations were found between DNA
adduct levels and atherogenic risk factors, such as
age, number of currently smoked cigarettes, blood
cholesterol and blood pressure. A limitation of these
two studies is that samples from non-atherosclerotic
subjects were not used for comparison.
DNA adduct analysis
DNA from tunica media tissues was isolated according to
Gupta's procedure (Gupta, 1985) using RNases A and T1
and proteinase K treatment followed by phenol/chloro-
form/isoamylalcohol extraction and ethanol precipita-
tion, except that the incubation of homogenates with
proteinase K was for 5 h at 558C as described by De Flora
et al. (1997). DNA concentration was estimated spectro-
photometrically by measuring the UV absorbance at 260
nm. DNA samples were kept at 808C until DNA
adducts analysis. 32P-postlabelling analysis was performed
as previously described (Gupta, 1993; Binkova et al.,
1998; Phillips and Castegnaro, 1999). Briefly, DNA
samples (6 mg) were digested by a mixture of micrococcal
nuclease and spleen phosphodiesterase for 4 h at 378C.
The nuclease P1 procedure was used for adduct enrich-
ment. Adducted nucleotides were enzymatically labelled
using [g-32P]ATP (specific activity 3000 Ci/mmol) and T4
polynucleotide kinase and separated by multidirectional
polyethylenimine-cellulose thin layer chromatography
(TLC). After screen-enhanced autoradiography at
808C the DNA adduct maps (distinct spots and diagonal
radioactive zone) were evaluated by measuring 32P-radio-
activity in a liquid scintillation counter. To determine the
exact amount of DNA in each sample, aliquots of the
DNA enzymatic digest were analyzed for nucleotide
content by reverse-phase HPLC with UV detection, which
simultaneously allowed for controlling the purity of the
DNA. DNA adduct levels were expressed as adducts per
108 nucleotides. A B[a]P-derived DNA adduct standard
(4 Æ 0.6 adducts/108 nucleotides) was run in triplicate in
each postlabelling experiment to control for inter-assay
variability and to normalize the calculated DNA adduct
levels. The data presented here are average values of total
DNA adduct levels for each DNA sample analyzed in at
least three independent postlabelling experiments with a
variability of less than Æ 15%.
The objectives of our study are as follows: 1)
verifying new methods of molecular epidemiology
for better understanding of the processes that may be
involved in the development of atherosclerosis; 2)
evaluating bulky-aromatic DNA adducts in human
aortas taken at autopsy from sudden and accidental
death subjects; 3) assessing the relationship between
DNA adducts and atherosclerotic changes with
pathology, age, smoking and blood cholesterol.
Materials and methods
Subjects and collection of samples
Bodies of male subjects, aged between 30 ± 60 years, who
died suddenly or in an accident, and had been examined at
the Institute of Forensic Medicine within 24 h after death,
were included in this study. The fragments of thoracic
aorta, 5 cm above diaphragm were removed (sample size
approximately 30 cm2). Each fragment was examined and
Oppenlander
