Mutation Research 516 (2002) 2940

Enhanced frequency of micronuclei in individuals exposed

to arsenic through drinking water in West Bengal, India
A. Basu a , J. Mahata a , A.K. Roy b , J.N. Sarkar b , G. Poddar b , A.K. Nandy b ,
P.K. Sarkar b , P.K. Dutta c , A. Banerjee a , M. Das d , K. Ray a , S. Roychaudhury a ,
A.T. Natarajan e , R. Nilsson f , A.K. Giri a,
a

Division of Human Genetics and Genomics, Indian Institute of Chemical Biology, 4,

Raja S.C. Mullick Road, Calcutta 700032, West Bengal, India
b School of Tropical Medicine, Calcutta 700073, West Bengal, India
c Calcutta Medical College, Calcutta 700073, West Bengal, India
d Department of Zoology, University of Calcutta, Calcutta 700019, India
Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Leiden, The Netherlands
f Division of Molecular Toxicology and Risk Assessment, Stockholm University, Stockholm, Sweden
Received 24 July 2001; received in revised form 8 January 2002; accepted 9 January 2002

Abstract
In West Bengal, India arsenic in ground water has been found to be above the maximum permissible limit in seven districts
covering an area of 37,493 km2 . In the present study, evaluation of the micronuclei (MN) formation in oral mucosa cells,
urothelial cells and peripheral blood lymphocytes was carried out in the symptomatic individuals exposed to arsenic through
drinking water. Forty five individuals with cutaneous signs of arsenicism from four affected districts (368.11 g/l of As in
drinking water) were considered as the exposed group and 21 healthy individuals with no symptoms of arsenic poisoning and
residing in two unaffected districts (5.49 g/l of As) were considered as controls. The exposed and control groups had similar
age distribution and socioeconomic status. Standardised questionnaires were utilised and medical examination was conducted
to ascertain exposure history, sociodemographic characteristics, diet, health, medication, addiction and chief symptoms in the
study participants. Arsenic exposure was confirmed by measuring the arsenic content in the drinking water, nails, hair and
urine samples from the volunteers. Arsenic contents in the urine, nail and hair in the exposed group were 24.45 g/l, 12.58 and
6.97 g/g, respectively which were significantly high in comparison to corresponding control group values of 4.88 g/l, 0.51
and 0.34 g/g, respectively. Exposed individuals showed a statistically significant increase in the frequency of MN in oral
mucosa, urothelial cells and lymphocytes (5.15, 5.74 and 6.39/1000 cells, respectively) when compared with the controls (0.77,
0.56 and 0.53/1000 cells, respectively). Thus, the above results indicate that the symptomatic individuals exposed to arsenic
through drinking water in this region have significant cytogenetic damage. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Arsenic; Micronuclei; Oral mucosa; Urothelial cells; Human lymphocytes

1. Introduction
Corresponding author. Tel.: +91-33-473-0492/6793;
fax: +91-33-473-5197.
E-mail addresses: akgiri15@yahoo.com, akgiri@iicb.res.in
(A.K. Giri).

Incidents of arsenic contamination in the ground

water have been reported from widespread areas
through out the world such as Taiwan, Mexico, Chile,

1383-5718/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 1 8 ( 0 2 ) 0 0 0 1 4 - 1

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A. Basu et al. / Mutation Research 516 (2002) 2940

Argentina, Thailand, Bangladesh and India. Minor

cases of chronic arsenic toxicity have been reported
in Poland, USA (Minnesota and California), Canada
(Ontario), Hungary and Japan. Arsenic in groundwater has been found to be above the WHO permissible limit in seven districts of West Bengal, India,
encompassing an area of 37,493 km2 , with concentrations ranging from 200 to 800 g/l. More than
200,000 people have already shown different types
of arsenical skin lesions [1]. It has been regarded as
the biggest arsenic calamity in the world [2]. However, reports on the cytogenetic survey of arsenic
exposed population in West Bengal are very limited. The pollution by naturally occurring arsenic of
alluvial Ganges aquifers which are used for public
water supply in West Bengal has become a serious threat to human life. Most of the contaminated
water samples are found to contain a mixture of arsenite and arsenate [1]. All these water samples are
obtained from underground sources and not from
surface or rainwater. Chronic ingestion of high levels of arsenic in drinking water is associated with
increased incidence of cancer at various sites, such
as skin, lung, bladder and other internal organs [3].
A cross-sectional survey conducted in 19951996 to
investigate arsenic-associated skin lesions reported a
higher incidence of keratosis and hyperpigmentation
in West Bengal [4].
Many human epidemiological studies on arsenic
poisoning are available. Recently we have reviewed
and updated the mutagenic and genotoxic effects of
arsenic. It has been found that arsenic is not mutagenic
but genotoxic in both animal and human systems
[5]. As far as mutagenicity of arsenic is considered
it appears to be largely non-mutagenic in bacterial
and standard mammalian cell mutation assays which
measure mutation at single gene locus [6]. It can
induce DNA damage in multiple test systems [7].
Almost all the results of in vitro assays of chromosomal aberrations (CAs), sister chromatid exchanges
(SCEs) and micronuclei (MN) formation induced by
arsenic in mammalian cells showed positive clastogenic effects [8,9]. Studies on cytogenetic assays in
populations with arsenic exposure clearly indicate
positive genotoxic effects on human lymphocytes
in vivo [10]. The majority of exposed populations
who had a history of relatively long periods of exposure showed higher incidences of CAs [11], SCEs

[12] and MN formation [13]. Significantly increased

rates of CAs and MN formation were also found in
exfoliated cells of oral mucosa and urinary bladder
[8,14].
Considering the widespread reports of arsenic induced carcinogenicity in human beings, we recognise
the need to biomonitor the genotoxic effects of arsenic
and its compounds in human beings exposed to arsenic through drinking water. We have studied the MN
formation in oral mucosa cells, urothelial cells and in
lymphocytes in the symptomatic individuals exposed
to arsenic through drinking water in West Bengal, India as this cohort represents a unique population in the
world for such study.

2. Materials and methods

2.1. Study area and study subjects
Out of the seven arsenic affected districts of West
Bengal, i.e. South 24 Parganas, North 24 Parganas,
Nadia, Murshidabad, Malda, Bardhaman and Hooghly,
the four former districts constituted our study area
(Fig. 1). Two groups of volunteers were recruited.
Forty-five individuals (30 men and 15 women), who
showed cutaneous signs of arsenicism like hyperpigmentation, hypopigmentation, palmoplantar hyperkeratosis, raindrop pigmentation or ulcerative lesions
and were inhabitants of the study area were selected
for the exposed group from the Outpatient Department at the School of Tropical Medicine, Calcutta,
India. Figs. 25 present photographs of some arsenical skin lesions from arsenic exposed individuals.
Twenty-one individuals (17 men and 4 women) with
no symptoms of arsenic poisoning and residing in the
unaffected districts of West Bengal (Howrah, Midnapur) were selected as controls (Fig. 1). The exposed
and control groups had similar socioeconomic status.
The distribution of age (between 15 and 60 years)
among both the groups was similar. Each volunteer
was interviewed about smoking habits, consumption of alcohol and chewing of tobacco, etc. The
purpose of the questionnaire was to select healthy
individuals from the exposed and control groups so
that confounding factors such as medical treatment,
smoking, alcoholism, and chronic disease could be
eliminated.

A. Basu et al. / Mutation Research 516 (2002) 2940

31

Fig. 1. Map of West Bengal, India with neighbouring states, seven arsenic affected districts of West Bengal and neighbouring country
Bangladesh.

2.2. Biomarkers used

The biomarkers selected for studying arsenic exposure were nails and hair since the arsenic absorbed
in the body tends to get deposited in the hair and
nails where it is firmly bound to keratin. Urinary ar-

senic concentration is also considered to be a good

parameter for assessing recent arsenic exposure [14].
Genetic toxicology endpoints have also been utilised
as biomarkers. The frequency of MN observed in the
exfoliated cells of oral mucosa and urinary bladder
is used as an index to monitor the genetic damage

32

A. Basu et al. / Mutation Research 516 (2002) 2940

Fig. 2. Plantar discrete hyperkeratosis with few plaque formation,

raised and irregular margin, grade II keratosis.

induced by arsenic since these cells are in direct contact with the carcinogen. The bladder cell MN assay
has been suggested to be one of the most appropriate
biological marker of arsenic genotoxicity [15]. Bladder cell MN reflect damage to the urothelial tissue
which occurs 13 weeks prior to the exfoliated cells
appearing in urine [16]. The cytokinesis-block MN
(CBMN) technique in lymphocyte culture is widely
regarded as a sensitive and reliable method for assessing chromosome damage [17]. Hence, for the investigation of exposure to arsenic, samples of drinking
water, hair, nails, urine and blood were collected.

Fig. 3. Hyper and hypopigmentation anterior chest wall (raindrop

pigmentation). Bowens disease (carcinoma in situ) medial and
upper part of left nipple, guttate melanosis (like black mole) upper
part of left side of chest.

2.3. Chemicals
RPMI-1640, foetal calf serum, phytohemaglutinin
(M form), l-glutamine, penicillin, streptomycin were
purchased from Gibco BRL (USA), cytochalasin
B, TrisHCl and ethylene diamine tetra acetic acid
(EDTA ) from Sigma were used.
2.4. Collection of water, nail and hair samples
Water samples (100 ml), nails (250 mg) and
hair (300 mg) were collected, coded and sent to the

Fig. 4. Palmar melanosis (melasma) with areas of hyperkeratosis

(early features of arsenicosis).

A. Basu et al. / Mutation Research 516 (2002) 2940

33

arsenic estimation. This gives the best measure of the

current arsenic exposure [14]. Approximately 50 ml
of urine samples (spot samples) were also collected,
coded, kept at 24 C in a cooling device and immediately brought to the laboratory, where they were
centrifuged to obtain the bladder cells (2000 rpm for
15 min) and further processed for MN assay. First
morning voids were not used for MN assay because
exfoliated cells tend to degrade from overnight exposure to urine [20]. Conc. HCl (1 ml/100 ml urine) was
added in the urine samples to prevent bacterial growth.
2.6. Blood sampling
Approximately 45 ml of blood samples were obtained from each individual by venipuncture in heparinised sterile conical plastic tubes. The samples were
coded, kept at 4 C in a cooling device and brought to
the laboratory where they were immediately processed
for lymphocyte culture.
2.7. Buccal and urothelial exfoliated cells

Fig. 5. Classical raindrop pigmentation with plantar hyperkeratosismore prominent on heel.

Analytical Chemistry Department, School of Tropical Medicine, Calcutta for estimation of arsenic.
The water samples were collected in plastic bottles
pre-washed with nitric acidwater (1 + 1) and after
collection nitric acid (1.0 ml/l) was added as preservative [18]. Nail and hair samples were collected using
ceramic blade cutters [2]. Both the samples were
thoroughly washed with double distilled water and
acetone and then processed [2]. Hair samples were of
similar size and were taken from more or less similar
region of head (close to the scalp behind the ear with
a diameter of about 1 cm) [19].

Buccal cells were obtained by scraping the inside

of the mouth (both cheeks) with a toothbrush. Bladder cells were recovered by centrifuging urine samples, as mentioned previously. The cells were washed
three times by centrifugation at 1500 rpm for 10 min
in a buffer solution consisting 0.1 M EDTA, 0.01 M
TrisHCl and 0.02 M NaCl (pH 7.0). Volumes of 25 ml
of the buffer solution in a 50 ml conical tube was
used in every washing step. Fifty microlitres of the
cell suspension was laid and spread well on clean,
pre-heated (40 C) glass slides and allowed to air-dry
for 510 min. The slides were fixed in methanol (80%
(v/v)) at 0 C for 20 min and air dried [21]. Buccal
cells were analysed following the method of Tolbert
et al. [22] and urothelial cells were analysed according to Reali et al. [23]. At least 1000 cells were scored
per individual.
2.8. Lymphocyte culture and harvesting

2.5. Urine sampling

Subjects were supplied with pre-coded polypropylene bottles for urine collection. First morning voids
were collected and sent to the Analytical Chemistry
Department, School of Tropical Medicine, Calcutta for

Lymphocytes were cultured and MN assay was

conducted following the method of Fenech [17] and
Migliore et al. [24]. Replicate lymphocyte cultures
were prepared. A volume of 0.5 ml of blood were
placed in sterile conical plastic tubes containing 6 ml

34

A. Basu et al. / Mutation Research 516 (2002) 2940

of RPMI-1640 medium supplemented with foetal calf

serum, phytohemaglutinin, l-glutamine and antibiotics. The samples were incubated for 72 h at 37 C.
At 44 h of incubation, cytochalasin B (at final concentration of 3 g/ml) was added to each culture to
inhibit cytokinesis. After 72 h, the cells were centrifuged at approximately 1000 rpm for 5 min. Culture
media was removed and cells were treated for 5 min
with a weak hypotonic solution (0.075 M KCl:saline,
1:9). After centrifugation the cells were fixed in fresh
fixative (methanol:glacial acetic acid, 3:1). Fixative
was removed by centrifugation and two more changes
of fixative were performed. Samples for microscopic
observations were obtained by carefully dropping
cell suspension from a Pasteur pipette onto wet clean
slides. For analysing MN, the slides were air-dried,
stained with aqueous Giemsa and at least 2000 binucleated cells were scored under the microscope.
2.9. Arsenic analysis in nails, hair, water and urine
Flow injection-hydride generation-atomic absorption spectrometry (FI-HG-AAS) was used for arsenic
analysis in various samples. A Perkin-Elmer model
analyst 100, Fias 100 AAS was utilised for the purpose. Nails, hair and urine samples were subjected to
nitric acid and perchloric acid pre-treatment for digestion [25]. Water was not subjected to any chemical
treatment before analysis.

the control group are presented in Table 2. Table 3

shows the summary of all the results with statistical
interpretation. Mean arsenic contents of nail, hair and
urine were significantly high in the symptomatic individuals when compared with the arsenic content in
the individuals of non-affected area. Similarly the arsenic content in the water samples was also significantly higher in the arsenic contaminated area when
compared with non-affected area. The arsenic content
in the water samples of the individuals code numbers
ST38, ST39 and ST41ST45 were same since these
individuals belong to the same family.
4.1. MN in exfoliated epithelial cells
A comparison was made between the MN frequency in urothelial cells of 40 exposed (26 males and
14 females) individuals and 21 control (17 males and
4 females) volunteers. For oral mucosa cells, the incidence of MN in 45 (30 males and 15 females) buccal
smears were compared with that in 21 (17 males and
4 females) control buccal smears. Individuals living
in the different arsenic affected districts and showing
arsenic induced skin lesions had higher frequencies of
micronucleated cells than those residing in the unaffected districts. This was true for buccal and bladder
cells. These differences were statistically significant
using a Students t-test (P < 0.01).
4.2. MN in lymphocytes

3. Statistical analysis
Results of the estimated arsenic content in water,
nail, hair and urine of the symptomatic individuals
were compared with respective controls by paired
Students t-test. MN assays were also analysed using
paired Students t-test and the level of significance is
presented in the Table 3.

A similar analysis was carried out with the data

obtained in lymphocytes. Twenty-one successful lymphocyte cultures were analysed in the control group
and 44 in the exposed group. There was a statistically
significant increase in the incidence of MN in lymphocytes of exposed individuals when compared to
controls.

5. Discussion
4. Results
Data on the total arsenic content in drinking water, and in the nails, hair and urine of the exposed
individuals and frequencies of MN in exfoliated cells
as well as the lymphocytes of symptomatic individuals are presented in Table 1. The same parameters in

Figs. 68 present the photographs of MN in oral

mucosa cells, urothelial cells and lymphocytes, respectively of arsenic exposed individuals. Symptomatic individuals showed a statistically significant
increase in the frequency of MN in oral mucosa,
urothelial cells and lymphocytes when compared with

A. Basu et al. / Mutation Research 516 (2002) 2940

35

Table 1
Arsenic content in water, nail, hair and urine and the frequencies of MN in exfoliated epithelial cells and blood lymphocytes from the
symptomatic individuals exposed to arsenic through drinking water
Code

ST1
ST3
ST4
ST5
ST7
ST10
ST 11
ST14
ST16
ST17
ST20
ST22
ST24
ST26
ST27
ST28
ST29
ST30
ST33
ST35
ST38
ST39
ST41
ST42
ST43
ST44
ST45
ST46
ST47
ST48
ST49
ST51
ST52
ST53
ST54
ST58
ST60
ST71
ST81
ST89
ST90
ST117
ST118
ST119
ST120
a

Age/sex

30/F
46/M
35/M
48/M
49/M
32/M
38/M
17/M
22/M
43/M
35/F
20/M
22/F
22/M
58/M
16/M
17/M
16/F
40/F
30/M
28/M
16/M
15/M
16/M
22/F
35/F
33/F
35/F
26/M
23/M
26/M
37/M
35/F
35/M
33/M
18/M
39/M
34/F
20/M
37/M
15/F
25/F
40/F
35/F
41/M

Exp pera

10
10
16
20
15
10
15
10
4
4
12
10
12
8
20
8
8
6
12
8
12
10
10
14
10
15
15
13
5
5
12
11
12
12
20
10
7
15
2
6
6
15
22
15
15

Skin
lesionsb

As in waterc
(g/l)

As in nail
(g/g)

As in hair
(g/g)

As in urine
(g/l)

MN/1000 cellsd
Urine

Buccal cells

Lymphocytes

2,
1,
1,
1,
1,
2
5
1,
1,
1,
2,
1,
2
1
2
1,
1,
1,
1
2,
1,
1,
1,
5
1,
1,
1,
1,
3
1
1
1
1
1
1
1
2
1
1
1,
1
1,
1,
1,
5

25
340
490
250
200
40
60
800
380
200
370
375
50
200
710
200
200
15
180
290
800
800
800
800
800
800
800
70
100
100
70
800
360
800
360
70
260
710
150
340
340
350
400
140
170

5.46
4.50
0.87
2.41
30.90
2.00
1.20
40.17
26.60
1.83
12.50
2.00
29.10
16.20
4.46
24.30
33.08
2.17
10.90
5.50
27.20
52.40
12.80
15.50
26.20
38.80
18.00
3.50
4.60
2.80
6.70
26.70
2.33
6.40
2.70
3.50
1.44
4.00
3.50
12.68
17.40
5.00
6.50
5.00
4.50

2.50
4.00
3.50
8.19
13.3
5.50
1.50
10.15
7.80
2.50
10.70
3.60
25.10
1.66
4.16
5.30
6.38
3.55
9.51
3.60
6.00
2.50
3.00
3.50
6.20
16.60
53.30
4.00
1.63
3.50
3.80
10.20
3.74
10.40
3.20
5.50
0.91
2.45
4.00
5.00
7.50
6.00
9.06
4.50
5.00

8.00
5.00
5.00
10.00
13.8
4.20
5.30
75.70
9.10
17.50
8.70
8.70
8.70
23.30
16.60
16.60
17.50
12.50
15.40
8.00
50.50
17.50
24.50
10.50
10.50
5.00
40.80
7.00
11.50
12.00
10.00
73.00
35.00
70.00
6.50
80.00
8.00
10.00
12.00
28.00
63.00
18.60
12.00
115.00
80.00

7.31
6.73
4.03
5.61
7.23
6.17
NCS
4.00
5.97
7.59
11.22
4.50
4.62
NCS
4.44
4.56
5.70
4.00
6.20
3.95
6.21
6.99
4.69
5.00
6.91
NCS
7.93
4.64
6.93
8.58
3.50
4.18
7.52
3.50
4.63
3.92
3.00
4.27
NCS
5.10
6.13
7.35
8.67
6.14
NCS

5.84
3.70
4.14
3.50
4.95
5.31
4.14
11.91
5.50
3.79
4.84
3.81
5.60
4.90
10.40
8.88
5.80
6.91
3.50
5.45
5.00
6.24
4.15
5.01
6.31
4.00
5.50
2.93
4.13
3.85
3.96
2.91
2.90
3.80
4.76
8.70
4.38
4.00
3.69
2.50
8.50
4.83
8.33
5.42
3.25

3.80
6.00
NCSe
5.20
7.50
7.32
5.80
8.50
6.50
4.00
7.20
4.90
8.00
4.90
6.20
8.00
8.50
4.50
5.00
6.50
7.00
8.50
6.00
5.31
5.00
11.00
10.50
7.00
4.64
6.50
4.50
5.11
5.80
7.00
6.50
5.00
5.50
10.00
6.50
7.74
5.92
4.98
5.34
4.87
6.71

3
2, 3
3
2
2

2,
2,
2,
4,
2

4, 5
3
3
5

2
2
2, 4
5
5
2
2, 5
2
2
2
2

2
2, 5
2, 5
4

Potential exposure period in years.

Types of skin lesions: 1, raindrop pigmentation; 2, palmoplantar hyperkeratosis; 3, depigmentation; 4, hypopigmentation and 5,
hyperpigmentation.
c Arsenic content in drinking water.
d Frequency of MN/1000 cells.
e No cell scored.
b

36

A. Basu et al. / Mutation Research 516 (2002) 2940

Table 2
Arsenic content in water, nail, hair and urine and the frequencies of MN in exfoliated epithelial cells and blood lymphocytes from control
individuals
Code

Age/sex

ST83
ST86
ST88
ST99
ST100
ST102
ST103
ST111
ST112
ST113
ST114
ST93
ST101
ST109
ST110
ST84
ST105
ST106
ST108
ST92
ST107
a
b

35/M
25/M
40/M
30/M
56/F
40/F
30/M
37/M
32/M
24/M
33/M
60/M
27/F
40/F
37/M
19/M
59/M
23/M
23/M
40/M
23/M

As in watera
(g/l)

As in nail
(g/g)

As in hair
(g/g)

As in urine
(g/l)

MN/1000 cellsb
Urine

Buccal cells

Lymphocytes

5.30
6.00
5.00
3.00
4.20
3.60
3.00
3.30
4.00
5.00
5.00
3.00
3.50
3.50
3.00
10.00
8.00
12.00
7.00
8.00
10.00

0.76
1.03
0.63
0.55
0.52
0.50
0.48
0.42
0.37
0.45
0.50
0.36
0.55
0.38
0.40
0.45
0.24
0.31
0.61
0.80
0.42

0.17
0.53
0.56
0.34
0.33
0.35
0.30
0.38
0.27
0.36
0.32
0.19
0.35
0.31
0.33
0.43
0.21
0.30
0.35
0.50
0.36

5.00
7.00
9.00
2.00
7.00
2.00
5.00
3.00
3.50
4.00
4.20
8.00
4.00
2.50
2.30
7.00
7.00
3.00
5.00
4.00
8.00

1.55
0.55
0.25
0.25
1.00
0.50
0.00
1.00
2.00
0.00
0.25
0.45
0.50
0.30
1.00
0.25
1.00
0.50
1.00
0.25
0.25

0.50
0.25
1.00
0.00
0.50
0.90
0.25
0.25
0.30
1.50
0.50
1.00
0.50
1.90
1.00
0.80
0.00
0.90
1.80
1.50
0.90

0.45
1.00
1.00
0.00
0.30
0.75
0.25
0.00
0.50
0.25
1.00
1.00
0.50
0.40
0.40
1.09
0.30
0.25
0.25
1.00
0.50

Arsenic content in drinking water.

Frequency of MN/1000 cells.

controls. Exposed individuals on an average, exhibited

a 12-fold increase in the proportion of lymphocytes
with MN compared to that observed in control lymphocyte cultures, while the frequency of MN in oral
and urothelial cells of exposed individuals was higher
by 7 and 10 times, respectively of that observed in
controls. Our results are in agreement with the earlier report of Gonsebatt et al. [8] in two populations

(3035 individuals) inhabiting a region with endemic

hydro-arsenicism in Mexico. In their study, the exposed population consumed water with mean arsenic
concentration of 408.17 g/ml while the mean level
of arsenic in the drinking water of the control population was 29.88 g/ml. They observed a definite correlation between the arsenic content in drinking water
samples and urinary arsenic estimates. Our study

Table 3
Mean values of the arsenic content in water, nail, hair and urine, and the frequency of MN in exfoliated epithelial cells and blood
lymphocytes of the symptomatic and control individuals
Biomarkers/MN

Mean S.D. (control individuals)

As
As
As
As

5.49
0.51
0.34
4.88

in
in
in
in

drinking water (g/l)

nail (g/g)
hair (g/g)
urine (g/l)

MN/1000 urothelial cells

MN/1000 oral cells
MN/1000 lymphocytes

P < 0.01.

2.62
0.18
0.09
2.12

(n
(n
(n
(n

= 21)
= 21)
= 21)
= 21)

0.56 0.45 (n = 21)

0.77 0.53 (n = 21)
0.53 0.34 (n = 21)

Mean S.D. (symptomatic individuals)

368.11
12.58
6.97
24.45

275.75 (n = 45)
12.66 (n = 45)
8.23 (n = 45)
26.07 (n = 45)

5.74 1.73 (n = 40)

5.15 1.99 (n = 45)
6.39 1.65 (n = 44)

A. Basu et al. / Mutation Research 516 (2002) 2940

37

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A. Basu et al. / Mutation Research 516 (2002) 2940

Fig. 7. Photographs of MN in urothelial cells of arsenic exposed

symptomatic individuals (a) and (b).

subjects are inhabitants of different districts with a

wide range of arsenic exposure. As all our samples
were collected from hospital patients, most of the
subjects were already aware of their chronic arsenic
exposure problem. Consequently some of them had
switched over to safer, uncontaminated or less contaminated sources of drinking water before our sample collection. Hence, correlation between the arsenic
content in drinking water sample and corresponding
urinary arsenic concentration may be lacking in such
cases. However, the arsenic concentration in their nails
and hair is high due to the long duration of exposure.
Unlike previous works [8], our results are not supportive of higher MN frequencies in males than in females. The heterogeneity and ex-user status of some
of the study subjects could be its probable cause. Although MN levels in lymphocytes of both exposed and
control individuals in our present study were much

Fig. 8. Photographs of MN in lymphocytes of arsenic affected

symptomatic individuals (a) and (b).

less compared to those previously reported for Andean populations [13] but the MN frequencies in oral
mucosa and urothelial cells in our populations were
much higher than the Mexican populations reported by
Gonsebatt et al. [8]. Despite the higher exposure to arsenic through drinking water in West Bengal the total
arsenic levels in urine is also lower in our population
relative to the levels in Andean population studied by
Vahter et al. [26] and Dulout et al. [13]. Further studies
are in progress in our laboratory using samples from
populations living in the same area and exploiting the
same water source to bring homogeneity to establish
dose-response relationship.
There are reports of inter-individual variations in
susceptibility to clastogenic and co-clastogenic effects
of arsenic in lymphocyte cultures [27,28]. We noted
variations in clastogenic effects among members
of the same family (code no. ST38ST45) exposed

A. Basu et al. / Mutation Research 516 (2002) 2940

to the same concentration of arsenic (800 g/l) for

similar duration. Clastogenic effects varied among
tissues also. Due to the longer life-span of lymphocytes compared to epithelial cells, the MN observed
in this tissue should not necessarily be correlated with
the MN observed in cells that have different turnover
rates [15]. The lack of correlation between urothelial
and buccal epithelial cells could be due to different
target tissue and individual sensitivity [8].
During the past 30 years there has been heavy
ground water withdrawal for irrigation in West
Bengal. This may have mobilised phosphate derived
from fertilisers and from the decay of natural organic
materials. The increase in phosphate concentration
could promote arsenic leaching from its source causing natural groundwater arsenic contamination [29].
Malnutrition, poor socio-economic conditions and
illiteracy have aggravated the arsenic toxicity in the
exposed population in West Bengal. The prevalence
of high frequencies of MN in the studied target cells
among arsenic exposed individuals calls for immediate remedial and preventive measures against arsenic
induced carcinogenicity. The vast surface and rain
water resource of West Bengal should be judiciously
used to combat the menace of arsenic toxicity. Our
study is one of the earliest reports of the assessment
of the cytogenetic damage in the symptomatic individuals exposed to arsenic through drinking water
from West Bengal, India. Further studies are required
to determine the extent of genetic damage induced
by arsenic through drinking water particularly from
this arsenic affected region of West Bengal which is
regarded as the biggest arsenic calamity in the world.
Acknowledgements
Authors are extremely grateful to all the staff
members of the Skin Outpatient Department, School
of Tropical Medicine for their kind co-operation
and help to collect the biological samples from the
arsenic skin lesion patients. Authors are also grateful to the Director, Indian Institute of Chemical
Biology for his kind help and co-operation to continue this study. This study was partly supported by
grants the National Swedish Environment Protection
Agency as well as the Commission of the European
Community, 5th Framework Programme, Contract
No. QLK-1999-01142.

39

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