Comparison of health effects between individuals with and without ...

Journal of Exposure Science and Environmental Epidemiology (2007) 17, 215–223 r 2007 Nature Publishing Group All rights reserved 1559-0631/07/$30.00

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Comparison of health effects between individuals with and without skin lesions in the population exposed to arsenic through drinking water in West Bengal, India PRITHA GHOSHa, MAYUKH BANERJEEa, SUJATA DE CHAUDHURIa, RAJDEEP CHOWDHURYa, JAYANTA K. DASb, ANGSHUMAN MUKHERJEEc, AJOY K. SARKARd, LAKSHMIKANTA MONDALe, KALIPADA BAIDYAe, TANMOY JYOTI SAUf, APURBA BANERJEEa, ARINDAM BASUa, KEYA CHAUDHURIa, KUNAL RAYa AND ASHOK K. GIRIa a

Division of Molecular and Human Genetics, Indian Institute of Chemical Biology, Kolkata, India Department of Dermatology, West Bank Hospital, Howrah, India c Department of Neurology, Vivekananda Institute of Medical Sciences, Ramakrishna Mission Seva Pratishthan, Kolkata, India d Peerless Hospital and B.K Roy Research Centre, Kolkata, India e Regional Institute of Ophthalmology, Calcutta Medical College Campus, Kolkata, India f Department of Medicine, Calcutta National Medical College, Kolkata, India b

A study was conducted to explore the effect of arsenic causing conjunctivitis, neuropathy and respiratory illness in individuals, with or without skin lesions, as a result of exposure through drinking water, contaminated with arsenic to similar extent. Exposed study population belongs to the districts of North 24 Parganas and Nadia, West Bengal, India. A total of 725 exposed (373 with skin lesions and 352 without skin lesions) and 389 unexposed individuals were recruited as study participants. Participants were clinically examined and interviewed. Arsenic content in drinking water, urine, nail and hair was estimated. Individuals with skin lesion showed significant retention of arsenic in nail and hair and lower amount of urinary arsenic compared to the group without any skin lesion. Individuals with skin lesion also showed higher risk for conjunctivitis ((odd’s ratio) OR: 7.33, 95% CI: 5.05–10.59), peripheral neuropathy (OR: 3.95, 95% CI: 2.61–5.93) and respiratory illness (OR: 4.86, 95% CI: 3.16–7.48) compared to the group without any skin lesion. The trend test for OR of the three diseases in three groups was found to be statistically significant. Again, individuals without skin lesion in the exposed group showed higher risk for conjunctivitis (OR: 4.66, 95% CI: 2.45–8.85), neuropathy (OR: 3.99, 95% CI: 1.95–8.09), and respiratory illness (OR: 3.21, 95% CI: 1.65–6.26) when compared to arsenic unexposed individuals. Although individuals with skin lesions were more susceptible to arsenicinduced toxicity, individuals without skin lesions were also subclinically affected and are also susceptible to arsenic-induced toxicity and carcinogenicity when compared to individuals not exposed to arsenic. Journal of Exposure Science and Environmental Epidemiology (2007) 17, 215–223. doi:10.1038/sj.jes.7500510; published online 12 June 2006

Keywords: arsenic, drinking water, skin lesions, neuropathy, respiratory diseases, eye problem.

Introduction Arsenic contamination is a major health problem of global concern. In West Bengal, India, more than 7 million people, encompassing an area of 38,865 sq km, are exposed to arsenic through drinking water. Arsenic content in the drinking water of these areas is much higher than the acceptable limit of 10 mg/l (WHO, 1996; Frost et al., 2003). More than 300,000 people show arsenic-induced skin lesions 1 Address all correspondence to: Dr. Ashok K. Giri, Molecular and Human Genetics Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India. Tel: þ 91 33 2473 3491/0492/6793; Fax: þ 91 33 2473 5197, 91 33 2472 3967. E-mail: [email protected]; [email protected] Received 21 March 2006; accepted 19 May 2006; published online 12 June 2006

(Chakraborti et al., 2002) and a larger number of people are at risk. This is regarded as the greatest arsenic calamity in the world. Epidemiological investigations have demonstrated associations between arsenic ingestion and hyper-pigmentation, keratosis of skin, anemia, burning sensation of the eyes, solid edema of the legs, liver fibrosis, chronic lung disease, gangrene of the toes (Blackfoot disease) and neuropathy (Guha Mazumder et al., 2001) as also, cancers of skin, lung, liver, bladder, kidney, and prostate (Tseng 1977; Chen et al., 1985, 1992; Wu et al., 1989). Skin lesions are recognized as the most sensitive end points of chronic arsenicism. Investigations led to identification of raindrop pigmentation, hypo- and hyper- pigmentation and palmo-plantar hyperkeratosis, as the hallmark signs of chronic arsenic toxicity. These skin lesions generally develop with latency period spanning more than 10 years from first exposure (Haque et al., 2003), although the duration between first exposure

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Arsenic in drinking water and its health effects

Materials and methods

administrative blocks (Gaighata, Habra, Deganga and Baduria) of North 24 Parganas and two villages from Haringhata block of Nadia were selected for our study area (Figure 1). Our previous study already showed that drinking water was the principal source of arsenic exposure in this region (Ghosh et al., 2006). In brief, four trained volunteers were initially sent to the villages for door-to-door survey for identifying skin lesions among the villagers. All the villagers were requested to attend the medical camp irrespective of the presence of arsenic-induced skin lesions. Response rate was high (93% in North 24 Parganas and 92% in Nadia) among the participants. Unexposed subjects with no history of arsenic contaminated drinking water and age group between 15 and 70 years were selected from the East and West Midnapur districts. Response rate was 95% in case of unexposed individuals. Arsenic exposed and unexposed individuals were matched with age, sex and socio-economic status. Seven hundred and twenty-five individuals (373 with skin lesions, 352 without skin lesion) from arsenic exposed area and 389 individuals from unexposed area were recruited in this study. Individuals ranging from 15 to 70 years of age with at least 10 years of exposure were included as exposed study participants. Individuals within similar age group and no history of arsenic exposure were included as unexposed study participants. The entire study was conducted between January 2003 and May 2005. The non-physician interviewer, blind to the clinical status of the participants, interviewed them on the basis of a structured questionnaire that elicited information about lifetime residential history, occupation, diet, and smoking habit. The detailed information on current and lifetime smoking was obtained, including number of cigarettes or bidi smoked per day along with duration of smoking. No exsmoker was included in this study. Of The study population 10% was randomly reinterviewed in the field to check the accuracy of the information provided. Then, a team of expert physicians consisting of specialists in fields of dermatology, neurology, ophthalmology and respiratory and chest diseases, with at least 15 years of experience, interviewed each participant as regards the clinical aspects. All the study participants provided informed consent. Water, urine, nail, hair, and blood samples were collected from these subjects on the same day in respective coded containers. Information from questionnaire-sourced data from the individuals was not revealed to the technician who performed arsenic analysis. This study was conducted in accord with the Helsinki II Declaration and approved by the institutional ethics committee.

Study Design and Population The present work is a cross-sectional study involving individuals with arsenic exposure, from districts of North 24 Parganas and Nadia. These districts are severely affected by arsenic (Mandal et al., 1996). Five villages from four

Physical Examination of Skin A careful examination of skin was conducted under natural daylight to reveal the presence of typical arsenic-induced skin lesions like raindrop, hypo and hyper-pigmentation, palmoand planter-hyperkeratosis. Visible or palpable dermal

and skin lesions manifestation might be as low as 6 months. The appearance of skin lesions depends on the concentration of arsenic in drinking water, volume of intake and the health and nutritional status of individuals (Rahman et al., 2001). Arsenicism is a complex disease involving multisystem disorder. Prolonged arsenic ingestion leads to its accumulation in the liver, kidneys, heart and lungs and in smaller amounts in the muscles, nervous system, gastrointestinal tract and spleen (Benramdane et al., 1999). Respiratory disease is also common in arsenic toxicity (Mazumder et al., 2000). Recently, ophthalmologist from our group has also showed association of persistent conjunctivitis with arsenic contaminated drinking water (Baidya et al., 2006). A delayed sensorimotor peripheral neuropathy may occur after acute arsenic poisoning. Commonly reported symptoms include numbness, tingling and ‘‘pins and needles’’ sensation in hands and feet, often in symmetrical ‘‘stocking glove’’ distribution, and muscular tenderness in the extremities (Mukherjee et al., 2003). Clinical involvement spans the spectrum from mild parasthesia to distal weakness, quadriplegia and in rare instances, respiratory muscle insufficiency. It has been shown that, respiratory disease is more common in patients with characteristic skin lesions of arsenic toxicity (Guha Mazumder, 2003). Ingestion of inorganic arsenic results in chronic pulmonary effects, as manifested by, cough, chest sound in lungs and shortness of breath. We have been working on the assessment of the genotoxic effects of arsenic in the exposed population in West Bengal (Basu et al., 2001, 2002, 2004; Mahata et al., 2003). Presence of arsenic specific skin lesions along with arsenic estimation in water and other biological samples serve as the excellent marker for identifying affected individuals. However, not all individuals drinking arsenic contaminated water develop skin lesions. Although a large number of individuals are exposed to arsenic through drinking water but only 15% to 20% individuals show arsenic-induced skin lesions (Ghosh et al., 2006). Considering the role of arsenic in the etiology of human cancer, we recognized the need to assess the prevalence of neuropathy, respiratory illnesses and eye problems in both the groups (i.e. with and without skin lesion) as a result of arsenic exposure. Attempts have also been made to compare the results with a group of unexposed individuals drinking minimum or no arsenic contaminated water.

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Figure 1. Map of West Bengal, India, study area, nine arsenic affected districts of West Bengal and neighboring country Bangladesh.

lesions were documented. Physical examination of skin identified three types of cancerous lesions, such as, squamous cell carcinoma, bowen’s disease and basal cell carcinoma. Two dermatologists jointly diagnosed any ‘‘probable or doubtful’’ skin lesion. The cases that still remained doubtful were not considered. Arsenic-induced skin lesions served as a biological marker of exposure. We divided our exposed population into two subpopulations that is individuals with arsenic-induced skin lesions and with no arsenic specific skin lesions. Journal of Exposure Science and Environmental Epidemiology (2007), 17(3)

Identification of Eye Problems Till date much effort has been given to dermatological and neurological complications due to arsenic toxicity. However, we also observed recurrent eye problems in our population. Individuals frequently reporting irritation, watering and redness in both eyes, actually prompted us to focus our attention towards studying the probable association of eye problems with arsenic intake. Conjunctivitis ultimately emerged as the most consistent eye symptom related to arsenic toxicity (Baidya et al., 2006), which has also been 217

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reported from Bangladesh population (Hossain et al., 2005). Cases having history of mucopurulent discharge (characteristic of bacterial conjunctivitis), history of severe watering and photophobia (characteristic of viral conjunctivitis), and history of severe itching and ropy discharge (characteristic of allergic conjunctivitis) were not considered.

Identification of Neurological Symptoms Observations were recorded by expert neurologist for items considered consistent with peripheral motor and sensory neuropathy and for other neurologic abnormalities as well. The criteria recorded for neurological problems were (i) pain and paraesthesias in stocking and glove distribution, (ii) numbness, (iii) weakness, and (iv) muscle cramp. The different signs of clinical neuropathy were anaesthesia or hypoaesthesia (no or reduced sensation) to touch, pain, temperature, pressure, vibration, calf tenderness, power and deep tendon reflexes. Criteria for the exclusion of neurological problem, not due to arsenic exposure, were followed as described earlier (Mukherjee et al., 2003). Electrophysiologic studies (Nerve Conduction Velocity (NCV) and Electromyograph (EMG) test) were performed to clinically confirm the probable as well as the doubtful cases, at National Neuroscience Centre, Kolkata. Respiratory Diseases Ingestion of inorganic arsenic results in chronic pulmonary effects as manifested by cough, chest sounds in lungs and shortness of breathe (SOB). Respiratory tract irritations including cough, hoarseness of voice, and irritation of throat etc are observed in arsenic-induced laryngitis. SOB along with crepitations and ronchi was found in interstitial lung disease due to arsenicosis. Individuals with history of seasonal cough or bronchial asthma or family history with chronic bronchitis were excluded from the study. Chemicals Ascorbic acid, acetone, extran (phosphate free), nitric acid (69% GR), potassium iodide, sodium hydroxide, sodium borohydride (96% pure), obtained from Merck (Hohenbrunn, Germany), arsenic (III) and arsenic (V) AAS (Atomic Absorption Spectrometry) standards were obtained from Qualigens (Shanon Co. Clare, Ireland) and hydrochloric acid (35%) was bought from Rankem (Ranbaxy, New Delhi, India). Sample Collection and Exposure Analysis Study participants were provided with acid-washed (nitric acid–water (1 þ 1)) plastic bottles for collection of drinking water (approximately 100 ml) samples into which nitric acid (1.0 ml/l) was added later on as preservative (Chatterjee et al., 1995). First morning voids (approximately 100 ml) were collected in precoded polypropylene bottles for arsenic estimation as these give the best measure of the recent arsenic 218


exposure (Biggs et al., 1997). After collection, water and urine samples were stored in the icebox and brought to the laboratory and immediately kept at 201C until arsenic estimation was carried out. In order to avoid any contamination during sample collection, a tube with distilled water was taken along with other sample vials to the spot and again returned to the laboratory. It was treated similarly with other sample vials during analytical test. Nail (approximately 250– 500 mg) and hair (approximately 300–500 mg) samples were collected using blade/nail cutter. Hair samples were taken from very close to the scalp of the more or less similar region of head (Das et al., 1995). The arsenic content in water and other biological samples was analyzed in our laboratory at Indian Institute of Chemical Biology, Kolkata and also at the School of Environmental Studies (SOES), Jadavpur University, Kolkata. Of the samples 5% were reanalyzed at the SOES, Jadavpur University, Kolkata. Reagent blank was taken for negative control. Nail and hair samples had to be digested prior to estimation of arsenic, but water and urine samples required no digestion. Moreover, both the nail and hair samples were thoroughly cleaned for removal of exogenous arsenic, as described earlier (Basu et al., 2004). Before estimation, the nail and hair samples were dried in a hot oven after treating them with concentrated nitric acid (Das et al., 1995). Flow injection-hydride generation-atomic absorption spectrometry (FI-HG-AAS) was used for the estimation of arsenic in the collected samples. A PerkinElmer Model-3100 spectrometer at SOES, Kolkata and Model Analyst-700 spectrometer at our own Institute equipped with a Hewlett-Packard Vectra computer with GEM software, Perkin-Elmer EDL System-2, arsenic lamp (lamp current 400 mA), illuminating the cuvette at 193.7 A˚ (specific for arsenic) were utilized for this purpose (Ghosh et al., 2006). Instrument was calibrated at regular intervals and also prior to the estimation of arsenic. Background correction and standard curve preparation have been carried out properly before sample analysis. One ppb is the lowest amount of arsenic that can be accurately measured by this instrument, as per manufacturer’s specification. For each sample, reading was taken in duplicate. Repeat sampling and rechecking in both the laboratories of same sample gave consistent results.

Statistical Analysis All individuals with and without skin lesions, with a history of arsenic in drinking water were defined as exposed population. Similar parameters obtained from participants residing in unexposed area were compared with exposed population to find the baseline information. p-values were calculated for the distribution of categorical variables among the study groups by one-way ANOVA. Standard twosample t-test (large sample approximations) was performed to test for significant differences in mean arsenic contents in drinking water, urine, nail, and hair among three different Journal of Exposure Science and Environmental Epidemiology (2007), 17(3)


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study groups. Logistic regression analysis was performed to calculate odds ratio (OR) and 95% confidence intervals (CI), adjusted for age, sex and smoking habit. OR and 95% CI for diseases were calculated in skin lesion as well as without skin lesion group, taking unexposed individuals as referent group. Again, OR and 95% CI for the diseases were calculated in skin lesion group, taking without skin lesion group as a reference. MS excel and R (version 2.1.1) statistical software were used for the analysis. The trend test for OR of the three diseases in three groups was also calculated.

groups. Distribution of different type of skin lesions in the arsenic exposed skin lesion group is furnished in Table 2. The data revealed 77.68% raindrop pigmentation, 85.59% palmar/plantar hyperkeratoses, and 40.63% hypo/hyperpigmentation in studied male individuals. Females with arsenic-induced skin lesion also showed 80.54% raindrop pigmentation, 85.24% palmar and plantar hyperkeratoses, and 47.65% hypo/hyper pigmentation (Table 2). The skin lesion subtypes were not found to be dependent on age of the affected individual (data not shown). Among 373 individuals with skin lesions, 17 were affected with squamous cell carcinoma (SCC), and one with basal cell carcinoma (BCC) (Figure 2). Among 17 SCC cases, 15 had Bowen’s disease. Arsenic content in drinking water and other biological samples of exposed population, for individuals with and without skin lesion, were significantly higher (Po0.001) when compared with the unexposed group (Table 3). Within the exposed group (i.e. with and without skin lesion), the

Results General characteristics of the study participants, which include unexposed, exposed skin lesions and without skin lesion individuals, are summarized in Table 1. Age, sex and socio-demographic characteristics are similar in three study Table 1. Descriptive characteristics of study population Unexposed (N ¼ 389)

Parameters

Male % (N) Age distribution Total o20 years 20–39 years 40–59 years 460 years Smoking habit Never smoker Ever smoker

9.21 39.33 34.72 16.74

239 (22) (94) (83) (40)

Female % (N)

7.33 44.67 37.33 10.67

150 (11) (67) (56) (16)

Without skin lesion Group (N ¼ 352) Male % (N)

10.15 40.61 35.03 14.21

Female % (N)

197 (20) (80) (69) (28)

7.74 43.87 37.42 10.96

155 (12) (68) (58) (17)

Exposed skin lesion Group (N ¼ 373) Male % (N)

7.59 43.30 37.50 11.60

224 (17) (97) (84) (26)

P-valuea

Female % (N)

8.05 37.58 42.95 11.41

149 (12) (56) (64) (17)

0.98

44.76 (107) 55.24 (132)

70.60 (106) 29.40 (44)

46.70 (92) 53.30 (105)

75.48 (117) 24.52 (38)

37.95 (85) 62.05 (139)

70.46 (105) 29.54 (44)

0.84

Type of smoking Bidi/cigarette Pan with tobacco Both

46.97 (62) 31.82 (42) 21.21 (28)

25.00 (11) 59.09 (26) 15.91 (7)

66.67 (70) 18.09 (19) 15.24 (6)

15.79 (6) 84.21 (32) 0

53.24 (74) 21.58 (30) 25.18 (35)

2.27 (1) 95.46 (42) 2.27 (1)

0.72

Occupation Cultivation Business Daily wage earner Service Student Unemployed Housewife

68.20 (163) 10.88 (26) 5.02 (12) 5.44 (13) 7.53 (18) 2.93 (7) F

11.33 (17) 1.33 (2) 2.67 (4) F 7.33 (11) F 77.34 (116)

68.54 (135) 5.58 (11) 15.22 (30) 4.06 (8) 4.06 (8) 2.54 (5) F

7.10 (11) 3.87 (6) 3.23 (5) F 2.58 (4) F 83.22 (129)

64.73 (145) 10.72 (24) 12.05 (27) 4.02 (9) 5.36 (12) 3.12 (7) F

9.40 (14) 4.70 (7) 0.67 (1) 1.34 (2) 3.36 (5) F 80.53 (120)

0.98

a

One-way ANOVA.

Table 2. Distribution of different skin lesions in arsenic exposed skin lesion individuals Exposed skin lesion group Male Female

Raindrop pigmentation % (N)

Palmar/planter hyperkeratosis % (N)

Hypo/hyper pigmentation % (N)

77.68 (174) 80.54 (120)

85.59 (192) 85.24 (127)

40.63 (91) 47.65 (71)


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Figure 2. (a–f): Different types of arsenic-induced skin lesions. (a) Classical raindrop pigmentation on both legs. (b) Raindrop pigmentation on right leg. (c) Hyperkeratosis on palm and sole. (d) Spotty hyperkeratosis on soles. (e) Bowen’s disease (carcinoma in situ) on anterior abdominal wall. (f) Squamous cell carcinoma on toe of left foot.

Table 3. Comparison of arsenic content from water, nail, hair and urine in the study groups Variables

Unexposed N ¼ 389 (Mean7SD)

Exposed without Exposed skin lesion skin lesion group group N ¼ 373 N ¼ 352 (Mean7SD) (Mean7SD)

Water (mg/l) Urine (mg/l) Nail (mg/g) Hair (mg/g)

6.9772.10 27.69720.96 0.5570.39 0.3370.247

186.897124.67a 238.217246.79a 2.8972.19a 1.9371.52a

200.837145.83a 175.257114.04a,b 4.4174.27a,b 2.6772.33a,b

Two sample T-test (large sample approximation). a Po0.001, compared to unexposed group. b Po0.001, compared to the group without any skin lesion.

difference in the arsenic content in their drinking water was not significant. However, arsenic content in nail, hair and urine was found to be significantly different between these two groups. Significantly higher amount of urinary arsenic was detected in the group without skin lesion, while significant retention of arsenic in nail and hair was found in the skin lesion group (Table 3). Arsenic content in the drinking water of our exposed study area ranged from 50 to 1188 mg/l, while that of unexposed area ranged from 0 to 10 mg/l. Table 4 shows a relative distribution of three diseases in our study groups. Arsenic exposed individuals, irrespective of skin lesion manifestation showed significant risk of develop220

ing all three diseases compared to unexposed group (referent). Within the exposed group, the individuals who already developed skin lesions, showed considerably higher risk of developing conjunctivitis (OR: 7.33, 95% CI: 5.05– 10.59), peripheral neuropathy (OR: 3.95, 95% CI: 2.61– 5.93) and respiratory illness (OR: 4.86, 95% CI: 3.16–7.48) compared to those without skin lesion with similar exposure history. The trend test for OR of the three diseases in three groups was found to be statistically significant.

Discussion Long-term exposure to arsenic causes changes in skin pigments and hyperkeratosis. This promotes ulceration of skin and accelerates the risk of cancer of skin, liver, bladder, and kidney. In spite of all the individuals drinking arsenic contaminated water, only 15–20% of arsenic exposed population develops arsenic-induced skin lesions (Ghosh et al., 2006). Duration and exposure level ultimately determines the latency period between exposure and skin lesion outcome. Raindrop pigmentation, palmar and planter hyperkeratosis, hypo or hyper pigmentation are the most common skin lesions. Interestingly, we have shown that, there is no significant difference in the level of arsenic in drinking water used by the group with skin lesion and the other group without any skin lesion. This observation obviously leads to the speculation that, genetic variation might be a strong guiding factor in susceptibility to arsenic Journal of Exposure Science and Environmental Epidemiology (2007), 17(3)


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Table 4. Odd’s ratio for conjunctivitis, peripheral neuropathy and respiratory illness in unexposed and arsenic exposed study populations in West Bengal, India Parameters

Unexposed group (N ¼ 389) Male (N) Female (N) OR (Ref)

Conjunctivitis Present Absent

Exposed no-skin lesion group (N ¼ 352) Male (N)

Female (N)

OR (95%CI)

Exposed skin lesion group (N ¼ 373)a Male (N) Female (N)

P-valueb

OR (95%CI)

6 144

1.0

25 172

19 136

4.66 (2.45–8.85)

126 98

82 67

37.22 (20.56–67.36)

o0.001

Peripheral neuropathy Present 4 Absent 235

7 143

1.0

18 179

15 140

3.99 (1.95–8.09)

58 166

56 93

15.61 (8.2–29.71)

o0.001

Respiratory illness Present 8 Absent 231

5 145

1.0

14 183

18 137

3.21 (1.65–6.26)

77 147

41 108

13.54 (7.45–24.62)

o0.001

7 232

a

OR and 95% CI was calculated for exposed skin lesion group with reference to exposed no skin lesion group; conjunctivitis: 7.33 (5.05–10.59), peripheral neuropathy: 3.95 (2.61–5.93) and respiratory: 4.86 (3.16–7.48) b Test for trend of OR. OR: Odd’s ratio; CI: Confidence Interval. All OR values are adjusted for age, sex and smoking.

toxicity and paves the way for gene-environment studies that are already being preformed by our group (De Chaudhuri et al., 2006; Ghosh et al., 2006). Previous studies identified neuropathy and respiratory effects in arsenic exposed individuals with skin lesion, worldwide, as well as, in West Bengal population. According to the published literature little or no attention had been given to those individuals who were free of skin lesion despite arsenic exposure. Till date, only a single study from our group focused association of conjunctivitis with arsenic (Baidya et al., 2006). Conjunctivitis was found to be strongly associated with arsenicism. Present study provides strong evidence that ingestion of inorganic arsenic through drinking water results in significant increase in peripheral neuropathy, conjunctivitis and respiratory illness even in those individuals who were free of skin lesion despite arsenic exposure (Table 4). The trend test for OR of the three diseases in three groups was found to be statistically significant (Table 4). However, we did not find any association of these diseases with age, sex, or smoking status (data not shown). Hence, we calculated the OR and 95% CI values adjusted for these three parameters. The similarity between exposed and unexposed study subjects with respect to socio-demographic characteristics, lifestyles, and diet made it unlikely that confounding could explain the association. A number of major studies investigated prevalence of arsenicism based on arsenic concentration in drinking water, as a principal parameter. In a population-based survey, the dose response analysis has some limitations. Arsenic concentration might vary considerably from well to well in the same area and even in the same well during different seasons. The likely fluctuation in Journal of Exposure Science and Environmental Epidemiology (2007), 17(3)

exposure depends on precipitation and other circumstances. Currently, filters are provided with many tube wells in village to provide safer water. The time frame of individuals’ switch over from contaminated to safer water also varies considerably. Hence, despite our attempt, it was not possible to find correlation between arsenic content in drinking water, urine, nail and hair with the disease outcome in study subjects. Urine arsenic serves as better indicator and assesses current arsenic exposure. Estimation of arsenic from hair and nail can provide the arsenic exposure over past 6–12 months (Buchet et al., 1981; Biggs et al., 1997). The basic difference between the two groups (i.e. individuals with and without skin lesion) due to arsenic sensitivity lies in the distribution of arsenic in their body parts; those with skin lesions retained much higher level of arsenic in their nail and hair while they excreted much lower amount of arsenic through urine compared to the other group. This observation is consistent with the phenotypic expression since higher retention of arsenic would be expected to cause more damage to the cells. Although the molecular mechanism for this observed difference has not yet been deciphered, it is reasonable to propose that those individuals with lower capacity to methylate the inorganic arsenic, an obligatory step for excretion of this metalloid, might retain it in their system and as a result develop skin lesions. Although arsenic and its association with cancer is well established, we could not explore this correlation in our study group, consisting mostly of villagers, since only the participants with cancer could be recorded while information on those who died in the community due to arsenic related cancer could not be retrieved due to lack of proper death certificate citing real cause of death. 221

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Several possible modes of action of arsenic carcinogenesis have been proposed, such as, chromosomal abnormalities, oxidative stress, altered DNA repair, altered methylation patterns, altered growth factors, enhanced cell proliferation, promotion/progression, gene amplification, and suppression of p53 gene (Kitchin, 2001; Basu et al., 2005; Mahata et al., 2003). It is evident from the available literature that arsenic directly or indirectly induces many steroid related genes, cytokine genes, cell cycle related genes, DNA repair and stress genes, transcription and translation regulatory genes, cell differentiation and apoptosis related genes and a few oncogenes (Shimizu et al., 1998; Chen et al., 2004). Therefore, it is not surprising that arsenicosis displays a complex pathology which include conjunctivitis, peripheral neuropathy and respiratory illness. However, whether a underlying common pathophysiology plays any role is not yet clear based on the current state of knowledge in this area. Understanding this chronic disease necessitates the exploration of gene–environment interaction. Varying individual susceptibility to common environment may depend on underlying genetic make-up. If gene and environment interact to generate risk synergistically, then, eliminating either the genetic or the environmental influence represents an effective strategy for disease prevention.

Conclusion The overall results of this comprehensive study provide conclusive evidence that arsenic exposure is a risk factor for conjunctivitis, peripheral neuropathy and respiratory diseases. Our study demonstrates that although the individuals with skin lesions are more susceptible to arsenic-induced toxicity and carcinogenicity, individuals without skin lesion in exposed group are substantially affected and are also significantly susceptible to arsenic-induced toxicity when compared to unexposed individuals.

Acknowledgements The authors are grateful to the Council of Scientific and Industrial Research (CSIR), Government of India for funding the study (project no. CMM 003) and for providing research fellowships to PG, MB, SDC and RC.

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