Introduction

Arsenic (As) is a well documented human toxicant and contamination with this heavy metal is a global concern presenting a major issue in environmental health (¹). Recent studies suggested that a vast area of 9 districts of West Bengal, India is severely affected by this toxicant of geological origin and the exposed population is about 42.7 million (²). Besides dermatological manifestations like pigmentation and keratosis, its exposure is associated with various systemic manifestations (³). Arsenic induced non-cirrhotic portal hypertension in two patients was first reported from Chandigarh (⁴). It was also reported that non-cirrhotic portal lesion is the predominant lesion in chronic arsenic toxicity (⁵).Exposure of arsenic through drinking of contaminated water may cause conjugated hyperbilirubinemia with simultaneous rise in serum alkaline phosphatase (ALKP) (⁶).Elevation of ALKP, AST and ALT was noticed as an effect of arsenic exposure in human (⁷). Our laboratory also observed arsenic induced stress which may cause hepatic injury. Little information is available on stress which mirrors such damage in hepatic tissues at risk of arsenic exposure. The studies of arsenic toxicity involving human subjects were carried out only on exposed individuals although reports of arsenic induced hepatic toxicity are available on murine model using control. The aim of the present study is to assess bio indicators of hepatocellular injury, cholestasic injury, liver biosynthetic capacity, diabetogenic probability in relation to clinical manifestation of the arsenic exposed subjects and compare it with control.

Materials and Methods

Sampling: A team of scientists visited the area and chosen five exposed and two control wards out of 22 wards of Ashokenagar- Kalyangarh municipal area of 24 Paraganas, West Bengal, India. This district was chosen for its proximity to Kolkata, the consequent convenience of transporting samples from the study site to the laboratory and also because it is reported to be severely affected by arsenic (⁸). For the study, it has been decided to examine 150 exposed and 50 control subjects. A proportional allocation was made taking one family as unit depending on the population of ward. Basing on the proportional allocation, adult males and adult females, males below 25 years and females below 25 years were chosen using random sampling (⁹) taking one family as one unit. Thus 150 exposed subjects and 50 control subjects were covered for medical examination. Average duration of consuming arsenic contaminated water for tested population was 7-8 years. Approval of the ethical committee at the Regional Occupational Health Centre (Eastern) was obtained for the study. Simultaneously written and informed consent was obtained from each of the participants. Water used by these subjects was also collected.

Water Collection: Tube well water samples were collected in 100ml pre-washed polythene bottles (in 10% HNO3) and the samples were acidified with nitric acid (1ml/l). The water presently being consumed by the subjects were collected for each individual house for analysis purpose.

Estimation of arsenic in water: Estimation of arsenic in water was done by atomic absorption spectrophotometer (model-Avanta, GBC Scientific Equipment PVT. Ltd., Australia) attached with hydride generation system (GBC HG 3000, hydride generator) as previously described (¹⁰). Stock solutions of arsenic (1000mg/l) prepared from 99% AS₂O₃ (Sigma, USA) in deionised water and working standards were prepared daily from the stock solution. Before measurement all As present was reduced to As III by acidifying the samples with two molar hydrochloric acid (HCL) (2M HCl) and 0.2% potassium iodide (KI). Time period for complete reduction at room temperature was one hour. Arsenic concentration in the water samples was estimated against the working standards prepared daily from the stock solution at an absorbance at 193.7nm by using a AAS-HG technique.

Collection of serum and plasma samples

Biochemical evaluation

Statistical analysis

Result

Mean arsenic concentration in drinking water was detected 15.17±2.07 μg/L in control population (range 0.01-44.44) and 121.95±11.30 μg/L (range 59.26-190.81) in exposed population . Average age group of tested males and females were as follows:

Exposed : male (>25 years) = 45.94 years; female ((>25 years) = 42.47 years; Control male (>25 years) = 50.25 years; female ((>25 years) = 47.94 years; Exposed male (≤ 25 years)= 19.00; female (≤ 25 years)= 17.89 years; Control male (≤25 years) = 17.50 years; female ((≤25 years) = 14.20 years [ unpublished data].

Table 1 represents the percentage of cases showing above normal range of biochemical parameters. It has been noticed that about 60% of both male (65%) and female (55%) exposed subjects of age group ≤ 25 years showed higher values of AST. About 82% of both male (81%) and female (83%) exposed subjects of age group > 25 years showed higher values of AST. ALT showed similar pattern in tested population. Our study indicated that ALKP level increases with increase in age (29% at ≤ 25 years and 32% at > 25 years) in case of tested male. The corresponding figure was 20% (at ≤ 25 years) and 28% (at > 25 years) in case of female. It was observed that the enlargement of livers showed some similarity with it.

Figure 1

Table I: Percentage of cases of arsenic exposure induced deviation from normal range of biochemical parameters

Table 2A indicates the effect of arsenic exposure on biochemical parameters of male. AST and ALT, as indicator of hepatocellular injury, of exposed subjects of both age group increased significantly (p<0.001) in comparison to control. ALKP, as biomarker of cholestasic injury, showed similar trend. Total Protein, as indicator of liver biosynthetic capacity, of the tested males elevated significantly in comparison to control but was within normal range. Albumin of both the age group declined significantly (p<0.001) compared to control resulting in increase in quantity of globulin. Urea and blood glucose of exposed males (age group > 25 years) elevated significantly (p<0.001) with respect to control.

Figure 2

Table 2A: Effect of arsenic exposure on biochemical parameters in males

Number in parenthesis indicating subjects

Table 2B indicates the effect of arsenic exposure on biochemical parameters of female. Significant elevation (p<0.001) of AST, ALT and ALKP was noted compared to control population. Increase in TP was observed in case of tested females of age group ≤ 25 years in comparison to control. Alb decreased significantly [age group ≤ 25 years (p<0.01) and age group > 25 years (p<0.001)] as compared to control. Significant change (p<0.001) in glucose and urea was also noted in case of tested population of female of both age group with respect to control of comparable age .

Figure 3

Table 2B: Effect of arsenic exposure on biochemical parameters in females

Clinical examination for detection of exposure induced palpability of liver, pigmentation, keratosis of the subjects is shown in figure 1. Arsenic induced palpability of liver was observed in female (30% cases) and male (11.70% cases) of age group ≤ 25 years. On the other hand, tested population of age group of >25 years showed liver palpability in 13% females and 10% males. It has been noted that 6.80 % male and 15% female of age group ≤ 25 years showed skin pigmentation whereas the corresponding figure was 35% in male and 23% in female for age group >25 years. Arsenic induced keratosis was observed in 6.80% male of age group ≤ 25 years and the rise in percentage of cases (20.7%) was noticed among subjects of age group >25 years. This trend was not seen among female.

Figure 4

Fig I Effect of arsenic on Clinical Manifestations

Discussion

It has been observed that the biological indicators of hepatic effect parameters like AST, ALT, and ALKP rise as an effect of arsenic exposure in drinking water. Reports indicated that elevation was also observed as ALKP in 51.3%, ALT in 11.8% and AST in 27.6% cases with enlargement of liver in 76.9% cases (³) Other reports of elevation of liver enzymes also support our observation (⁷). The murine model, relevant to epidemic human toxicity in areas of arsenic concentration, also showed that continued arsenic feeding resulted in fatty liver with serum ALT, AST elevated at 12 months and hepatic fibrosis at 15 months. (⁵).Our studies also indicated that level of serum albumin, as biological indicator of liver biosynthetic capacity, declines although the effect of exposure on TP was not consistent with various age groups of tested population. Consequently, high level of serum globulin is expected. This observation is supported by other workers (¹⁸). Although blood glucose level was noted within the normal limit, significant elevation was observed in blood glucose of exposed subject (age group >25 years) in comparison to control. Reports are available on the normal level of blood glucose of arsenic affected patients. (¹⁹). Arsenic exposure is a risk factor for diabetes mellitus (²⁰). Increasing trends of plasma sugar with indices of arsenic exposure in drinking water was found to be independent of the presence of skin lesions associated with arsenic exposure (²¹). The present study revealed the probability of exposed individuals to be diabetogenic. Level of serum urea was within the normal range but exposure dependent elevation was noticed in females compared to control. Other studies also showed that serum urea of arsenic affected patients was within normal range (⁷). The association between arsenic exposure and diabetes mellitus is a new finding (²¹). Our preliminary observation showed the elevation of glucose upon exposure. Studies in Bangladesh also supported our finding (²⁰). The present study showed some similarity of clinical manifestation including palpapility of liver of both arsenic exposed males and females of tested population to the percentage of cases where elevated level of biomarkers were noticed. This observation was also supported by other workers (⁷).

All the studies so far carried out in different laboratories was involving patients and including effects of arsenic exposure in concentration dependent manner. To our knowledge, this is the first report where control subjects are included in the study to observe the effect of arsenic. Further studies are needed to identify and characterize the stress factors causing elevation of biomarkers induced by arsenic.

Acknowledgement

Correspondence to

B. Dalal
Scientist B
Regional Occupational Health Centre (Eastern)
Indian Council of Medical Research
Block DP, Sector V, Salt Lake
Kolkata 700 091
India
e-mail : dalal.basudeb@gmail.com
Telephone No. +91 33 2367 4390
Fax +91 33 2367 6683