1-s2.0-S1383571802000141-main

Mutation Research 516 (2002) 29–40

Enhanced frequency of micronuclei in individuals exposedto arsenic through drinking water in West Bengal, India

A. Basua, J. Mahataa, A.K. Royb, J.N. Sarkarb, G. Poddarb, A.K. Nandyb,P.K. Sarkarb, P.K. Duttac, A. Banerjeea, M. Dasd, K. Raya, S. Roychaudhurya,

A.T. Natarajane, R. Nilssonf , 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, Indiab School of Tropical Medicine, Calcutta 700073, West Bengal, India

c Calcutta Medical College, Calcutta 700073, West Bengal, Indiad Department of Zoology, University of Calcutta, Calcutta 700019, India

e Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Leiden, The Netherlandsf 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 districtscovering 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 throughdrinking water. Forty five individuals with cutaneous signs of arsenicism from four affected districts (368.11�g/l of As indrinking water) were considered as the exposed group and 21 healthy individuals with no symptoms of arsenic poisoning andresiding in two unaffected districts (5.49�g/l of As) were considered as controls. The exposed and control groups had similarage distribution and socioeconomic status. Standardised questionnaires were utilised and medical examination was conductedto ascertain exposure history, sociodemographic characteristics, diet, health, medication, addiction and chief symptoms in thestudy participants. Arsenic exposure was confirmed by measuring the arsenic content in the drinking water, nails, hair andurine samples from the volunteers. Arsenic contents in the urine, nail and hair in the exposed group were 24.45�g/l, 12.58 and6.97�g/g, respectively which were significantly high in comparison to corresponding control group values of 4.88�g/l, 0.51and 0.34�g/g, respectively. Exposed individuals showed a statistically significant increase in the frequency of MN in oralmucosa, 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 arsenicthrough 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

∗ Corresponding author. Tel.:+91-33-473-0492/6793;fax: +91-33-473-5197.E-mail addresses: [email protected], [email protected](A.K. Giri).

1. Introduction

Incidents of arsenic contamination in the groundwater have been reported from widespread areasthrough out the world such as Taiwan, Mexico, Chile,

1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S1383-5718(02)00014-1

30 A. Basu et al. / Mutation Research 516 (2002) 29–40

Argentina, Thailand, Bangladesh and India. Minorcases of chronic arsenic toxicity have been reportedin Poland, USA (Minnesota and California), Canada(Ontario), Hungary and Japan. Arsenic in ground-water has been found to be above the WHO permis-sible limit in seven districts of West Bengal, India,encompassing an area of 37,493 km2, with concen-trations ranging from 200 to 800�g/l. More than200,000 people have already shown different typesof arsenical skin lesions [1]. It has been regarded asthe biggest arsenic calamity in the world [2]. How-ever, reports on the cytogenetic survey of arsenicexposed population in West Bengal are very lim-ited. The pollution by naturally occurring arsenic ofalluvial Ganges aquifers which are used for publicwater supply in West Bengal has become a seri-ous threat to human life. Most of the contaminatedwater samples are found to contain a mixture of ar-senite and arsenate [1]. All these water samples areobtained from underground sources and not fromsurface or rainwater. Chronic ingestion of high lev-els of arsenic in drinking water is associated withincreased incidence of cancer at various sites, suchas skin, lung, bladder and other internal organs [3].A cross-sectional survey conducted in 1995–1996 toinvestigate arsenic-associated skin lesions reported ahigher incidence of keratosis and hyperpigmentationin West Bengal [4].

Many human epidemiological studies on arsenicpoisoning are available. Recently we have reviewedand updated the mutagenic and genotoxic effects ofarsenic. It has been found that arsenic is not mutagenicbut genotoxic in both animal and human systems[5]. As far as mutagenicity of arsenic is consideredit appears to be largely non-mutagenic in bacterialand standard mammalian cell mutation assays whichmeasure mutation at single gene locus [6]. It caninduce DNA damage in multiple test systems [7].Almost all the results of in vitro assays of chromo-somal aberrations (CAs), sister chromatid exchanges(SCEs) and micronuclei (MN) formation induced byarsenic in mammalian cells showed positive clasto-genic effects [8,9]. Studies on cytogenetic assays inpopulations with arsenic exposure clearly indicatepositive genotoxic effects on human lymphocytesin vivo [10]. The majority of exposed populationswho had a history of relatively long periods of ex-posure showed higher incidences of CAs [11], SCEs

[12] and MN formation [13]. Significantly increasedrates of CAs and MN formation were also found inexfoliated cells of oral mucosa and urinary bladder[8,14].

Considering the widespread reports of arsenic in-duced carcinogenicity in human beings, we recognisethe need to biomonitor the genotoxic effects of arsenicand its compounds in human beings exposed to ar-senic through drinking water. We have studied the MNformation in oral mucosa cells, urothelial cells and inlymphocytes in the symptomatic individuals exposedto arsenic through drinking water in West Bengal, In-dia as this cohort represents a unique population in theworld for such study.

2. Materials and methods

2.1. Study area and study subjects

Out of the seven arsenic affected districts of WestBengal, 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), whoshowed cutaneous signs of arsenicism like hyperpig-mentation, hypopigmentation, palmoplantar hyperk-eratosis, raindrop pigmentation or ulcerative lesionsand were inhabitants of the study area were selectedfor the exposed group from the Outpatient Depart-ment at the School of Tropical Medicine, Calcutta,India. Figs. 2–5 present photographs of some arseni-cal skin lesions from arsenic exposed individuals.Twenty-one individuals (17 men and 4 women) withno symptoms of arsenic poisoning and residing in theunaffected districts of West Bengal (Howrah, Midna-pur) were selected as controls (Fig. 1). The exposedand control groups had similar socioeconomic status.The distribution of age (between 15 and 60 years)among both the groups was similar. Each volunteerwas interviewed about smoking habits, consump-tion of alcohol and chewing of tobacco, etc. Thepurpose of the questionnaire was to select healthyindividuals from the exposed and control groups sothat confounding factors such as medical treatment,smoking, alcoholism, and chronic disease could beeliminated.

A. Basu et al. / Mutation Research 516 (2002) 29–40 31

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

2.2. Biomarkers used

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

senic concentration is also considered to be a goodparameter for assessing recent arsenic exposure [14].Genetic toxicology endpoints have also been utilisedas biomarkers. The frequency of MN observed in theexfoliated cells of oral mucosa and urinary bladderis used as an index to monitor the genetic damage

32 A. Basu et al. / Mutation Research 516 (2002) 29–40

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 con-tact with the carcinogen. The bladder cell MN assayhas been suggested to be one of the most appropriatebiological marker of arsenic genotoxicity [15]. Blad-der cell MN reflect damage to the urothelial tissuewhich occurs∼1–3 weeks prior to the exfoliated cellsappearing in urine [16]. The cytokinesis-block MN(CBMN) technique in lymphocyte culture is widelyregarded as a sensitive and reliable method for assess-ing chromosome damage [17]. Hence, for the inves-tigation of exposure to arsenic, samples of drinkingwater, hair, nails, urine and blood were collected.

2.3. Chemicals

RPMI-1640, foetal calf serum, phytohemaglutinin(M form), l-glutamine, penicillin, streptomycin werepurchased from Gibco BRL (USA), cytochalasinB, Tris–HCl 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) andhair (∼300 mg) were collected, coded and sent to the

Fig. 3. Hyper and hypopigmentation anterior chest wall (raindroppigmentation). Bowen’s disease (carcinoma in situ) medial andupper part of left nipple, guttate melanosis (like black mole) upperpart of left side of chest.

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

A. Basu et al. / Mutation Research 516 (2002) 29–40 33

Fig. 5. Classical raindrop pigmentation with plantar hyperkerato-sis—more prominent on heel.

Analytical Chemistry Department, School of Trop-ical Medicine, Calcutta for estimation of arsenic.The water samples were collected in plastic bottlespre-washed with nitric acid–water (1+ 1) and aftercollection nitric acid (1.0 ml/l) was added as preserva-tive [18]. Nail and hair samples were collected usingceramic blade cutters [2]. Both the samples werethoroughly washed with double distilled water andacetone and then processed [2]. Hair samples were ofsimilar size and were taken from more or less similarregion of head (close to the scalp behind the ear witha diameter of about 1 cm) [19].

2.5. Urine sampling

Subjects were supplied with pre-coded polypropy-lene bottles for urine collection. First morning voidswere collected and sent to the Analytical ChemistryDepartment, School of Tropical Medicine, Calcutta for

arsenic estimation. This gives the best measure of thecurrent arsenic exposure [14]. Approximately 50 mlof urine samples (spot samples) were also collected,coded, kept at 2–4◦C in a cooling device and im-mediately brought to the laboratory, where they werecentrifuged to obtain the bladder cells (2000 rpm for15 min) and further processed for MN assay. Firstmorning voids were not used for MN assay becauseexfoliated cells tend to degrade from overnight expo-sure to urine [20]. Conc. HCl (1 ml/100 ml urine) wasadded in the urine samples to prevent bacterial growth.

2.6. Blood sampling

Approximately 4–5 ml of blood samples were ob-tained from each individual by venipuncture in hep-arinised sterile conical plastic tubes. The samples werecoded, kept at 4◦C in a cooling device and brought tothe laboratory where they were immediately processedfor lymphocyte culture.

2.7. Buccal and urothelial exfoliated cells

Buccal cells were obtained by scraping the insideof the mouth (both cheeks) with a toothbrush. Blad-der cells were recovered by centrifuging urine sam-ples, as mentioned previously. The cells were washedthree times by centrifugation at 1500 rpm for 10 minin a buffer solution consisting 0.1 M EDTA, 0.01 MTris–HCl and 0.02 M NaCl (pH 7.0). Volumes of 25 mlof the buffer solution in a 50 ml conical tube wasused in every washing step. Fifty microlitres of thecell suspension was laid and spread well on clean,pre-heated (40◦C) glass slides and allowed to air-dryfor 5–10 min. The slides were fixed in methanol (80%(v/v)) at 0◦C for 20 min and air dried [21]. Buccalcells were analysed following the method of Tolbertet al. [22] and urothelial cells were analysed accord-ing to Reali et al. [23]. At least 1000 cells were scoredper individual.

2.8. Lymphocyte culture and harvesting

Lymphocytes were cultured and MN assay wasconducted following the method of Fenech [17] andMigliore et al. [24]. Replicate lymphocyte cultureswere prepared. A volume of 0.5 ml of blood wereplaced in sterile conical plastic tubes containing 6 ml

34 A. Basu et al. / Mutation Research 516 (2002) 29–40

of RPMI-1640 medium supplemented with foetal calfserum, phytohemaglutinin,l-glutamine and antibi-otics. The samples were incubated for 72 h at 37◦C.At 44 h of incubation, cytochalasin B (at final con-centration of 3�g/ml) was added to each culture toinhibit cytokinesis. After 72 h, the cells were cen-trifuged at approximately 1000 rpm for 5 min. Culturemedia was removed and cells were treated for 5 minwith a weak hypotonic solution (0.075 M KCl:saline,1:9). After centrifugation the cells were fixed in freshfixative (methanol:glacial acetic acid, 3:1). Fixativewas removed by centrifugation and two more changesof fixative were performed. Samples for microscopicobservations were obtained by carefully droppingcell suspension from a Pasteur pipette onto wet cleanslides. For analysing MN, the slides were air-dried,stained with aqueous Giemsa and at least 2000 binu-cleated cells were scored under the microscope.

2.9. Arsenic analysis in nails, hair, water and urine

Flow injection-hydride generation-atomic absorp-tion spectrometry (FI-HG-AAS) was used for arsenicanalysis in various samples. A Perkin-Elmer modelanalyst 100, Fias 100 AAS was utilised for the pur-pose. Nails, hair and urine samples were subjected tonitric acid and perchloric acid pre-treatment for diges-tion [25]. Water was not subjected to any chemicaltreatment before analysis.

3. Statistical analysis

Results of the estimated arsenic content in water,nail, hair and urine of the symptomatic individualswere compared with respective controls by pairedStudent’st-test. MN assays were also analysed usingpaired Student’st-test and the level of significance ispresented in the Table 3.

4. Results

Data on the total arsenic content in drinking wa-ter, and in the nails, hair and urine of the exposedindividuals and frequencies of MN in exfoliated cellsas well as the lymphocytes of symptomatic individu-als are presented in Table 1. The same parameters in

the control group are presented in Table 2. Table 3shows the summary of all the results with statisticalinterpretation. Mean arsenic contents of nail, hair andurine were significantly high in the symptomatic in-dividuals when compared with the arsenic content inthe individuals of non-affected area. Similarly the ar-senic content in the water samples was also signifi-cantly higher in the arsenic contaminated area whencompared with non-affected area. The arsenic contentin the water samples of the individuals code numbersST38, ST39 and ST41–ST45 were same since theseindividuals belong to the same family.

4.1. MN in exfoliated epithelial cells

A comparison was made between the MN fre-quency in urothelial cells of 40 exposed (26 males and14 females) individuals and 21 control (17 males and4 females) volunteers. For oral mucosa cells, the inci-dence of MN in 45 (30 males and 15 females) buccalsmears were compared with that in 21 (17 males and4 females) control buccal smears. Individuals livingin the different arsenic affected districts and showingarsenic induced skin lesions had higher frequencies ofmicronucleated cells than those residing in the unaf-fected districts. This was true for buccal and bladdercells. These differences were statistically significantusing a Student’st-test (P < 0.01).

4.2. MN in lymphocytes

A similar analysis was carried out with the dataobtained in lymphocytes. Twenty-one successful lym-phocyte cultures were analysed in the control groupand 44 in the exposed group. There was a statisticallysignificant increase in the incidence of MN in lym-phocytes of exposed individuals when compared tocontrols.

5. Discussion

Figs. 6–8 present the photographs of MN in oralmucosa cells, urothelial cells and lymphocytes, re-spectively of arsenic exposed individuals. Symp-tomatic individuals showed a statistically significantincrease in the frequency of MN in oral mucosa,urothelial cells and lymphocytes when compared with

A. Basu et al. / Mutation Research 516 (2002) 29–40 35

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

Code Age/sex Exp pera Skinlesionsb

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

ST1 30/F 10 2, 3 25 5.46 2.50 8.00 7.31 5.84 3.80ST3 46/M 10 1, 2, 3 340 4.50 4.00 5.00 6.73 3.70 6.00ST4 35/M 16 1, 3 490 0.87 3.50 5.00 4.03 4.14 NCSe

ST5 48/M 20 1, 2 250 2.41 8.19 10.00 5.61 3.50 5.20ST7 49/M 15 1, 2 200 30.90 13.3 13.8 7.23 4.95 7.50ST10 32/M 10 2 40 2.00 5.50 4.20 6.17 5.31 7.32ST 11 38/M 15 5 60 1.20 1.50 5.30 NCS 4.14 5.80ST14 17/M 10 1, 2, 4, 5 800 40.17 10.15 75.70 4.00 11.91 8.50ST16 22/M 4 1, 2, 3 380 26.60 7.80 9.10 5.97 5.50 6.50ST17 43/M 4 1, 2, 3 200 1.83 2.50 17.50 7.59 3.79 4.00ST20 35/F 12 2, 4, 5 370 12.50 10.70 8.70 11.22 4.84 7.20ST22 20/M 10 1, 2 375 2.00 3.60 8.70 4.50 3.81 4.90ST24 22/F 12 2 50 29.10 25.10 8.70 4.62 5.60 8.00ST26 22/M 8 1 200 16.20 1.66 23.30 NCS 4.90 4.90ST27 58/M 20 2 710 4.46 4.16 16.60 4.44 10.40 6.20ST28 16/M 8 1, 2 200 24.30 5.30 16.60 4.56 8.88 8.00ST29 17/M 8 1, 2 200 33.08 6.38 17.50 5.70 5.80 8.50ST30 16/F 6 1, 2, 4 15 2.17 3.55 12.50 4.00 6.91 4.50ST33 40/F 12 1 180 10.90 9.51 15.40 6.20 3.50 5.00ST35 30/M 8 2, 5 290 5.50 3.60 8.00 3.95 5.45 6.50ST38 28/M 12 1, 5 800 27.20 6.00 50.50 6.21 5.00 7.00ST39 16/M 10 1, 2 800 52.40 2.50 17.50 6.99 6.24 8.50ST41 15/M 10 1, 2, 5 800 12.80 3.00 24.50 4.69 4.15 6.00ST42 16/M 14 5 800 15.50 3.50 10.50 5.00 5.01 5.31ST43 22/F 10 1, 2 800 26.20 6.20 10.50 6.91 6.31 5.00ST44 35/F 15 1, 2 800 38.80 16.60 5.00 NCS 4.00 11.00ST45 33/F 15 1, 2 800 18.00 53.30 40.80 7.93 5.50 10.50ST46 35/F 13 1, 2 70 3.50 4.00 7.00 4.64 2.93 7.00ST47 26/M 5 3 100 4.60 1.63 11.50 6.93 4.13 4.64ST48 23/M 5 1 100 2.80 3.50 12.00 8.58 3.85 6.50ST49 26/M 12 1 70 6.70 3.80 10.00 3.50 3.96 4.50ST51 37/M 11 1 800 26.70 10.20 73.00 4.18 2.91 5.11ST52 35/F 12 1 360 2.33 3.74 35.00 7.52 2.90 5.80ST53 35/M 12 1 800 6.40 10.40 70.00 3.50 3.80 7.00ST54 33/M 20 1 360 2.70 3.20 6.50 4.63 4.76 6.50ST58 18/M 10 1 70 3.50 5.50 80.00 3.92 8.70 5.00ST60 39/M 7 2 260 1.44 0.91 8.00 3.00 4.38 5.50ST71 34/F 15 1 710 4.00 2.45 10.00 4.27 4.00 10.00ST81 20/M 2 1 150 3.50 4.00 12.00 NCS 3.69 6.50ST89 37/M 6 1, 2 340 12.68 5.00 28.00 5.10 2.50 7.74ST90 15/F 6 1 340 17.40 7.50 63.00 6.13 8.50 5.92ST117 25/F 15 1, 2, 5 350 5.00 6.00 18.60 7.35 4.83 4.98ST118 40/F 22 1, 2, 5 400 6.50 9.06 12.00 8.67 8.33 5.34ST119 35/F 15 1, 4 140 5.00 4.50 115.00 6.14 5.42 4.87ST120 41/M 15 5 170 4.50 5.00 80.00 NCS 3.25 6.71

a Potential exposure period in years.b 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.

36 A. Basu et al. / Mutation Research 516 (2002) 29–40

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

Code Age/sex 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

ST83 35/M 5.30 0.76 0.17 5.00 1.55 0.50 0.45ST86 25/M 6.00 1.03 0.53 7.00 0.55 0.25 1.00ST88 40/M 5.00 0.63 0.56 9.00 0.25 1.00 1.00ST99 30/M 3.00 0.55 0.34 2.00 0.25 0.00 0.00ST100 56/F 4.20 0.52 0.33 7.00 1.00 0.50 0.30ST102 40/F 3.60 0.50 0.35 2.00 0.50 0.90 0.75ST103 30/M 3.00 0.48 0.30 5.00 0.00 0.25 0.25ST111 37/M 3.30 0.42 0.38 3.00 1.00 0.25 0.00ST112 32/M 4.00 0.37 0.27 3.50 2.00 0.30 0.50ST113 24/M 5.00 0.45 0.36 4.00 0.00 1.50 0.25ST114 33/M 5.00 0.50 0.32 4.20 0.25 0.50 1.00ST93 60/M 3.00 0.36 0.19 8.00 0.45 1.00 1.00ST101 27/F 3.50 0.55 0.35 4.00 0.50 0.50 0.50ST109 40/F 3.50 0.38 0.31 2.50 0.30 1.90 0.40ST110 37/M 3.00 0.40 0.33 2.30 1.00 1.00 0.40ST84 19/M 10.00 0.45 0.43 7.00 0.25 0.80 1.09ST105 59/M 8.00 0.24 0.21 7.00 1.00 0.00 0.30ST106 23/M 12.00 0.31 0.30 3.00 0.50 0.90 0.25ST108 23/M 7.00 0.61 0.35 5.00 1.00 1.80 0.25ST92 40/M 8.00 0.80 0.50 4.00 0.25 1.50 1.00ST107 23/M 10.00 0.42 0.36 8.00 0.25 0.90 0.50

a Arsenic content in drinking water.b Frequency of MN/1000 cells.

controls. Exposed individuals on an average, exhibiteda 12-fold increase in the proportion of lymphocyteswith MN compared to that observed in control lym-phocyte cultures, while the frequency of MN in oraland urothelial cells of exposed individuals was higherby 7 and 10 times, respectively of that observed incontrols. Our results are in agreement with the ear-lier report of Gonsebatt et al. [8] in two populations

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

Biomarkers/MN Mean± S.D. (control individuals) Mean± S.D. (symptomatic individuals)

As in drinking water (�g/l) 5.49 ± 2.62 (n = 21) 368.11± 275.75∗∗ (n = 45)As in nail (�g/g) 0.51± 0.18 (n = 21) 12.58± 12.66∗∗ (n = 45)As in hair (�g/g) 0.34± 0.09 (n = 21) 6.97± 8.23∗∗ (n = 45)As in urine (�g/l) 4.88 ± 2.12 (n = 21) 24.45± 26.07∗∗ (n = 45)

MN/1000 urothelial cells 0.56± 0.45 (n = 21) 5.74± 1.73∗∗ (n = 40)MN/1000 oral cells 0.77± 0.53 (n = 21) 5.15± 1.99∗∗ (n = 45)MN/1000 lymphocytes 0.53± 0.34 (n = 21) 6.39± 1.65∗∗ (n = 44)

∗∗ P < 0.01.

(30–35 individuals) inhabiting a region with endemichydro-arsenicism in Mexico. In their study, the ex-posed population consumed water with mean arsenicconcentration of 408.17�g/ml while the mean levelof arsenic in the drinking water of the control popula-tion was 29.88�g/ml. They observed a definite corre-lation between the arsenic content in drinking watersamples and urinary arsenic estimates. Our study

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Fig. 7. Photographs of MN in urothelial cells of arsenic exposedsymptomatic individuals (a) and (b).

subjects are inhabitants of different districts with awide range of arsenic exposure. As all our sampleswere collected from hospital patients, most of thesubjects were already aware of their chronic arsenicexposure problem. Consequently some of them hadswitched over to safer, uncontaminated or less con-taminated sources of drinking water before our sam-ple collection. Hence, correlation between the arseniccontent in drinking water sample and correspondingurinary arsenic concentration may be lacking in suchcases. However, the arsenic concentration in their nailsand hair is high due to the long duration of exposure.Unlike previous works [8], our results are not sup-portive of higher MN frequencies in males than in fe-males. The heterogeneity and ex-user status of someof the study subjects could be its probable cause. Al-though MN levels in lymphocytes of both exposed andcontrol individuals in our present study were much

Fig. 8. Photographs of MN in lymphocytes of arsenic affectedsymptomatic individuals (a) and (b).

less compared to those previously reported for An-dean populations [13] but the MN frequencies in oralmucosa and urothelial cells in our populations weremuch higher than the Mexican populations reported byGonsebatt et al. [8]. Despite the higher exposure to ar-senic through drinking water in West Bengal the totalarsenic levels in urine is also lower in our populationrelative to the levels in Andean population studied byVahter et al. [26] and Dulout et al. [13]. Further studiesare in progress in our laboratory using samples frompopulations living in the same area and exploiting thesame water source to bring homogeneity to establishdose-response relationship.

There are reports of inter-individual variations insusceptibility to clastogenic and co-clastogenic effectsof arsenic in lymphocyte cultures [27,28]. We notedvariations in clastogenic effects among membersof the same family (code no. ST38–ST45) exposed

A. Basu et al. / Mutation Research 516 (2002) 29–40 39

to the same concentration of arsenic (800�g/l) forsimilar duration. Clastogenic effects varied amongtissues also. Due to the longer life-span of lympho-cytes compared to epithelial cells, the MN observedin this tissue should not necessarily be correlated withthe MN observed in cells that have different turnoverrates [15]. The lack of correlation between urothelialand buccal epithelial cells could be due to differenttarget tissue and individual sensitivity [8].

During the past 30 years there has been heavyground water withdrawal for irrigation in WestBengal. This may have mobilised phosphate derivedfrom fertilisers and from the decay of natural organicmaterials. The increase in phosphate concentrationcould promote arsenic leaching from its source caus-ing natural groundwater arsenic contamination [29].Malnutrition, poor socio-economic conditions andilliteracy have aggravated the arsenic toxicity in theexposed population in West Bengal. The prevalenceof high frequencies of MN in the studied target cellsamong arsenic exposed individuals calls for immedi-ate remedial and preventive measures against arsenicinduced carcinogenicity. The vast surface and rainwater resource of West Bengal should be judiciouslyused to combat the menace of arsenic toxicity. Ourstudy is one of the earliest reports of the assessmentof the cytogenetic damage in the symptomatic indi-viduals exposed to arsenic through drinking waterfrom West Bengal, India. Further studies are requiredto determine the extent of genetic damage inducedby arsenic through drinking water particularly fromthis arsenic affected region of West Bengal which isregarded as the biggest arsenic calamity in the world.

Acknowledgements

Authors are extremely grateful to all the staffmembers of the Skin Outpatient Department, Schoolof Tropical Medicine for their kind co-operationand help to collect the biological samples from thearsenic skin lesion patients. Authors are also grate-ful to the Director, Indian Institute of ChemicalBiology for his kind help and co-operation to con-tinue this study. This study was partly supported bygrants the National Swedish Environment ProtectionAgency as well as the Commission of the EuropeanCommunity, 5th Framework Programme, ContractNo. QLK-1999-01142.

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