Arsenic is a paradoxical human carcinogen affecting millions of people around the world. At present, people in more than 35 countries across the globe are affected by drinking arsenic-contaminated ground water. In India, West Bengal is the worst affected state where more than 26 million people in 9 out of 18 districts are drinking heavily contaminated ground water through hand-pumped tube-wells [1]. The arsenic concentrations in these districts are far above the current maximum contaminant level (MCL) established by WHO and US EPA i.e. 10 μg/l [2, 3]. This is regarded as the greatest arsenic calamity in the world [4]. Long term exposure to arsenic-contaminated water causes a wide range of adverse health effects, including cancer [5], cardiovascular disease [6], diabetes mellitus [7], neuropathies [8], liver disease [9], ocular diseases [8], and skin lesions [5, 10]. The skin lesions include rain drop pigmentation, hypopigmentation, hyperpigmentation, keratoses and skin cancers including Bowen’s disease, basal cell carcinoma and squamous cell carcinoma [11, 12]. More than 300,000 people in West Bengal have these skin lesions that are hallmarks of chronic arsenic toxicity. 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 [13]. Respiratory disease is also common in arsenic toxicity [8, 11, 14].

Among the various internal organs affected by chronic exposure to arsenic in humans, liver is one of the important targets. Epidemiological studies have shown association between chronic arsenic exposure and liver disease including hepatomegaly, hepatoportal sclerosis, liver fibrosis and cirrhosis of liver [9, 15–17]. Abnormal liver functions as manifested by severe gastrointestinal problems and clinical elevations of liver enzymes in plasma including alanine amino transferase (ALT), asparatate amino transferase (AST), alkaline phosphatase (ALP) also are associated with chronic arsenic exposure [9, 15, 18]. Exposure of mice to arsenic in drinking water causes elevation of liver enzymes in plasma [19] and capilarization of liver sinusoidal endothelium [20]. Infact, liver is the major site of arsenic metabolism [21] and hence arsenic exposure causes liver disease in exposed humans [9].

Antinuclear antibodies (ANA) are a diverse group of autoantibodies that are found in autoimmune disorders like systemic lupus erythomatosus, Rheumatoid arthritis, Sjorgen’s syndrome, systemic sclerosis and inflammatory myositis. Arsenic exposure increases the incidence of auto-immune diseases like diabetes mellitus [7]. ANA are present in lower titers in liver diseases, leprosy, multiple sclerosis and juvenile rheumatoid arthritis [22].The association of chronic arsenic exposure and autoimmune disorders has received only minimal attention [23, 24].

Inflammation has been found to be one of the most important factors that contribute to cardiovascular disease. Elevated levels of monocyte chemoattractant protein (MCP-1), interleukin 6 (IL6), and tumor necrosis factor alpha (TNFα), produced by the immune system play important roles in increasing the risk of cardiovascular disease. Both IL6 and TNFα play important roles in the regulation of the synthesis of other acute phase proteins which are established risk factors for atherosclerosis [25]. Exposure of mice to arsenic in drinking water causes the induction of inflammatory cytokines associated with increased atherosclerosis [26].

We have been studying health effects, cytogenetic damage, genetic variants, and immunological changes in the population exposed to arsenic through drinking water in West Bengal [5, 10, 12, 27–32]. During our epidemiological survey, we found that the arsenic exposed population complains of muscular cramps and joint pains, which are characteristics symptoms of rheumatoid arthritis. In the present study we assessed bioindicators of hepatocellular injury as indicated by the serum levels of liver enzymes, determined the autoimmune status as indicated by the amounts of serum ANA and anti-dsDNA and measured circulating inflammatory cytokines related to cardiovascular diseases in the population exposed to very high arsenic content in their drinking water. The results have been compared with a group of arsenic unexposed individuals (controls). We show that the arsenic exposed population exhibits increased liver enzymes, increased serum ANA and inflammatory cytokines indicating liver injury, heightened autoimmunity, and increased likelihood of cardiovascular disease.

Chronic arsenic exposure is well established as carcinogenic but interest in the non-cancer disease endpoints of arsenic exposure is of great interest. The non-cancer disease endpoints include cardiovascular disease [6] and immune dysfunction [34], as well as neuropathies and ocular diseases [8, 35, 36]. In the present study, we focused on biological markers of liver disease, autoimmunity and cardiovascular disease in a population in West Bengal exposed to high levels of arsenic in drinking water. Increased bilirubin and liver enzyme levels in serum indicated the presence of liver injury in the arsenic exposed individuals. Increased serum ANA indicated increased likelihood of autoimmunity, and increased serum inflammatory cytokine levels indicated increased systemic inflammation and risk of cardiovascular diseases in the arsenic exposed individuals.

Liver function tests are a helpful screening tool to detect hepatic dysfunction. Liver has to perform different kinds of biochemical, synthetic and excretory functions, so no single biochemical test can detect the global functions of liver. To detect the proper functioning of the liver, various tests are performed to detect specific liver activities. Among various parameters, serum bilirubin is the marker the liver’s capacity to transport organic anions and to metabolize drugs. Aminotransferases (ALT, AST), alkaline phosphatase (ALP), aminopeptidase are the tests to detect injury to hepatocytes. Total protein is the marker for liver’s biosynthetic capacity [37]. Bilirubin is an endogenous anion derived from hemoglobin degradation. Underlying liver disease is indicated when the liver function tests are abnormal and serum bilirubin levels are elevated. Hyperbilirubinemia seen in acute viral hepatitis is directly proportional to the degree of histological injury of hepatocytes and the longer course of the disease [38]. The aminotransferases, AST and ALT are the most frequently utilized indicators of hepatocellular necrosis. ALT is primarily localized to the liver but the AST is present in a wide variety of tissues like the heart, skeletal muscle, kidney, and brain as well as liver. The AST and ALT levels are increased to some extent in almost all liver diseases. The highest elevations occur in severe viral hepatitis, drug or toxin induced hepatic necrosis and circulatory shock [38]. Higher levels of alkaline phosphatase occur in cholestatic disorders. Elevations occur as a result of both intrahepatic and extrahepatic obstruction to bile flow and the degree of elevation does not help to distinguish between the two. The mechanism by which alkaline phosphatase reaches the circulation may be due to leakage from the bile canaliculi into hepatic sinusoids via leaky tight junctions [39]. In healthy people most circulating alkaline phosphatase originates from liver or bone [40]. In liver disease, glutamyl transpeptidase activity correlates well with alkaline phosphatase levels [41]. So, clinicians are often confused when they see elevated alkaline phosphatase levels and are unable to differentiate between liver diseases and bony disorders and in such situations measurement of glutamyl transferase helps as it is raised only in cholestatic disorders and not in bone diseases. Elevated levels of these liver function enzymes have been associated with hepatic dysfunctions in lead [42, 43] and cadmium [44] exposure. In our study, we have found that serum levels of bilirubin, ALT, AST, and ALP have increased significantly in the arsenic exposed population when compared to the unexposed group with similar socio-economic status. These increases clearly indicate that chronic arsenic exposure causes injury to hepatocytes with damage to the liver’s capacity to transport organic anions and to metabolize drugs, cholestatic injury and impairment of the liver’s biosynthetic capacity. Our results are consistent with many findings of chronic exposure to arsenic associated with hepatomelagy, hepatoportal sclerosis, liver fibrosis and cirrhosis with concomitant increase in serum bilirubin, ALT, AST, and ALP [9, 45, 46].

ANA and anti-ds DNA are frequently found in serum of patients with different types of autoimmune disorders and are biomarkers of autoimmune disorders [47–50]. Rheumatic diseases that affect connective tissue, including the joints, bone, and muscle are associated with these antibodies. Autoimmune and rheumatic diseases can be difficult to diagnose. People with the same disease can have very different symptoms. A helpful strategy in the diagnosis of these diseases is to find and identify an autoantibody in the person's blood. ANA bind to several nuclear antigens. It is useful as a screen for many autoantibodies associated with systemic diseases. Presence of anti-dsDNA is one of the diagnostic criteria for systemic lupus erythomatosus, which is an autoimmune disorder. IgG class antibodies, antibodies to single stranded DNA (ssDNA) and IgM antibodies to DNA are found in number of connective diseases as well as in rheumatoid disorders [51, 52]. Increased ANA titres have also been reported in arsenic exposed population earlier [23]. Thus, ANA can be utilized as a biomarker in early risk assessment of arsenic induced autoimmune disease in high risk arsenic zones, since most of the individuals did not show any symptoms of rheumatoid arthritis although they were ANA positive. We have also found a significant increase in the serum levels of ANA and anti ds DNA in arsenic exposed population compared to the unexposed population in West Bengal. Therefore ANA appears to be a useful biomarker in early risk assessment of arsenic exposure induced autoimmune disease, which might lead to rheumatoid arthritis or other types of autoimmune diseases.

In order to find out the dose response of the LFT parameters and autoimmune markers with the increase of arsenic concentration in urine, we have stratified the data of the exposed population into four sub-groups depending upon the arsenic content in urine as mentioned in the results section. Our findings show that an increasing trend is observed in both LFT parameters and autoimmune markers with increasing urinary arsenic concentration. The dose response increase is significant in case of AST, ALT, ANA and anti-dsDNA indicating that with increase in arsenic exposure (as urine is the best current exposure marker), the levels of hepatic damage and autoimmune response increases in the exposed individuals. Although no dose response is observed in bilirubin and alkaline phosphatase levels with urinary arsenic content compared to unexposed group , the significant increase in levels of other hepatic parameter indicates that increase in arsenic exposure causes hepatic injury since all the parameters together contribute to hepatic damage.

Cytokines play pivotal roles in systemic inflammation and thus in cardiovascular diseases [52]. In atherosclerotic plaque, inflammatory cytokines (IL6, IL8, MCP-1, TNFα, IL1b) secreted by macrophages, dendritic cells, T cells and smooth muscle cells, aggravate plaque instability by inhibiting extracellular matrix synthesis and promoting smooth muscle cell apoptosis. Elevated levels of IL8 found in atherosclerotic plaques suggest that it acts as important mediator of angiogenesis in this tissue contributing to plaque formation [53]. Elevated plasma levels of IL6 and TNFα were detected in patients with stable or unstable angina and myocardial infarction [54, 55]. It was also found that increased plasma levels of IL6, IL8 and MCP-1 were associated with cardiovascular risk in patients with systemic lupus erythematosus [56]. Arsenic exposure has been connected with a host of cardiovascular risk factors and endpoints including peripheral arterial disease (blackfoot disease), atherosclerosis, coronary heart disease, stroke, and hypertension [57]. It has been found that the expression of several cytokine related genes is increased in human subjects with increase in arsenic exposure, including heme oxygenase-1 (HO-1), MCP-1 and IL6 [58]. The secretion of theses cytokines are regulated by reactive oxygen species (ROS) and they are involved in modulating the biological functions of vascular smooth muscle cells are thus are involved in atherosclerosis [59]. In our previous study we have found increased generation of ROS [10] in the arsenic exposed population of West Bengal. The increased oxidative stress due to arsenic exposure may influence inflammatory responses in the vascular cells. We can conclude that increased serum concentrations of these cytokines and chemokines (IL6, IL8 and MCP-1) may act as early biomarkers of increased cardiovascular risk in the arsenic exposed subjects. In this context it is worthwhile to discuss that Kupffer cells in the liver are a major source of the inflammatory cytokines upon hepatotoxic insult [60]. Our results show that liver injury by arsenic exposure. Thus, the injured hepatic cells may induce increased secretion of the inflammatory cytokines (IL6, IL8, TNFα) in the arsenic exposed individuals which in turn contributes to cardiovascular risk.

Chronic arsenic exposure causes a wide variety of human diseases. Exposed individuals are at higher risk of developing liver and cardiovascular disease, as indicated by elevated serum levels of liver injury biomarkers and inflammatory cytokines. Increase of autoimmune markers in the serum suggests that arsenic exposure also induces autoimmune diseases such as rheumatoid arthritis. Both rheumatoid arthritis and liver disease are risk factors for cardiovascular disease. Thus, the arsenic exposure induced systemic inflammatory disease and the hepatic injury likely contributes to increased cardiovascular risk observed in arsenic exposed populations.

Dr. Ashok K. Giri is a Chief Scientist at the CSIR-Indian Institute of Chemical Biology. His work includes various epidemiological and molecular biological aspects of the highly arsenic exposed population of West Bengal. He has more than 20 peer reviewed publications projecting research work of more than 10 years in this area.