Arsenic is a major toxic environmental contaminant and a human carcinogen. Prolonged drinking of arsenic-contaminated water leads to chronic arsenic toxicity (arsenicosis) and it results in pathophysiological changes like alteration of skin colour, hard patches on palms and soles of feet, cancers of the skin, bladder, kidney and lung, and vascular diseases in humans. Besides cancers, arsenic ingestion is associated with other diseases such as blackfoot disease, atherosclerosis, hypertension, diabetes mellitus, skin lesions and liver injury [1]. Arsenic is also immunotoxic and renders the host immune-compromised [2]. Arsenic toxicity has become a public health issue and has been reported from many countries; however, the severity of arsenic contamination in West Bengal in India and Bangladesh, the deltaic region in Ganga-Brahmaputra-Meghna basin, is unprecedented. Around 1 million people of West Bengal are at risk due to consumption of water having arsenic concentration higher than the maximum permissible limit [1, 3]. Occurrence of arsenic in groundwater have also been reported from other states in India, which include Bihar, Jharkhand, Chhattisgarh, Uttar Pradesh, Assam, Tripura, Arunachal Pradesh, Manipur and Nagaland [4, 5]. Human exposure to arsenic is caused mainly through arsenic-contaminated underground drinking water, it is now understood that food-chain is another contributor to arsenicosis problem. Widespread use of arsenic-contaminated groundwater for irrigation of rice and other crops leads to accumulation of arsenic in rice plants, grains and other crops and vegetables. The domestic animals and birds are exposed to arsenic through intake of contaminated feed [6]. Fish and other aquatic animals are naturally the worst affected ones as they feed, breed and grow in the contaminated aquatic habitat, thereby getting lifelong exposure. For understanding the molecular toxicology of arsenicosis, which could provide clues for developing mitigation measures, fish is an important model. Understanding the effect of arsenic load on fish is not only important for its implications on human health [7], but it is important from fish health and aquaculture angle also as aquaculture is one of the fastest growing food production sectors; freshwater aquaculture alone contributes over 95 % of the total production [8].

Accumulation of arsenic in different tissues and organs of different species of fishes grown in arsenic-contaminated waters have been reported [9, 10]; however, typical signs and symptoms of arsenicosis in fishes need to be better understood. We report here the pathophysiological and stress protein (heat shock proteins, Hsps) expression changes in the Indian major carp rohu (Labeo rohita) experimentally exposed to arsenic.

Apparently healthy fishes (L. rohita fingerlings; (n = 150; length 13–15 cm, weight 24–25 g) were procured from a local hatchery and were acclimatized in the laboratory for 15 days before the exposure study. Fishes were provided with commercially available feed and tubifex daily.

Experimental arsenic exposure was carried out for 12 days in the laboratory at room temperature (25–27 °C). Fishes were divided into seven groups and there were 16 fishes in each treatment group. Arsenic (NaAsO2; Hi-Media) at concentrations—0.0 (control), 0.5, 1, 2.5, 5, 10 and 15 ppm (mg/l) were added to the glass aquaria (30 l capacity). Water was changed and freshly prepared solution of arsenic was added to each aquarium on daily basis. Aerators were connected to all the aquaria to maintain dissolved oxygen level at 6–7 ppm and a constant source of power supply and light were also provided. Fishes were provided with commercially available feed and tubifex, as before. Appearance of gross lesions in the body, behavioral changes and mortality occurring at different time were recorded. The experimental exposure was repeated after a gap of 2 months to verify the reproducibility of the gross lesions.

After the 12 days exposure period, fishes were sacrificed by euthanizing with MS 222 (200 mg/l) and liver and kidneys were aseptically collected. For histopathological examination, tissues were washed in PBS and fixed with buffered formalin (10 % of buffer) for 24 h and were then processed following routine histochemical technique [11]. The sections were stained with hematoxylin–eosin staining, mounted with DPX for investigating the microscopic changes in the tissues.

Soluble liver protein extracts were prepared [12]. Protein concentration in the supernatants was determined, using BSA as the standard [13]. SDS-PAGE was carried out using 12 % separating gel [14]. Immunoblot analysis was carried out to identify the Hsps (Hsp60, Hsp70 and Hsp90) in the liver protein extracts of L. rohita exposed to different concentrations of arsenic. Transfer of proteins from polyacrylamide gel to NC membranes (0.2 µm) was carried out on iBlot (Invitrogen) and transfer was confirmed by staining a piece of membrane with Ponceau S (P7170, Sigma). Anti Hsp70, Hsp60 and Hsp90 (Sigma H5147, H4149, H1775, respectively) were used as the primary antibodies and anti mouse IgG-peroxidase conjugate (A2304, Sigma) was used as the secondary antibody. Besides, immunoblotting for β-actin (as an internal control) was done using mouse anti-β actin primary antibody (Sigma A2228). The blots were developed using a SNAP i.d. protein detection system (Millipore). DAB (Sigma D-8001) and H2O2 were used as substrates. Bands on the immunoblot were quantified using ImageQuant TL 7.0 (GE Health Care).

In the present study, clearly distinguishable skin/scale discolouration with dark and white patches, were prominent in the exposed fishes at concentration ≥10 ppm arsenic. Other gross morphological changes included pigmentation and depigmentation around buccal cavity and opercular region, lesions on the upper surface, near gill region with reddish tinge, distortion of the upper lip area and aberration of the pectoral fin (Fig. 1). There was skin lesion development at 15 ppm arsenic exposure. At the end of the 12 days exposure period, fishes (11/16) were sacrificed and organs were collected for analysis. From this exposure study, it was observed that fishes developed cataract as the first response against arsenic exposure followed by development of skin lesions.

Fig. 1
figure 1

Gross lesions observed in Labeo rohita on exposure to arsenic. Development of scar mark on the upper body surface and aberration of caudal fin included pigmentation and depigmentation around the area are shown. Damage of dorsal fin with conspicuous white spot encircling the region and pigmentation around buccal cavity were found. Cataract was observed in the As-exposed fishes

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At ≥5 ppm concentration, fifth day onwards with the continued exposure, exposed fishes progressively became sluggish, lethargic and restless. Caudal bending was noticed in the fishes exposed ≥10 ppm, with time, which greatly retarded the normal swimming pattern. The behavioral alteration due to arsenic toxicity in zebrafish (Danio rerio) has been reported earlier [15]. Hyper excitability, loss of equilibrium and sinking to the bottom of aquarium and staying motionless are the prime behavioral changes observed, which corroborates with the observations made in earlier reports [15, 16]. In an earlier study it was suggested that caudal bending, which greatly retarded the normal swimming pattern in the experimental fishes, could be due to the inhibition of acetylcholinesterase (AChE) [16].

Eye lens plays an important role in vision and any damage of the eye-lens in fish is taken as a pathological lesion and has negative impact on consumer acceptability. At 15 ppm arsenic exposure, fishes showed eye damage gradually, starting from fifth day post-exposure. It was observed that there was cataract development in 15 ppm arsenic exposed fishes (Fig. 1). Interestingly, at <10 ppm arsenic concentration, there was no cataract development till the last i.e. 12th day of exposure. Cataract, a disease of protein aggregation, leads to clouding and loss of transparency of lens and is a leading cause of human-blindness and visual impairment worldwide. It has been earlier reported that lens damage is significantly related to exposure of fish to environmental contaminants like pesticides [17] and heavy metals [18]. In this study, typical eye lesions like cataract of lens were observed in L. rohita following arsenic exposure. Dose–response relationship between ingested arsenic and cataracts among human subjects living in arseniasis-hyperendemic villages in Southwestern Taiwan have been reported [19]. In this study, cataract resulting from arsenic exposure has been shown in fish [20] and leads us to believe that arsenic could be another predisposing factor for cataract development.

Liver sections of arsenic exposed L. rohita were examined and histopathological changes were noted (data not shown). Increase in cytoplasmic granularity was noticed at 5 ppm exposure; congestion of central vein and portal vessels, vacuolar degeneration and diffused necrosis of hepatocytes were observed at ≥10 ppm exposure. Acute and extensive necrosis of liver cells and hypertrophy of hepatocytes were also observed. Hepatocyte cytoplasmic vacuolation was the most evident pathological alteration observed, thus the cellular structure was totally obscured. Varied degree of necrosis and degenerative changes in the hepatocyte’s syncytial arrangements and central vein were seen at arsenic concentration ≥10 ppm which corroborates with the previous studies reporting the necrotic changes, gross lesions and apoptosis of liver cells due to oxidative stress induced by arsenic [21, 22].

Microscopic examination of cross sections of kidney exposed to different doses of arsenic revealed enhanced glomerular necrosis and tubular degeneration (Fig. 2). The shrunken glomeruli, shrinkage and degenerated renal tubules are shown in the exposed fishes (Fig. 2). The renal corpuscles exhibited severe damage including breakdown of glomerular blood capillaries. The glomeruli appeared shrunken with densely basophilic nuclei and there was an increased space within the Bowman’s capsule indicated by arrow marks (Fig. 2), which could be due to infiltration of liquid material from glomeruli to Bowman’s spaces as reported in an earlier study [22]. Damaged glomerulus with increased mesangial matrix and severe vacuolation of tubular epithelium (shown with roundhead arrow marks) were also visible (Fig. 2).

Fig. 2
figure 2

Cross sections of posterior-kidney of Labeo rohita exposed to different doses of arsenic are shown. Control (×100) kidney showing normal glomeruli, tubules and hematopoietic tissue. Glomerular necrosis seen at arsenic concentration of 15 ppm (×50). Arrow mark shows shrinkage of glomeruli and expansion of Bowman’s capsule. Round head arrow mark shows cytoplasmic vacuolation and cytoplasmic blebbing. Tubular degeneration and necroses seen at 15 ppm of arsenic (×100). Square head arrow mark shows degenerative changes in the distal and proximal tubules

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Representative western blots showing stress protein expressions in arsenic-exposed fish livers are shown (Fig. 3). Up regulation in expression of Hsps could be due to protective effect of these proteins against arsenic stress as Hsps have cytoprotective function. Hsp90 is ubiquitously present in prokaryotic and eukaryotic cells. In both stressed and normal cells, Hsp90 coordinates the trafficking and protein folding [23]. It is an abundant cytosolic protein in normal cells. There was gradual decrease in Hsp90 expression (0.6 fold at 2.5 ppm); however at 15 ppm arsenic exposure, there was up regulation (1.17 fold) of this stress protein which corroborates with earlier observations where inductions of Hsp90 have been reported in liver of mice treated with inorganic arsenicals [24]. There was not much change (slight down regulation) in Hsp70 expression at all level of arsenic exposure, as compared to control. There was also little change (0.2 fold up regulation) in Hsp60 expression at 15 ppm of arsenic conc.

Fig. 3
figure 3

Immunoblot analysis of heat shock proteins (Hsps) in liver of Labeo rohita. Changes in expression of Hsp70, Hsp60 and Hsp90 proteins were analyzed. Densitometry analyses of immunoblots were carried out to find out the fold changes in stress protein expression at different levels of As-exposure

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In summary, in this study we have reported the typical skin lesions (pigmentation and depigmentation of skin to those seen in human subjects in arsenicosis) and cataract development following arsenic exposure, for the first time. This study is of predictive value for human health as the human-subjects residing in arsenic endemic areas spend their life span there getting naturally exposed to arsenic through food chain which could possibly be predisposing them to early onset of cataract.