Genome damage in oropharyngeal cancer patients treated by radiotherapy.

Abstract

To estimate genome damage in oropharyngeal cancer patients before, during, and after radiotherapy and to measure the persistence of caused genome damage relevant in the evaluation of secondary cancer risk.
DNA damage was evaluated in peripheral blood lymphocytes of 10 oropharyngeal cancer patients using alkaline comet assay, analysis of structural chromosome aberrations, and micronucleus assay. Blood samples were taken 2 hours before irradiation on day 1 of the first radiotherapy cycle, 2 hours after the application of the first dose, in the middle of the radiotherapy cycle, within 2 hours after the last received radiotherapy dose, and after 6 and 12 months after radiotherapy.
In most participants, the highest level of primary DNA damage was recorded in blood samples collected after the administration of first radiation dose (mean tail length 25.04+/-6.23 mum). Most patients also had increased frequency of comets with long tail-nucleus (LTN comets) after the administration of the first radiation dose (mean, 10.50+/-7.71 per 100 comets), which remained increased in the middle of radiotherapy (mean, 18.30+/-27.62 per 100 comets). Later on, the levels of primary DNA damage as recorded by the comet assay, slightly diminished. The frequency of structural chromosome aberrations in lymphocytes gradually increased during the radiation cycle (26.50+/-27.72 per 100 metaphases at the end of the therapy), as well as the frequency of micronuclei (mean total number of micronuclei 167.20+/-35.69 per 1000 binuclear cells).
Oropharyngeal cancer patients had relatively high levels of primary DNA damage in their peripheral blood lymphocytes even before therapy. The frequency of complex structural chromosome aberrations and the frequency of micronuclei increased with the progression of the radiation cycle and the doses delivered. As the frequency of chromosomal aberrations a year after radiotherapy mostly did not return to pre-therapy values, it represents an important risk factor related to the onset of second cancer.

Full text

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Genome Damage in Oropharyngeal Cancer Patients Treated by
Radiotherapy
Aim To estimate genome damage in oropharyngeal cancer patients be-
fore, during, and after radiotherapy and to measure the persistence of
caused genome damage relevant in the evaluation of secondary cancer
risk.
Methods DNA damage was evaluated in peripheral blood lymphocytes
of 10 oropharyngeal cancer patients using alkaline comet assay, analysis
of structural chromosome aberrations, and micronucleus assay. Blood
samples were taken 2 hours before irradiation on day 1 of the first ra-
diotherapy cycle, 2 hours after the application of the first dose, in the
middle of the radiotherapy cycle, within 2 hours after the last received
radiotherapy dose, and after 6 and 12 months after radiotherapy.
Results In most participants, the highest level of primary DNA damage
was recorded in blood samples collected after the administration of first
radiation dose (mean tail length 25.04 ± 6.23 μm). Most patients also had
increased frequency of comets with long tail-nucleus (LTN comets) after
the administration of the first radiation dose (mean, 10.50 ± 7.71 per 100
comets), which remained increased in the middle of radiotherapy (mean,
18.30 ± 27.62 per 100 comets). Later on, the levels of primary DNA dam-
age as recorded by the comet assay, slightly diminished. The frequency of
structural chromosome aberrations in lymphocytes gradually increased
during the radiation cycle (26.50 ± 27.72 per 100 metaphases at the end
of the therapy), as well as the frequency of micronuclei (mean total num-
ber of micronuclei 167.20 ± 35.69 per 1000 binuclear cells).
Conclusion Oropharyngeal cancer patients had relatively high levels of
primary DNA damage in their peripheral blood lymphocytes even before
therapy. The frequency of complex structural chromosome aberrations
and the frequency of micronuclei increased with the progression of the
radiation cycle and the doses delivered. As the frequency of chromosomal
aberrations a year after radiotherapy mostly did not return to pre-therapy
values, it represents an important risk factor related to the onset of sec-
ond cancer.
1Department of Oncology,
Zagreb University Hospital
Center, Zagreb, Croatia
2Institute for Medical Research
and Occupational Health,
Mutagenesis Unit, Zagreb,
Croatia
3The University Hospital for
Tumors, Zagreb, Croatia
Marija Gamulin1, Nevenka Kopjar2, Mislav Grgić1, Snježana Ramić3, Vesna Bišof1,
Vera Garaj-Vrhovac2
515
www.cmj.hr
Marija Gamulin
Department of Oncology
University Hospital Center Zagreb
Kišpatićeva 12
10000 Zagreb, Croatia
marija.gamulin@zg.t-com.hr
> Received: June 15, 2008
> Accepted: July 22, 2008
> Croat Med J. 2008;49:515-27
> Correspondence to:
> doi:10.3325/cmj.2008.4.515
Clinical Science
Clinical Science

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Croat Med J 2008;49:515-527
516
Squamous cell carcinoma of the oropharynx
is an uncommon disease, the prevalence of
which is about 5-10% of all newly diagnosed
cancer cases in Europe and the United States
(1). Pharyngeal carcinomas, like other tumors
of the head and neck, are sentinel diseases of
exposure to environmental factors. The de-
velopment of oropharyngeal cancer is strong-
ly associated with tobacco use and alcohol
consumption, as well as with the exposure to
several occupational carcinogens, vitamin de-
ficiencies, and poor oral hygiene (2). Only a
fraction of exposed individuals develop can-
cer in the head and neck region, which sug-
gests that individual sensitivity to mutagens
is an important endogenous risk factor that
significantly contributes to the development
of the disease (3-5). Although radiation is the
mainstay of current therapy for oral cancer,
the variability in intrinsic radiosensitivity sig-
nificantly contributes to the outcome of the
disease control. In general, >15% of nasopha-
ryngeal cancer patients develop acute or late
symptoms of enhanced radiosensitivity (6). It
has been well documented that patients with
head and neck malignancies are at consider-
able risk of developing a second primary tu-
mor either within or outside the head and
neck area (7).
Apart from the beneficial effect of radio-
therapy, adverse consequences on normal tis-
sue are almost always present. Following γ-
irradiation, different types of lesions can be
detected in the nuclear DNA. DNA single-
and double-strand breaks and a plethora of
modified nucleotides are induced by direct
ionization of DNA and by free radical-medi-
ated reactive oxidative species developed by
the radiolysis of water (8). Although unre-
paired DNA damage is useful in killing can-
cerous cells, it can be detrimental to normal
cells, leading to the onset of secondary can-
cer (9). Growing evidence suggests that de-
layed radiation-induced damage, ie, induced
genomic instability may also significant-
ly contribute to the onset of secondary neo-
plasms (10).
The most extensively used biomarkers for
the assessment of genotoxic and carcinogen-
ic risks in humans involve cytogenetic end-
points such as chromosomal aberrations, sis-
ter chromatid exchanges, and micronuclei in
mitogen-stimulated peripheral blood lym-
phocytes. In the last decade, the alkaline com-
et assay, as a relatively new biomarker, has
also gained an increased application in clini-
cal medicine (11,12). The majority of reports
were concerned with either biomonitoring of
increased levels of basal DNA damage in can-
cer patients or excess DNA damage caused by
treatment with radiation or antineoplastic
drugs (13-15).
Background genome damage in oropharyn-
geal cancer before radiotherapy has never been
correlated with the rate of caused genome
damage during and elimination of genome
damage after radiotherapy. As in most cases,
heavy drinkers with oropharyngeal cancer rep-
resent a unique population with genome bur-
den related to alcohol consumption and pos-
sible genome instability related to cancer. Due
to significant improvement of therapy efficien-
cy in this type of cancer, the secondary cancer
risk follow-up should be included in medical
surveillance of these patients.
In this study, the levels of primary and
residual DNA/chromosome damage in pa-
tients with oropharyngeal cancer were stud-
ied using the alkaline comet assay, analysis of
structural chromosome aberrations, and cy-
tokinesis-block micronucleus (CBMN) assay
in peripheral blood lymphocytes. This study
also investigated the susceptibility of can-
cer patients to radiation doses received in the
course of radiotherapy, as well as possible in-
ter-individual differences in the persistence of
lymphocyte genome damage one year after ir-
radiation.

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Gamulin et al: Radiotherapy and Genome Damage in Oropharyngeal Cancer
517
Participants and methods
Participants
This study was performed in accordance with
high standards of ethics and was approved by
the Ethical Committee of the Zagreb Uni-
versity Hospital for Tumors, Zagreb, Croatia
where the study participants were recruited.
They were cancer patients who had not previ-
ously been treated with cytotoxic drugs or ra-
diotherapy. Before entering the study, all par-
ticipants were informed about the aim and the
experimental details and gave their informed
consent for voluntarily participation. Alto-
gether 10 men (age range, 48-62 years) who
had undergone excision of the primary oro-
pharyngeal tumor and the unilateral neck dis-
section were selected. All of them were sub-
jected to similar diagnostic procedures before
operation (including chest x-ray) and to simi-
lar post-surgical treatments. All patients were
regular and heavy smokers (they had smoked
more than 20 cigarettes a day for over 10 years)
and reported long-term alcohol consumption
(average daily intake of 2-3 l of wine and oth-
er alcoholic drinks). They were free from other
pathology and were not on medication that is
known to cause DNA damage. None of them
reported cancer cases in their family history.
Histopathologically, all patients suffered from
squamous cell carcinoma of the oropharynx,
with positive nodes in the neck and negative
borders. Patients were also classified according
to the current Tumor Nodes Metastasis classi-
fication (16).
Adjuvant radiotherapy was scheduled to
start within 4 to 6 weeks following surgery,
when patients were in good clinical condi-
tion. Postoperative irradiation is recommend-
ed based on the tumor stage, tumor histology,
and surgical findings after tumor resection. All
patients were irradiated with photon beams
from a 60Co source in the area of the primary
tumor and the neck. Total tumor dose applied
after surgery was 62 Gy in 31 daily fractions.
Two opposite lateral fields and one direct field
(50Gy/25 fractions) to the neck were applied.
Higher doses of radiation are required for mi-
croscopic disease to decrease the chance of lo-
coregional failure because of interruption of
the normal vasculature, scarring, and relative
hypoxia in the tumor bed. Radiation doses re-
ceived in the course of therapy were as follows:
2 Gy (second sampling, after the application
of the first dose), 30 Gy (third sampling, in
the middle of the radiotherapy cycle), and 62
Gy (fourth sampling, at the end of the radio-
therapy cycle).
Methods
Blood sampling. Blood was taken by venipunc-
ture. Further laboratory manipulations with
blood samples and all investigations have been
carried out in accordance with high standards
of ethics.
Samples of venous blood (5 mL) were col-
lected in heparinized vacutainer tubes (Bec-
ton Dickinson, New Jersey, NJ, USA) under
sterile conditions. Altogether 6 blood sam-
ples were collected from each donor. Pre-
treatment blood sample (sample 1) was col-
lected on day 1 of the first radiotherapy cycle,
two hours before irradiation. Response of pe-
ripheral blood leukocytes to radiotherapy was
evaluated on the blood sample taken with-
in two hours after the application of the first
dose (sample 2), in the middle of the radio-
therapy cycle (sample 3), and within 2 hours
after the last received radiotherapy dose (sam-
ple 4). One blood sample was taken 6 months
(sample 5) and 12 months (sample 6) after ra-
diotherapy.
All blood samples were handled in the
same manner. After venipuncture they were
coded, cooled to +4°C in the dark, and trans-
ferred to our laboratory. They were analyzed
immediately after the arrival (one hour after
collection at the latest) using the alkaline com-

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Croat Med J 2008;49:515-527
518
et assay, the analysis of structural chromosome
aberrations, and the CBMN assay.
Alkaline comet assay. All chemicals were
purchased from Sigma Chemicals (St. Lou-
is, MO, USA). The comet assay was carried
out under alkaline conditions, as described
by Singh et al (17). Fully frosted slides were
covered with 1% normal melting point aga-
rose. After solidification, the gel was scraped
off from the slide. The slides were then coat-
ed with 0.6% normal melting point agarose.
When this layer had solidified, a second lay-
er, containing the whole blood sample (4 µL)
mixed with 0.5% low melting point agarose,
was placed on the slides. After 10 minutes of
solidification on ice, the slides were covered
with 0.5% low melting point agarose. After-
wards, the slides were immersed for 1 hour
in ice-cold freshly prepared lysis solution (2.5
M NaCl, 100 mM Na2EDTA, 10 mM Tris-
HCl, 1% Na-sarcosinate, pH 10) with 1% Tri-
ton X-100 and 10% dimethyl sulfoxide add-
ed fresh to lyse cells to allow DNA unfolding.
The slides were then randomly placed side by
side in the horizontal gel-electrophoresis tank,
facing the anode. The unit was filled with
freshly prepared electrophoretic buffer (300
mM NaOH, 1 mM Na2EDTA, pH 13.0)
and the slides were set in this alkaline buffer
for 20 minutes to allow DNA to unwind and
express alkali-labile sites. Denaturation and
electrophoresis were performed at 4°C under
dim light. Electrophoresis was carried out for
the next 20 minutes at 25 V (300 mA). After
electrophoresis, the slides were washed gently
three times at 5-minute intervals with a neu-
tralization buffer (0.4 M Tris-HCl, pH 7.5) to
remove excess alkali and detergents. Each slide
was stained with ethidium bromide (20 μg/
mL) and covered with a coverslip. Slides were
stored at 4°C in humidified sealed containers
until analysis.
To prevent additional DNA damage, han-
dling blood samples and all the steps included
in the preparation of the slides for the comet
assay were conducted under yellow light or in
the dark. Furthermore, to avoid possible posi-
tion effects during electrophoresis, two parallel
replicate slides per sample were prepared. Each
replicate was processed in a different electro-
phoretic run.
The slides were examined at 250 × magnifi-
cation using a fluorescence microscope (Zeiss,
Gottingen, Germany), equipped with an ex-
citation filter of 515-560 nm and a barrier fil-
ter of 590 nm. The microscope was connected
through a black and white camera to a com-
puter-based image analysis system (Comet As-
say II, Perceptive Instruments Ltd, Suffolk,
UK). A total of 100 comets per patient were
scored (50 from each of two replicate slides).
Comets were randomly captured at a constant
depth of the gel, avoiding the edges of the gel,
occasional dead cells, and superimposed com-
ets. To avoid potential variability, one well-
trained scorer performed all scorings of com-
ets. Tail length (calculated from the midpoint
of the head and presented in micrometers) and
tail moment (calculated by the computer pro-
gram) were used in this study as measures of
DNA damage.
Analysis of structural chromosome aberra-
tions. The analysis of structural chromosome
aberrations was carried out using the standard
procedure proposed by current International
Programme on Chemical Safety guidelines for
the monitoring of genotoxic effects of carcin-
ogens in humans (18). Lymphocyte cultures
were incubated in vitro in F-10 medium for 48
hours. To arrest dividing lymphocytes in meta-
phase, colchicine (0.004%) was added 3 hours
prior to the harvest. Cultures were centrifuged
at 1000 rpm for 10 minutes, the supernatant
was carefully removed, and the cells were re-
suspended in a hypotonic solution (0.075 M
KCl) at 37°C. After centrifugation, the cells
were fixed with a freshly prepared fixative of
ice-cold methanol/glacial acetic acid (3:1, v/

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Gamulin et al: Radiotherapy and Genome Damage in Oropharyngeal Cancer
519
v). Fixation and centrifugation were repeated
several times until the supernatants were clear.
Cells were pelleted and resuspended in a mini-
mal amount of fresh fixative to obtain a homo-
geneous suspension. The cell suspension was
dropped onto microscope slides and left to air-
dry. Preparations were made according to the
standard procedure. Slides were stained with
5% Giemsa solution (Sigma). All slides were
coded and scored blindly at 1000 × magnifi-
cation under oil immersion. Structural chro-
mosome aberrations were classified based on
the number of sister chromatids and breakage
events involved. Only metaphases containing
45-47 centromeres were analyzed. Microscope
slides were coded and scored blindly. One
hundred metaphases per sample (50 from each
of two replicate cultures) were analyzed for to-
tal numbers and types of aberrations, as well as
the percentage of aberrant cells.
CBMN assay. Lymphocyte cultures were
incubated in F-10 medium for 72 hours, ac-
cording to standard protocol for CBMN assay
(19) with slight modifications. Cytochalasin B
in final concentration 6 µg/mL was added into
the culture after 44 hours. Following incuba-
tion, cultures were centrifuged at 600 rpm for
10 minutes, the supernatant was carefully re-
moved, and the cells were gently resuspend-
ed in physiological saline at room tempera-
ture. After centrifugation, the cells were fixed
with a freshly prepared fixative of ice-cold
methanol/glacial acetic acid (3:1, v/v). Fixa-
tion and centrifugation were repeated several
times until the supernatants were clear. Cells
were pelleted and resuspended in a minimal
amount of fresh fixative to obtain a homoge-
neous suspension. The cell suspension was
dropped onto microscope slides and left to
air-dry. Preparations were made according to
standard procedure. Slides were stained with
5% Giemsa solution (Sigma). For micronuclei
identification, the criteria of Fenech et al (20)
were used. Scoring of micronuclei in 1000 bi-
nucleated cells was performed on coded slides
at 1000 × magnification under oil immersion.
Total number of micronuclei and their distri-
bution were determined, along with the num-
ber of micronucleated cells.
Statistical analysis
Statistical analyses were carried out using Sta-
tistica 7.0 software (StatSoft, Tulsa, OK,
USA). In alkaline comet assay, the extent of
DNA damage was measured by the medi-
an (range) comet tail lengths and moments.
Moreover, cells were classified as either “un-
damaged” or “damaged” by considering thresh-
old levels indicating the LTN comets, ie, com-
ets with the length over the 95th percentile of
the distribution of the tail lengths among con-
trols (21).
Since the distribution of data was not nor-
mal, statistical analysis was made using non-
parametric methods.
Data on the comet assay, structural chro-
mosome aberrations, and the CBMN assay
gathered in different sampling times were eval-
uated by Friedman ANOVA test and Wilcox-
on matched pairs test with downward adjust-
ment of the α-level for multiple comparisons
between pairs of measurements points. Dif-
ferences in the prevalence of individual condi-
tions were measured using χ2 test and Cochran
Q test. The level of statistical significance was
set at P<0.05.
Results
Baseline DNA damage in peripheral blood
leukocytes
Our results showed inter-individual differ-
ences in pre-therapy DNA damage in periph-
eral blood leukocytes of cancer patients (sam-
ple 1). Mean individual DNA migration ±
standard deviation, ranged from 14.93 ± 2.57
μm to 32.02 ± 21.61 μm. For the whole stud-
ied group, mean tail length of 22.54 ± 4.58 μm

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Croat Med J 2008;49:515-527
520
was recorded. Mean frequency of LTN comets
among cancer patients was 4.80 ± 0.79, where-
as their individual values varied between 3 and
6 per 100 comets measured in total. Mean
individual tail moment values ranged from
2.99 ± 0.75 to 5.98 ± 4.39. Mean tail moment
recorded for the whole group was 4.38 ± 0.99.
Post-irradiation DNA damage in peripheral blood
leukocytes
The assessment of comet parameters in blood
samples collected after administration of the
first fraction of radiotherapy (sample 2) con-
firmed a positive response to the therapy in
almost all patients. Among the patients in-
ter-individual differences were observed.
Mean individual DNA migration ranged
from 17.94 ± 0.34 µm to 41.27 ± 2.40 µm.
For the whole group, the mean tail length of
25.04 ± 6.23 μm was recorded. Mean frequen-
cy of LTN comets among cancer patients
was 10.50 ± 7.71, while their individual val-
ues varied between 2 and 24 per 100 comets
measured in total. Mean individual tail mo-
ment values were in range from 3.77 ± 0.10 to
7.39 ± 0.54. Mean tail moment recorded for
the whole group was 4.80 ± 1.16.
The third blood sampling, which was per-
formed in the middle of radiotherapy, also
showed increased level of primary DNA dam-
age in almost all patients. Mean individual
DNA migration ranged from 19.13 ± 0.39 µm
to 39.83 ± 2.32 µm. For the whole group, mean
tail length of 26.81 ± 8.74 μm was recorded.
Mean frequency of LTN comets among cancer
patients was 18.30 ± 27.62, while their indi-
vidual values varied between 0 and 90 per 100
comets measured in total. Mean individual tail
moment values were in range from 3.87 ± 0.08
to 7.10 ± 0.52. Mean tail moment recorded for
the whole group studied was 4.94 ± 0.96.
The values of comet parameters record-
ed in most of the blood samples collected af-
ter administration of the last fraction of ra-
diotherapy (sample 4) did not substantially
differ or were even lower than pre-therapy val-
ues. Mean individual DNA migration was in
range from 15.85 ± 0.36 µm to 34.41 ± 0.92
µm. For the whole group, mean tail length of
22.37 ± 5.21 μm was recorded. Mean frequen-
cy of LTN comets among cancer patients was
13.90 ± 21.70, while their individual values
varied between 0 and 66 per 100 comets mea-
sured in total. Individual tail moment values
were in range from 3.55 ± 0.10 to 5.40 ± 0.22.
Mean tail moment recorded for the whole
group studied was 4.51 ± 0.63.
Six months following
(sample 5), relatively high levels of prima-
ry DNA damage in peripheral blood leuko-
cytes of cancer patients were detected. Mean
individual DNA migration was in range
from 20.13 ± 0.27 µm to 29.64 ± 1.68 µm.
For the whole group, mean tail length of
24.86 ± 3.56 µm was recorded. Mean frequen-
cy of LTN comets among cancer patients
was 11.00 ± 14.97, while their individual val-
ues varied between 0 and 35 per 100 comets
measured in total. Mean individual tail mo-
ment values were in range from 3.86 ± 0.09 to
6.66 ± 0.41. Mean tail moment recorded for
the whole group studied was 4.87 ± 1.22.
Decline of primary DNA damage in pe-
ripheral blood leukocytes of cancer patients
was evident in blood samples taken one year
following radiotherapy, when the values of
all comet parameters returned to the baseline
level. Individual DNA migration was in range
from 14.67 ± 0.18 µm to 25.28 ± 1.25 µm. For
the whole group studied, mean tail length of
17.11 ± 4.04 μm was recorded. Mean frequen-
cy of LTN comets among cancer patients was
1.33 ± 3.27, while their individual values var-
ied between 0 and 8 per 100 comets measured
in total. Mean individual tail moment values
were in range from 2.96 ± 0.04 to 4.85 ± 0.23.
Mean tail moment recorded for the whole
group studied was 3.46 ± 0.70.
radiotherapy

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Gamulin et al: Radiotherapy and Genome Damage in Oropharyngeal Cancer
521
Taken together, the results obtained by the
alkaline comet assay indicated that administra-
tion of adjuvant radiotherapy in patients with
carcinoma of oropharynx caused an increase in
DNA damage in their peripheral blood leuko-
cytes up to the middle of the radiation cycle.
Later on, the levels of DNA damage gradually
decreased and a year after the end of radiother-
apy they returned to lower values.
However, Friedman ANOVA showed no
significant differences between six blood sam-
plings (for comet tail lengths – P = 0.063, co-
efficient of concordance = 0.349, R = 0.219;
for the frequency of LTN comets – P = 0.345,
coefficient of concordance = 0.187, R = 0.025;
and for the tail moments P = 0.224, coefficient
of concordance = 0.232, R = 0.078).
The distribution of data regarding total
number of long tailed nuclei recorded in dif-
ferent sampling times in peripheral blood lym-
phocytes of patients with carcinoma of oro-
pharynx is shown in Figure 1.
Analysis of structural chromosome aberrations
Pre-therapy chromosomal damage in peripheral
blood lymphocytes. There were inter-individual
differences in pre-therapy chromosomal dam-
age in peripheral blood lymphocytes of cancer
patients (sample 1). Individual values for the
total number of structural chromosome aber-
rations were in range from 1 to 5 aberrations
per 100 metaphases analyzed, with the mean
total number of structural chromosome aber-
rations 3.40 ± 1.43. Most of chromosomal ab-
errations recorded before radiotherapy were
chromatid breaks, chromosome breaks, and
acentric fragments. A presence of cells with
only one type of structural chromosome aber-
rations was also observed.
Post-irradiation chromosomal damage in
peripheral blood lymphocytes. After adminis-
tration of the first fraction of radiotherapy
(sample 2), an increase of chromosomal dam-
age was observed in all patients. However, in-
ter-individual differences among them were
also noticed. Individual values for the total
number of structural chromosome aberra-
tions were in range from 3 to 9 aberrations per
100 metaphases analyzed, with the mean total
number of structural chromosome aberrations
7.10 ± 2.08. The frequency of cells with more
than one type of structural chromosome aber-
rations was also increased.
In the middle of radiotherapy (sample 3),
an increase of chromosomal damage as com-
pared with the samples was observed in the
majority of the patients. Frequencies of acen-
tric fragments and dicentric chromosomes in-
creased. Considerable inter-individual dif-
ferences were noticed, accompanied with an
increased frequency of cells bearing more than
one structural chromosome aberration. In-
dividual values for the total number of struc-
tural chromosome aberrations were in range
from 4 to 34 aberrations per 100 metaphases
analyzed with mean total number of structural
chromosome aberrations 17.50 ± 10.50 (medi-
an: 15.50).
The highest frequency of structural chro-
mosome aberrations, as well as the frequency
of aberrant cells was observed in blood samples
collected after administration of the last frac-
Figure 1. Distribution of the total number of long tailed nuclei (LTN) recorded in
peripheral blood lymphocytes of patients with oropharyngeal carcinoma before
(sample 1), during (samples 2-4), and six (sample 5) and 12 months (sample 6) af-
ter adjuvant radiotherapy. Dot represents mean, box represents mean ± standard
deviation, and whiskers represent the range of the individual values measured
(minimum-maximum).

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Croat Med J 2008;49:515-527
522
tion of radiotherapy (sample 4). Individual val-
ues for the total number of structural chromo-
some aberrations were in range from 6 to 98
aberrations per 100 metaphases analyzed, with
the mean total number of structural chromo-
some aberrations of 26.50 ± 27.72. Frequency
of cells bearing more than one structural chro-
mosome aberration was increased and these
cells predominated over cells bearing only one
structural chromosome aberration.
Six months following radiotherapy (sam-
ple 5), in the majority of patients, a decrease of
chromosomal damage was detected. Individual
values for the total number of structural chro-
mosome aberrations were in range from 6 to
32 aberrations per 100 metaphases analyzed,
with the mean total number of structural
chromosome aberrations of 15.86 ± 9.34. Al-
though their frequency decreased as compared
with previous blood sampling, cells bearing
more than one structural chromosome aberra-
tion predominated over cells bearing only one
structural chromosome aberration.
In blood samples taken one year follow-
ing radiotherapy (sample 6) the levels of chro-
mosomal damage in peripheral blood lympho-
cytes were relatively high. Individual values for
the total number of structural chromosome
aberrations were in range from 5 to 49 aber-
rations per 100 metaphases analyzed, with the
mean total number of structural chromosome
aberrations of 19.83 ± 15.70. Chromosome
damage in blood cells of some cancer patients
did not return to baseline levels, both in terms
of total number of structural chromosome ab-
errations and total number of aberrant cells.
Friedman ANOVA revealed significant
differences between six blood samplings in
the total number of chromosome aberrations
(P = 0.006, coefficient of concordance = 0.540,
R = 0.448), frequency of acentric fragments
(P = 0.003, coefficient of concordance = 0.610,
R = 0.532), frequency of dicentric chro-
mosomes (P = 0.002, coefficient of concor-
dance = 0.650, R = 0.579), and number of cells
with chromosome aberrations (P = 0.033, co-
efficient of concordance = 0.404, R = 0.285).
Although the frequency of cells with 1 chro-
mosome aberration did not significantly dif-
fer when six samplings were compared, signifi-
cant difference was observed in the frequency
of cells with more than one chromosome ab-
erration (P = 0.007, coefficient of concor-
dance = 0.531, R = 0.438).
Total number of structural chromosome
aberrations recorded in different samplings in
peripheral blood lymphocytes of patients with
carcinoma of oropharynx is shown in Figure 2.
Cytokinesis-block micronucleus assay
Pre-therapy frequency of micronuclei in periph-
eral blood lymphocytes. There were inter-indi-
vidual differences in pre-therapy frequency of
micronuclei in peripheral blood lymphocytes
of cancer patients (sample 1). Individual val-
ues for the total number of micronuclei were
in range from 18 to 30 micronuclei per 1000
binuclear cells, with the mean total number
of micronuclei of 23.60 ± 3.63. The frequen-
cy of micronucleated cells ranged between 18
and 28 per 1000 binuclear cells, with the mean
total number of 23.00 ± 3.43 micronucleated
Figure 2. Distribution of the total number of structural chromosome aberrations
(CA) recorded in peripheral blood lymphocytes of patients with oropharyngeal car-
cinoma before (sample 1), during (samples 2-4), and six (sample 5) and 12 months
(sample 6) after adjuvant radiotherapy. Dot represents mean, box represents
mean ± standard deviation, and whiskers represent the range of the individual
values measured (minimum-maximum).

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Gamulin et al: Radiotherapy and Genome Damage in Oropharyngeal Cancer
523
cells per 1000 binuclear cells. Most of binucle-
ar cells contained a single micronucleus.
Post-irradiation frequency of micronuclei
in peripheral blood lymphocytes . After admin-
istration of the first fraction of radiotherapy
(sample 2) increased prevalence of micronuclei
was observed in all patients. However, inter-
individual differences among them were also
noticed. Individual values for the total num-
ber of micronuclei were in range from 24 to
36 micronuclei per 1000 binuclear cells, with
the mean total number of 29.00 ± 3.43 micro-
nuclei. The frequency of micronucleated cells
ranged between 24 and 32 per 1000 binucle-
ar cells, with the mean of 28.60 ± 2.32 micro-
nucleated cells per 1000 binuclear cells. Most
of binuclear cells contained a single micronu-
cleus.
In blood samples analyzed in the middle
of radiotherapy cycle (sample 3) an increase
in the total number of micronuclei was re-
corded as compared with the sample 2. There
were also inter-individual differences noticed,
accompanied with a higher rate of binucle-
ar cells with more than 1 micronucleus. Indi-
vidual values for the total number of micronu-
clei were in range from 68 to 164 micronuclei
per 1000 binuclear cells, with the mean to-
tal number of micronuclei of 114.20 ± 26.64.
The frequency of micronucleated cells ranged
between 64 and 144 per 1000 binuclear cells,
with the mean of 94.40 ± 22.17 (median: 90)
micronucleated cells per 1000 binuclear cells.
Most of binuclear cells contained only a single
micronucleus.
In all patients, the prevalence of micronu-
clei, as well as the frequency of micronucleated
cells was the highest in blood samples collect-
ed after administration of the last fraction of
radiotherapy (sample 4). Individual values for
the total number of micronuclei were in range
from 134 to 250 micronuclei per 1000 binu-
clear cells, with the mean total number of mi-
cronuclei of 167.20 ± 35.69. The frequency of
micronucleated cells ranged between 114 and
176 per 1000 binuclear cells, with the mean of
142.60 ± 17.99 micronucleated cells per 1000
binuclear cells. Most of binuclear cells con-
tained a single micronucleus, but some also
contained 2-4 micronuclei.
Six months following radiotherapy (sam-
ple 5), a decrease of micronuclei frequen-
cy was detected. Individual values for the to-
tal number of micronuclei were in range from
36 to 78 micronuclei per 1000 binuclear cells,
with the mean total number of micronuclei of
59.71 ± 14.02. The frequency of micronucle-
ated cells ranged between 34 and 76 per 1000
binuclear cells, with the mean of 55.71 ± 15.12
micronucleated cells per 1000 binuclear cells.
Most of binuclear cells contained a single mi-
cronucleus, but some also contained 2-4 mi-
cronuclei.
In blood samples taken one year follow-
ing radiotherapy, the micronuclei frequency
further decreased, reaching values compara-
ble to their pre-therapy values. Individual val-
ues for the total number of micronuclei were
in range from 22 to 36 micronuclei per 1000
binuclear cells, with the mean total number of
micronuclei of 29.00 ± 4.69. The frequency of
micronucleated cells ranged between 22 and
36 per 1000 binuclear cells, with the mean of
28.00 ± 4.73 micronucleated cells per 1000 bi-
nuclear cells. Most of binuclear cells contained
a single micronucleus, and some contained 2
micronuclei.
Friedman ANOVA revealed significant
differences between six blood samplings for
the total number of micronuclei (P<0.001, co-
efficient of concordance = 0.935, R = 0.922),
total number of cells with micronuclei
(P<0.001, coefficient of concordance = 0.929,
R = 0.915), total number of cells with 1 mi-
cronucleus (P<0.001, coefficient of concor-
dance = 0.928, R = 0.914), and number of cells
with 2 micronuclei (P<0.001, coefficient of
concordance = 0.835, R = 0.802).

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Croat Med J 2008;49:515-527
524
Total number of micronuclei recorded in
different sampling times in binuclear lympho-
cytes of patients with carcinoma of orophar-
ynx is shown in Figure 3.
Discussion
The results obtained in this study showed
higher levels of baseline genome damage in
leukocytes of patients with carcinoma of the
oropharynx than in Croatian general popu-
lation (22). In some, the DNA damage mea-
sured by the alkaline comet assay was com-
parable to background values of the healthy
population, but in others it was notably high-
er, even 2-fold higher than the normal values
(23). Increased endogenous DNA/chromo-
some damage could be to some extent related
to diagnostic exposures. However, considering
that blood sampling was done approximate-
ly one month after radiological examination
and the fact that cellular repair mechanisms
efficiently remove most of the primary DNA
damage induced by ionizing radiation within
a few hours, it is doubtful whether the elevat-
ed levels of primary DNA damage, as recorded
in some cancer patients before radiotherapy,
were caused by diagnostic examination alone.
Since the DNA damage detected by the alka-
line comet assay represents a steady state be-
tween the induction of lesions and their repair,
lower damage level in an individual may be the
result of an actually lower number of lesions
or of a high efficiency of repair. Therefore, it is
likely that cancer patients have impaired DNA
repair mechanisms, particularly those special-
ized for the removal of oxidative damage. For
this reason, their baseline DNA damage was
higher than in healthy participants. Other in-
vestigators also reported the probability that
the neoplastic disease itself is associated with
increased DNA damage, as well as that cancer
patients have a more fragile DNA than healthy
individuals (24,25). The background levels of
DNA damage in leukocytes are mostly influ-
enced by reactive oxygen species that are con-
tinuously generated under physiological con-
ditions as an outcome of cellular metabolism,
personal life-style, age, and daily environmen-
tal exposure to different mutagens (26). Po-
tential influence of occupational exposure in
our study is excluded, as the patients reported
no exposure to known occupational mutagens.
The contribution of smoking and alcohol con-
sumption to the background DNA/chromo-
some damage is reported in oropharyngeal
carcinoma as a sentinel disease of exposure to
different external factors (27). All patients in-
volved in our study were regular heavy smok-
ers and reported long-term alcohol consump-
tion. Alcohol consumption increases the level
of DNA and chromosomal damage, most-
ly due to the metabolic conversion of ethanol
to highly reactive acetaldehyde, an established
genotoxic agent by most of the short-term as-
says (28,29).
Chromosome aberration assay and micro-
nucleus assay showed a time-dependent oc-
currence and elimination of DNA and chro-
mosomal damage before, during the course,
and one year after radiotherapy. As comet as-
say did not show significant difference in mea-
Figure 3. Distribution of the total number of micronuclei (MN) recorded in binu-
clear lymphocytes of patients with carcinoma of oropharynx before (sample 1),
during (samples 2-4), and six (sample 5) and 12 months (sample 6) after adjuvant
radiotherapy. Dot represents mean, box represents mean ± standard deviation,
and whiskers represent the range of the individual values measured (minimum-
maximum).

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Gamulin et al: Radiotherapy and Genome Damage in Oropharyngeal Cancer
525
sured parameters during the sampling period,
it could be concluded that the applied version
of the comet assay is not sufficiently sensitive
for the purpose of such biomonitoring.
Inter-individual differences in response to
radiotherapy were obviously influenced by dif-
ferent mutagen sensitivity and DNA repair ca-
pacity in the participants. Similar was also re-
ported in previous investigations on cancer
patients (5,25,30).
This study showed that prolonged exposure
to therapy by ionizing radiation leads to some
kind of adaptive response in peripheral blood
leukocytes of the most of the treated patients.
Small acute single doses of ionizing radiation
produce damage in a very short time. Many of
these are double-strand breaks of the DNA.
The DNA double-strand breaks induced by
acute, low radiation dose may be sufficient to
activate induced resistance, which may develop
in protective mechanism, ie, adaptive response.
The adaptation induced by low doses of radia-
tion is attributed to the induction of a novel ef-
ficient chromosome break repair mechanism
that, if active at the time of challenge with high
doses, would lead to less residual damage. Pre-
vious investigations also indicated that the hu-
man population exhibited heterogeneity in the
adaptive response to ionizing radiations that
might be, at least in part, genetically deter-
mined (31,32). The results of our study are also
in agreement with these observations.
In patients with oropharyngeal carcinoma,
adjuvant radiotherapy caused a steady increase
in chromosomal damage throughout the radia-
tion cycle. Despite significant elimination rate,
a year after radiotherapy the levels of chromo-
somal damage mostly did not return to pre-
therapy values. It is also important to stress
that the frequency of complex structural chro-
mosome aberrations, such as dicentric, tricen-
tric, quadricentric, and ring chromosomes, in
this study was strongly time-dependent and
correlated well with radiation doses.
The results obtained by the micronucleus
assay correlated well with the levels of chro-
mosomal damage. Micronuclei originate from
chromosome fragments or whole chromosomes
that fail to engage with the mitotic spindle
and, therefore, lag behind when the cell divides
(20). Results of earlier micronucleus studies in
untreated and therapeutically exposed cancer
patients showed this method as useful and im-
portant biomarker of genomic instability and a
good cancer risk predictor (33).
In this study, peripheral blood lympho-
cytes were chosen as a model system as they
were previously established as suitable biodo-
simeters that integrate the effects of exposure
to exogenous and endogenous genotoxins, due
both to the amount of agent metabolic capaci-
ty and the DNA repair capacity of the individ-
ual (34). Lymphocytes are also favored because
of their easy availability, synchronous popula-
tion, low frequency of spontaneous chromo-
somal aberrations, convenient culture meth-
ods, and ease of sample collection. The latter
was particularly important in our study, as we
were able to collect the blood samples from
the patients using minimal invasive procedure,
without putting them in additional emotional
or physical stress.
It is known that lymphocytes consist
of subpopulations with different life spans.
About 90% are long-lived, with a half-life of
about 3 years (some even have a lifespan of sev-
eral decades), while the remaining 10% have a
half-life of 1-10 days (35).
The levels of DNA/chromosome damage,
as recorded in such surrogate cells, could indi-
cate the following: 1) comparable levels of un-
desirable DNA/chromosome damage in oth-
er non-target cells, most likely correlated with
secondary cancer risk and 2) possible levels of
desirable DNA/chromosome damage in target
tumor cells disseminated as micrometastases.
Our results showed that local fractionated
radiotherapy delivered to cancer patients crit-

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Croat Med J 2008;49:515-527
526
ically influenced the levels of primary DNA
damage, induced chromosomal aberrations,
and micronuclei in their peripheral blood
lymphocytes. The large variation in DNA/
chromosome damage observed in the course
of study may result from selective elimina-
tion or no proliferation of cells with multiple
chromosome aberrations and a subsequent re-
plenishment with newly formed cells without
aberrations. Frequencies of persistent DNA/
chromosome damage will depend on the bal-
ance between the cell killing and related in-
duction of proliferative responses in normal
cell precursors (35). The decrease in the fre-
quency of genetic damage, as detected during
the follow-up period in this study, could prob-
ably be due to the elimination of damaged
cells, dilution into the blood stream, or the in-
stability of these cells to complete mitotic divi-
sion; although radiation doses, sampling time,
cell kinetics, and the individual sensitivity are
factors that should be kept in mind when ex-
plaining this decline.
In conclusion, introduction of genotoxi-
cological methods in medical surveillance of
cancer patients before and after radiotherapy
could be important in evaluating secondary
cancer risk and, in case of cancers such as oro-
pharyngeal carcinoma which may have initial
genome burden, could be an important factor
for individual therapy adjustment. Chromo-
some aberration assay and micronucleus assay
are not expensive and time consuming meth-
ods, which could be run in any laboratory of
clinical cytogenetics. In the future, application
of fluorescent in situ hybridization will give
focused insight in permanent genome damage
of cancer patients after radio or chemotherapy
yielding toward individual monitoring related
with specific genomic rearrangements.
Acknowledgment
The authors gratefully acknowledge the cooperation of
all volunteers who participated in this study. We thank
Dr T. Poljičanin for her help with statistical analysis.
This investigation was supported by the Institute for
Medical Research and Occupational Health and Croa-
tian Ministry of Science, Education, and Sports (grant
No. 0022020).
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