Mutation Research 425 Ž1999.

1–8

Higher frequency of dicentrics and micronuclei in peripheral

blood lymphocytes of cancer patients
P. Venkatachalam a , Solomon F.D. Paul a , Mary N. Mohankumar a ,
B. Karthikeya Prabhu a , N. Gajendiran a , A. Kathiresan b, R.K. Jeevanram a,)

a
Health and Safety DiÕision, SHINE Group, Indira Gandhi Centre for Atomic Research, Kalpakkam 603 102 (TN), India
b
Tamilnad Hospitals, Cheran Nagar, Perumbakkam, Chennai 603 102 (TN), India
Received 26 August 1998; revised 27 October 1998; accepted 11 November 1998

Abstract

The presence of dicentric chromosome ŽDC. and micronuclei ŽMN. frequency in the peripheral blood lymphocytes of 25
cancer patients prior to chemo and radiotherapy and 21 healthy volunteers were studied. The overall DC and MN showed
significantly higher frequency compared to those obtained in normal healthy volunteers Ž p - 0.0001.. However, among 25
patients only 15 showed a higher frequency of DC aberration, nine patients showed the presence of minutes ŽM. and seven
patients showed chromatid breaks ŽChB.. The reasons for the higher frequency of aberration observed in these cancer
patients are discussed in this paper. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Cancer; Chromosome; Dicentrics; Micronucleus; Virus; Lymphocyte

1. Introduction neous chromosomal aberrations such as chromatid

breaks ŽChB., gaps, dicentrics ŽDC. and sister chro-
Chromosome rearrangements occurring over a pe-
matid exchanges ŽSCE. were shown to be higher
riod of time due to the exposure of various mutagens
than those of normal healthy individuals in periph-
from environment and life style are known to cause
eral blood lymphocytes w3x. Similarly higher inci-
cancer. The relationship between specific chromoso-
dence of breaks and SCE were reported in childhood
mal rearrangements and the type of cancer has been
leukaemia w4x and in chronic myeloid leukaemia
studied extensively. These studies show transloca-
ŽCML. patient’s w5x. Antoine et al. w6x have also
tion, deletion, gain or loss of chromosomes in tu-
reported an increased DC frequency in the peripheral
mour cells. The karyotype of various tumours have
blood lymphocytes of cervical cancer patients. On
been reported in recent years w1,2x. However, in
the contrary Diener et al. w7x and Kleinerman et al.
patients with Bloom’s syndrome, Fanconi’s anaemia
w8x did not find any increase in chromosome aberra-
and ataxia telangiectasia, the frequency of sponta-
tions in peripheral blood of cervical cancer patients
and in Morbus Hodgkin’s disease.
)
Corresponding author. Tel.: q91-4114-40352, 40362; Fax: The limited but controversial reports available on
q91-4114-40235; E-mail: rkj@igcar.ernet.in the frequency of chromosome aberrations in periph-

0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 7 - 5 1 0 7 Ž 9 8 . 0 0 2 3 8 - 3
2 P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

eral blood lymphocytes of cancer patients prompted
us to carry out a systematic study to measure the
frequency of chromosomal aberrations such as DC
and micronuclei ŽMN. in peripheral blood of cancer
patients prior to the initiation of chemo or radiother-
apy.
2. Materials and methods
average of 51.36 years. In this study among seven
male patients two were smokers and among 18 fe-
male patients one had the habit of chewing tobacco.
With respect to the nature of cancer, 48% had cancer
of cervix, 8% had cancer of breast and oesophagus
and the remaining 34% had cancer of thyroid, rec-
tum, bladder. All the 21 of normal healthy volunteers
were males and non-smokers. Their ages are given in
Table 3.

2.1. Study population 2.2. Sample collection

The blood samples were obtained from a total Blood samples of cancer patients were collected
number of 46 persons. Among these, 25 were cancer from Tamilnad Hospitals, Chennai, Tamilnadu, India
patients unexposed to any chemo or radiotherapy and and Government Arignar Anna Cancer Hospital,
the remaining 21 were from normal healthy volun- Kancheepuram, Tamilnadu, India. About 3 ml blood
teers. Details such as age, gender and type of the from each patient was collected in a sterile hep-
cancer of the 25 patients are given in Table 1. Age of arinised vial. The blood samples were then trans-
the cancer patients varied between 22 and 75 with an ported to the laboratory in an ice bath. These sam-
ples were brought back to room temperature prior to
setting up of the culture.
Table 1
Type of cancer, age and gender of patients
2.3. Culture for DC chromosome aberration
S. no. Code. Age Smokerr Gender Type of
no. Žyears. non-smoker cancer
The method for DC chromosome aberration assay
1 TNH-1 63 NS F Thyroid
is described elsewhere w9x. Briefly, the method is
2 TNH-2 46 NS F Breast
3 TNH-4 52 NS F Neck given below. To 0.5 ml of the blood sample, 5 ml
4 TNH-5 52 NS M Pyriformfossa RPMI-1640 ŽSigma. culture medium supplemented
5 TNH-6 68 NS M Lung with 7.5% NaHCO 3 , 20% fetal calf serum, 200 mM
6 TNH-7 22 NS M NHL L-glutamine, penicillin 100 unitsrml and strepto-
7 TNH-9 55 NS F Breast
mycin 100 mgrml, was added. A total of 200 ml of
8 TNH-10 55 NS F Cervix
9 TNH-13 47 NS F Cervix phytohaemagglutinin-M ŽPHA-M, Gibco, BRL. was
10 TNH-15 49 S M Oesophagus added to the culture to initiate cell division. At 46 h,
11 TNH-16 55 NS F Oesophagus the cells were blocked at metaphase by adding col-
12 TNH-17 57 NS M Rectum cemid to a final concentration 0.1 mgrml. The cul-
13 TNH-18 56 NS F Endometrium
ture was incubated for another 2 h. The cells were
14 TNH-19 35 NS F Cervix
15 TNH-20 45 NS F Cervix harvested by centrifuging the sample at 400 = g and
16 TNH-21 50 NS F Cervix the cell pellet obtained was given hypotonic treat-
17 TNH-22 54 S M Bladder ment with 0.45% KCl for 20 min at 378C. The cells
18 KAN-1 52 NS F Cervix were washed with a mixture of methanol and acetic
19 KAN-2 41 NS F Cervix
acid Ž3:1, Carnoy’s fixative. for three times. After
20 KAN-3 55 NS F Cervix
21 KAN-5 50 NS F Cervix the first wash, the cells were suspended in a mixture
22 KAN-6 50 NS F Cervix of methanol and acetic acid Ž1:1. for 10 min and
23 KAN-7 50 NS F Cervix centrifuged. After a final wash with Carnoy’s fixa-
24 KAN-8 75 T F Check tive, cell pellet was suspended in a known volume of
25 KAN-9 50 NS F Cervix
fixative Ž0.5 ml.. The cells were then cast by allow-
TNH, Tamilnad Hospital; KAN, Kancheepuram; F, female; M, ing three drops of the cell suspension to fall on a
male; S, smoker; NS, non-smoker; T, tobacco chewing. pre-cooled slide from a height of 10 cm. The slide
 P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8 3

was air dried and stained with 10% Giemsa solution
ŽpH 6.8., mounted with DPX and scored for DC
using light microscope ŽNikon Optiphot-2, Japan.
under 10 = 100x oil immersion.
2.4. Culture for MN
The method for cytokinesis-blocked MN assay
has been described by Fenech and Morely w10x. The
procedure is given briefly below. The culture was set
up the same way mentioned for DC aberration till
the addition of PHA-M. Cytochalasin-B ŽSigma. at a
final concentration of 3 mgr5 ml culture was added
at 44 h. The cells were incubated for another 28 h at
378C. The cells were then harvested, given hypotonic
treatment and washed with Carnoy’s fixative as men-
tioned for DC aberration. Three drops of cell suspen-
sion were allowed to fall on to a clear cooled slide
from a height of 1 cm, air dried and stained with
Giemsa. Cells with two daughter nuclei, surrounded
by cytoplasm were scored for the presence of MN
according to the modified criteria of Countryman
and Heddle w11x.
2.5. Statistical analysis
The DC, excess acentrics ŽAC., ChB, minutes
ŽM. and MN frequency obtained in cancer patients
were compared with those obtained from normal
healthy volunteers using Welch approximation un-
paired ‘t’-test.
3. Results
The aberrations such as DC, AC, ChB, M and
MN scored in cancer patients and healthy volunteers
are given in Tables 2 and 3, respectively. Table 4

Table 2
Frequency of various chromosomal aberration and MN observed in peripheral blood of cancer patients
Code CS DC AC M ChB DCr CB MN MNr
no. cell cells cell
TNH-1 136 1 1 1 – 0.0074 1000 39 0.039
TNH-2 106 0 1 – 1 ND 1000 45 0.045
TNH-4 196 0 2 1 – ND 1000 33 0.033
TNH-5 141 3 4 – 3 0.0213 1000 19 0.019
TNH-6 161 0 3 – 2 ND 1000 15 0.015
TNH-7 190 17 16 3 3 0.0895 1000 20 0.020
TNH-9 250 3 2 – 3 0.0120 1000 36 0.036
TNH-10 125 0 – 2 – ND 1000 28 0.028
TNH-13 100 0 – 2 4 ND 500 17 0.034
TNH-15 200 1 2 1 2 0.0050 1000 29 0.029
TNH-16 100 3 2 – 1 0.0300 1000 26 0.026
TNH-17 100 0 – – 2 ND 1000 18 0.018
TNH-18 100 1 1 – 1 0.0100 1000 10 0.010
TNH-19 126 0 – 2 2 ND CF – –
TNH-20 106 0 – – 2 ND 1000 15 0.015
TNH-21 150 1 – 1 1 0.0067 CF – –
TNH-22 100 2 – – – 0.0200 200 16 0.080
KAN-1 200 0 – 1 2 ND 1000 17 0.017
KAN-2 125 2 2 – – 0.0160 1000 18 0.018
KAN-3 150 4 – 2 3 0.0267 1000 21 0.021
KAN-5 150 5 2 – – 0.0333 1000 15 0.015
KAN-6 150 0 1 1 2 ND 1000 9 0.009
KAN-7 100 1 – – 1 0.0100 1000 39 0.039
KAN-8 100 5 2 – 5 0.0500 1000 123 0.123
KAN-9 100 1 3 – 2 0.0100 1000 12 0.012
Total 3462 50 44 17 42 21,700 620

CS, Metaphase cells scored; DC, dicentric chromosome; AC, excess acentrics; M, minutes; ChB, chromatid breaks; MN, micronuclei; ND,
not detected; CF, culture failed; CB cells, cytokinesis blocked binucleated cells.
4 P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

Table 3
Frequency of various chromosomal aberration and MN observed in peripheral blood of male healthy volunteers
S. Age CS DC AC M ChB DCr CB MN MNr
no. cell cells cell
1 46 239 – 1 – – ND 1000 11 0.011
2 28 727 1 – 2 – 0.0014 1000 13 0.013
3 38 200 1 – 1 – 0.005 1000 3 0.003
4 45 372 1 3 – – 0.0027 1000 21 0.021
5 35 171 – 1 – – ND 1000 15 0.015
6 43 295 1 1 1 – 0.0034 1000 10 0.010
7 32 203 1 – – – 0.0049 1000 20 0.020
8 39 239 – 5 – 4 ND 1000 19 0.019
9 37 200 – – – – ND 1000 8 0.008
10 29 192 – 1 1 – ND 1000 10 0.010
11 34 101 – 1 – 1 ND 1000 5 0.005
12 31 628 1 – 1 – 0.0016 1000 3 0.003
13 30 406 – 2 – – ND 1000 9 0.009
14 29 250 – 1 1 1 ND 1000 9 0.009
15 28 250 – – 1 – ND 1000 17 0.017
16 29 250 – – 1 1 ND 1000 14 0.014
17 33 250 – 2 – 2 ND 1000 22 0.022
18 35 250 1 1 – – 0.004 1000 14 0.014
19 37 250 – – 1 – ND 1000 7 0.007
20 29 250 – – – 1 ND 1000 12 0.012
21 28 400 – 1 – 3 ND 1000 13 0.013
Total 6123 7 20 10 13 21,000 255

CS, Metaphase cells scored; DC, dicentric chromosome; AC, excess acentrics; M, minutes; ChB, chromatid breaks; MN, micronuclei; ND,
not detected; CB cells, cytokinesis blocked binucleated cells.

compares the average frequencies of these aberra-
tions observed in cancer patients to those obtained in
healthy volunteers. The increase in the aberration
frequencies in cancer patients compared to healthy
volunteers were highly significant except for M,
where it was only significantly higher. Among these
aberrations DC frequency was nearly 13 times higher
compared to healthy volunteers as other aberrations
were only 2 to 5 times higher in cancer patients. The
average DC frequency in patients with cervical can-
Table 4
Comparison of various chromosomal aberrations and MN ob-
served between healthy individuals and cancer patients
Type of Healthy Cancer p-value
aberration individuals patients
cer Ž0.0113 " 0.0026. alone showed a significant
increase compared to that of healthy volunteers Ž p -
0.005, Table 5.. The DC frequency Ž0.0174 "
0.0031. observed in patients with cancer other than
the cervix, also showed a significant increase com-
pared to that of healthy volunteers Ž p - 0.0001.. In
one patient ŽTNH-7. remarkably high DC frequency
was observed Ž0.089rcell.. In this patient among 17
DC, a single cell alone had 10 DC Žone pentacentric,
one tricentric, four DC, Fig. 1.. The average DC
frequency Ž0.0101 " 0.0017. after excluding the re-
sult obtained from this sample, was significantly
Table 5
Frequency of DC in healthy individuals compared to that of
cervical cancer and patients other than cervical cancer

Dicentricsrcell 0.0011"0.0004 0.0144"0.0020 - 0.0001 Subjects Metaphase Dicentrics p-value

Acentricsrcell 0.0033"0.0007 0.0127"0.0019 - 0.0001 studied scored frequency
Ch. breakrcell 0.0021"0.0006 0.0121"0.0019 - 0.0001 Healthy individuals 6123 0.0011"0.0004
Minutesrcell 0.0016"0.0005 0.0049"0.0009 - 0.01 Cervical cancer patients 1682 0.0113"0.0026 - 0.005
MNrcell 0.0121"0.0008 0.0289"0.0018 - 0.0001 Other cancer patients 1880 0.0174"0.0031 - 0.0001
 P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8 5

Fig. 1. Multiple chromosome aberration observed in a lymphocyte of a non-hodgkins lymphoma patient prior to therapy.

higher compared to that of healthy volunteers Ž p -
0.0001..
The MN frequency observed in cancer patients
was also compared to that of age-matched healthy
individuals. The MN frequency in age-matched con-
trol was obtained using the equation Y s 0.00029 X
q 0.00171, where Y is the frequency of MN and X
is the age of the donor. This equation was generated
from the results obtained in this laboratory w12x. The
frequency of MN in cancer patients was significantly
higher than that of age-matched controls Ž p -
0.0001..
4. Discussion
Cytogenetic methods have been used extensively
for monitoring base-line chromosome aberration fre-
quency in population unexposed w13,14x, and ex-
6 P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

posed to low level radiation w15,16x. This technique not due to the uniform increase in aberration fre-
has also been used to monitor population exposed to quency among metaphase cells scored. Since the
industrial chemicals w17,18x and chemotherapeutic increase in DC was due to the presence of single
agents w5,19,20x. rogue cell, it was not included in the study as it was
With respect to the increase in DC, MN and felt that this may not result in corresponding increase
translocation frequency in the peripheral blood lym- in MN. TNH-21 was not included as the culture for
phocytes of cancer patients compared to those of MN had failed. The correlation study carried out in
healthy volunteers, it was suspected that these pa- the remaining 13 patients showed a ‘r ’ value of 0.55
tients had undergone radiotherapy or chemotherapy ŽFig. 2.. It is not known whether a better correlation
in the past. The medical records of these patients, would have be seen if the number of metaphases and
however, had indicated that they were not given any binucleated cells scored were large as the total num-
of these treatments prior to the collection of blood ber of DC observed in cancer patients was nearly 12
samples. If the patients had undergone radiotherapy, times higher than that of observed in normal sub-
the frequency of DC would have been much higher jects, whereas in the case of MN it was only 2.5
than that observed in the present study. It has been times higher in cancer patients compared to that of
reported that chemotherapeutic agents like bleomycin observed in normal subjects. With respect to MN
w19x, cyclophosphamide w17x and chemicals like ethy- frequency in cancer patients the comparison was
lene oxide w21x induce chromosome aberrations. made to that obtained in age matched healthy indi-
However, these patients were not treated any of these viduals as it is known that MN frequency increases
drugs, as evidenced from medical records. Perhaps with age. However, Fenech et al. w23x and Gatenberg
increase in chromosomal aberrations in blood lym-
phocytes of cancer patients could have been due to
the exposure of certain mutagenic agents capable of
inducing dicentric aberrations. Similar to our results,
Antoine et al. w6x have shown significantly higher
frequencies of DC in peripheral blood lymphocytes
of patients with cervical cancer. Honeycomb w5x has
shown AC and ChB in patients with CML. Aronson
et al. w4x also have observed AC and ChB in the
blood of children with leukaemia. At the same time
Diener et al. w7x, Kleinerman et al. w8x, and Catena et
al. w22x did not observe any increase in the frequency
of DC in blood lymphocytes of Morbus Hodgkin
lymphoma, cervical cancer and adenocarcinoma, re-
spectively.
In the present study, it has been observed that
even though the overall DC frequency observed in
cancer patients was higher compared to that of nor-
mal individuals, only 15 out of 25 cancer patients
had shown a higher frequency of DC. To confirm the
results further, the frequency of MN was measured.
A correlation between DC and MN was carried out
in patients who had detectable DC frequency. This
was studied only in 13 out of 15 patients who had
detectable DC frequency. The two patients who were
not included for the study were TNH-7 and TNH-21.
In the case of TNH-7 the number of DC was more Fig. 2. Correlation between DC and MN observed in cancer
because of the presence of single rogue cell and was patients Ž r s 0.55..
 P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8
et al. w24x have shown that frequency of MN in
cancer patients was same as that of normal persons.
The exact reason for the higher DC frequency
observed in 15 out of 25 cancer patients compared to
that of normal subjects is not known. However, one
can think that it could be due to chromosome insta-
bility as it has been reported that there exist genomic
instability at low frequency in peripheral blood lym-
phocytes in individuals affected by several types of
cancer w25,26x. An increased frequency of structural
chromosomal aberrations has also been reported in
families with high incidence of cancer w27x. Similar
to DC and MN, the frequency of acentrics, ChB and
M are also higher in cancer patients which could
either due to chromosome instability or due to some
agents such as virus inducing these aberrations.
The association between virus and cancer is well
established. The presence of Hepatitis-B virus in
primary hepatocellular carcinoma, Epstein Bar virus
in Burkitt’s lymphoma, human T cell lymphoma
virus in adult T cell lymphoma and leukaemia and
human papilloma virus in cervical cancer indicate
the involvement of these viruses in these cancers w2x.
It is known that certain viruses have the capacity to
induce a large number of DC, tricentrics and quadra-
centrics resulting in the formation of a rogue cell
w28x. In the present study, also one patient ŽTNH-7.
had a cell with multicentric aberration ŽRogue cell,
Fig. 1. indicating the possibility on the interaction of
such virus. Though it is well known that viruses play
an important role in the induction of cancer and
induce DC, the cytogenetic analysis was carried out
to look for specific translocation in a given tumour
cell w2x. Even if one looks for the presence of DC the
chances of seeing such aberrations are remote, as
they are unstable in nature and hence eliminated
during the fast division of tumour cells. Whereas
translocations remain in tumour cells as these aberra-
tions are of stable in nature. Since there is a strong
relationship between specific translocation and the
type of cancer most efforts had gone to identify
specific translocations in tumour cells and not for
other aberrations in blood lymphocytes. It is believed
that tumour triggering agents may interact with cells
of the body including blood cells causing chromo-
some aberration. Thus, one may expect presence of
DC in blood lymphocytes other than the organ which
has turned malignant.
7
5. Conclusion
The study shows that there is an increase in the
frequency of aberrations in the peripheral blood lym-
phocytes of cancer patients. It is believed that this
increase may be due to either chromosome instability
or some agents such as virus or the combination of
both factors.
Acknowledgements
Authors wish to thank Dr. S.M. Lee, Director,
SHINE Group, Shri L.V. Krishnan, Former Director,
Safety Research and Health Physics Group, Shri
A.R. Sundararajan, Former Associate Director, Safety
Research and Health Physics Group, Dr. A. Natara-
jan, Head, Health and Safety Division, IGCAR,
Kalpakkam, for their encouragement in carrying out
this work. The authors are grateful to Dr. M.
Veluswamy, CMD, Tamilnad Hospitals, Chennai, for
granting permission to irradiate the blood samples.
Thanks are due to Dr. A. Vaitheeswaran and J.
Thirulogachander, Medical Physicts, Tamilnad Hos-
pitals for their help during calibration of dose and
blood sample irradiation.
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