Cadmium Induced Histopathological Changes in the Intestine of Indian Flying Barb, Esomus danricus

Indian flying barb (Esomus danricus) was exposed to sublethal concentrations of 636.3, 63.6 and 6.3 µgl -1 Cadmium for 28 days and intestinal histopathology was observed by light microscopy after staining with Haematoxylin-Eosine. Exposed fishes showed severe to mild superficial erosion of mucosa, dense lamina propria, chronic inflammatory cell infiltration as well as vacuolation. With the increase in exposure dose, severity of effects was observed.


INTRODUCTION
Cd is a non essential heavy metal, mainly used for rechargeable nickel-cadmium batteries, pigments, coatings and plating, and as stabilizers for plastics 1 .Cadmium naturally occurs in the aquatic environment, but is of no known biological use and is considered one of the most toxic metals 2 .Inhalation of cadmium-containing fumes can result initially in metal fume fever but may progress to chemical pnemonitis, pulmonary edema 3 , Itai-itai disease, renal abnormalities, including proteinuria and glucosuria and finally death 4 .Cd has been shown to inhibit enzyme integrity, interfere with RNA and protein synthesis and to complex with DNA.In aquatic systems, cadmium quickly partitions to sediment, but is readily remobilized through a variety of chemical and biological processes 4,5 .The Indian flying barb, Esomus danricus (Hamilton-Buchanan) is an economically important cyprinid fish which mostly inhabits shallow water bodies of Northern India.This fish, owing to its particular habitat, runs the risk of being exposed to water borne toxicants including, Cd.It is very much essential to devise a rapid method to detect the effects of toxicants in various organs of fish and histopathology is one such effective tool 6 .Amongst various organs, very little is known about the effects of Cd on the fish intestine which is believed to be the first organ that come into contact with foodborne contaminants 7 .Also, the intestine is one of the most important sites where food enters and is assimilated.Thus, toxic substances, that enter the intestine, directly affect the vitality of the organism 8 .Therefore intestine can serve as a potent indicator for water borne Cd.The present study was thus, aimed to determine the histopathological effects of chronic doses of Cd to Indian flying barb intestine.

MATERIALS AND METHODS
Fishes of similar length (46.77 ± 4.30 mm) and weight (0.86 ± 0.16 g) were collected from unpolluted, freshwater ponds near Assam University campus, Barak valley, South Assam, India 9 .They were acclimatized under laboratory conditions seven days prior to experimentation.Temperature, pH, hardness and dissolved oxygen under laboratory condition were 29 °C, 6.8, 30 mg l - 1 and 5.5 mg l -1 respectively.A stock solution of actual concentration of Cd was prepared using double distilled water.Serial dilutions of stock solutions were prepared using tap water as per dilution techniques 10 .Three sub-lethal test concentrations viz., 636.3, 63.6 and 6.3 µgl -1 Cd were selected for inducing histological changes in fish intestine.Ten fish for each concentration of test chemical were kept separately in three litres of toxicant treated media for 28 days.Food was given during the study period.Test water was renewed every 24 hrs.After 28 days of exposure, fish were sacrificed and intestine was removed immediately and kept in 10% Formalin, as fixative, for 24 h, dehydrated, embedded in paraffin and sections cut at

RESULTS
The intestine of Esomus danricus shows four layers of tissues namely serosa, muscularis, submucosa and mucosa.The outermost serosa is followed by a well developed muscularis (longitudinal and circular muscle) embedded in loose connective tissue richly supplied with blood capillaries.It merges with tunica propria of the underlying mucosal coat.The mucosa is raised into several longitudinal folds (Fig. 1).In 636.3 µg l -1 of Cd administered E. danricus intestine after 28 days of exposure, superficial erosion of the mucosa, vacuolation and chronic inflammatory cell infiltration were seen.Lamina propria became dense (Fig. 2).For similar exposure duration, 63.6 µg l -1 of Cd exposed intestine showed mild mucosal lesion and infiltration of lymphocytes (Fig. 3).In 6.36 µg l -1 of Cd administered intestine, infiltrations of inflammatory cells (lymphocytes) were observed (Fig. 4).

DISCUSSION
In the present study, the result of the effect of Cd on the gastrointestinal system of Esomus danricus clearly show that this heavy metal exert toxic effects on the different layers of intestine.The alterations in the intestine of the flying barb were more severe in higher doses.Toxic lesions most common in the intestine of fishes exposed to cadmium chloride include hyperemia, degenerative changes in the tips of villi, loss of structural integrity of mucosal folds, degenerative mucosal epithelium (hypertrophy, vacuolation, hyper-chromasia) necrosis, desquamation of mucosal epithelium, cellular debris, excessive mucus in gut of lumen, necrosis of submucosa and inflammatory infiltration of submucosa [11][12][13] .Histopathological change due to Cd, like hypertrophy may lead to increased serum glucose in the intestine, as seen in estuarine teleost fish, and is possibly due to the fulfilment of extra energy requirement under stress condition 11 .In another study, cadmium exposure caused degenerative changes in the tips of villi like hydropic degeneration, cloudy swelling and necrosis of intestine of Ophiocephalus striatus 14 .In the intestine of Channa punctatus exposed to mercuric chloride, the cytoplasmic hyperchromasia, edema, loss of pepsinogen granules from chief cells, disintegration of glandular epithelium, desquamation of gastric mucosa in the stomach, hyperemia, degenerative changes in the tips of mucosal folds, hypertrophy and necrosis were observed 15 .Similarly, intestinal toxic lesions, includes hyperemia, loss of structural integrity of mucosal folds, necrosis, cellular debris, vacuolation in intestine of Mugil auratus exposed to inorganic and organic mercury were also observed in another study 16 .On exposure to another heavy metal, lead, hypersecretion of pepsin, leading to the degradation of tissue protein and increased ammonia and urea excretion by Channa punctutus was observed 17 .Such conditions are possibly due to the extremely adverse effects on stomach of fish due to lead nitrate toxicity.Although, the present study did not include these findings, yet we observed severe erosion of mucosa at highest sublethal exposure dose of Cd, which might hamper the normal gastrointestinal physiology.Besides, profuse infiltration of lymphocytes were observed in flying barb intestine, which was a sign of chronic inflammation.Thus, Indian flying barb intestine was adversely affected by Cd exposure which in turn might have impaired the growth rate of fishes.Hence, it is suggested that this heavy metal in higher dosage will be lethal to fish population in general.
5 µm thickness and stained with Harris Haematoxylin and Eosin.Changes induced by treatment in the intestinal tissues were photographed and analyzed by light microscope at 10X eye piece magnification and 40X objective magnification {Olympus (model U-CMAD3) with Camera attachment of Samsung (model SDC-313B)}.The work was done as per the Assam University Ethical guidelines on laboratory animal care.