Inhibition of Phosphatases in the Digestive Tract of Albino Rats (Rattus-rattus albino)

Florin Masih and R.K. Gautam

Dept. of Zoology, School of Life Sciences, Dr. B.R. Ambedkar University, Agra

ABSTRACT:

The toxic effects of copper nitrate and cadmium nitrate on the activity of two enzymes i.e. Alkaline phosphatase and Acid phosphatase in the digestive tract of albino rat- Rattus-rattus albino have been analysed histochemically. Inhibited enzyme reactions suggested damage to plasma membrane, lysosomes and endoplasmic reticulum. These observations from toxicological point of view have a great significance.

KEYWORDS: Rattus-rattus albino, digestive tract, copper nitrate, cadmium nitrate, Alkaline Phosphatase, Acid Phosphatase.

INTRODUCTION:

In recent years our knowledge about the presence, amount and distribution of heavy metals, like copper and cadmium in the environment has greatly advanced. Development of refined techniques has stimulated a great deal of research on these heavy metals not only in the environment but also in the tissues of the mammals like albino rats (Rattus- rattus albino), large number of albino rats have been reported to suffer from the exposure to the different toxic substances like copper nitrate and cadmium nitrate.

Comparatively very little information is available regarding the distribution of various enzymes in the digestive tract of the albino rats. Acid phosphatase is a hydrolytic enzyme by which the hydrolysis of esters is being done and also help in the autolysis of cells after death. It has been reported by some workers that the Acid phosphatase associated with nucleic acid synthesis and its reduction adversely affected the permeability process and nucleic acid synthesis. Alkaline phosphatase which is a brush border enzyme splits various phosphate ester bonds at alkaline pH and mediate membrane transport mechanism. The present study has been designed to understand the inhibition of these two enzymes i.e. acid and alkaline phosphatase in the digestive tract of albino rats (Rattus- rattus albino) when treated with copper nitrate and cadmium nitrate.

Live specimens of a common rodent the albino rat (Rattus-rattus albino) of Charles foster strain were selected as the test animal from M/S India Biologicals, Agra and were acclimatized to laboratory conditions for a week. The rats were divided into three groups each containing rats of the same age, sex and weight. (70 gm + 5 gm). Members of the (Group A) were treated as control rats. Members of the (Group B) were allowed to feed upon copper nitrate 0.1 gm per kg body weight on each day for thirty days. Rats of the third group (Group C) were allowed to feed on cadmium nitrate at 0.05 gm per kg body weight daily for thirty days. On the 31^st day all the rats of the three groups (A, B, C) were sacrificed with an overdose of Penta-Barbitol sodium followed by exsanguinations.

Table 1: Distribution of Alkaline Phosphatase in the stomach of Rattus-rattus albino after treatment of copper nitrate and cadmium nitrate

Treatment	(Serosa) Tunica Externa	Tunica Muscularis	Tunica submucosa	Tunica mucosa			Glands/ Cells
				Muscularis mucosa	Lamina Propria	Mucosal epithelium	Mucous cells	Oxyntic cells (Parietal)	Zymogenic (Chief)	Argentaffin cells
Control	+++	++	++	++	+	+	+	+	+	+
Copper nitrate	++	+	+	+	+	+	+	-	-	-
Cadmium Nitrate	+	+	+	+	-	-	-	-	-	-

+++ = Very strong activity, + = moderate activity- = Nil activity, ++ = Strong activity, + = Dull activity

Table 2: Distribution of Alkaline Phosphatase in the intestine of Rattus-rattus albino after treatment of copper nitrate and cadmium nitrate

Treatment	(Serosa) Tunica Externa	Tunica Muscularis	Tunica submucosa	Tunica mucosa			Glands/ Cells
				Muscularis mucosa	Lamina Propria	Mucosal epithelium	Mucous cells	Oxyntic cells (Parietal)	Zymogenic (Chief)	Argentaffin cells
Control	++	++	++	+	+	+	+	+	+	+
Copper nitrate	+	+	+	+	+	+	+	+	-	-
Cadmium Nitrate	+	+	+	-	-	-	-	-	-	-

++ = Strong activity, + = Dull activity, + = moderate activity, - = Nil activi

For the localization of target enzymes viz. acid and alkaline phosphatase, the stomach and intestine from the control and experimental rats were removed and fixed in chilled acetone for 24 hours. The tissues were cleared in benzene and embedded in paraffin wax. The paraffin sections were processed for acid and alkaline phosphatase (Gomori, 1952).

Results and Discussion:

Alkaline Phosphatase- (Phosphomonoesterase - I):

Stomach: The stomach of Rattus-rattus albino exhibited very strong positive reaction for alkaline phosphatase in tunica externa (serosa) and strong positive reaction in tunica muscularis, tunica submucosa, muscularis mucosa, while moderate reaction in lamina propria, mucosal epithelium, mucous cell and dull in oxyntic, zymogenic and argentaffin cells. Copper nitrate treatment gave a dull activity in lamina propria, mucosal epithelium, mucous cells, nil activity was observed in oxyntic, zymogenic and argentaffin cells. Serosa showed strong positive reaction while moderate positive reaction was observed in tunica muscularis, tunica submucosa and muscularis mucosa.

After the treatment of cadmium nitrate the activity was extremely affected and became dull in tunica externa (serosa), tunica muscularis, tunica submucosa and muscularis mucosa while it became highly negative in rest of the layers, gastric glands and cells. Details are given in table-1.

Intestine: The controlled intestinal tissue exhibited a strong positive reaction for tunica externa (serosa), tunica muscularis, tunica submucosa, muscularis mucosa, lamina propria, mucosal epithelium and mucous cells exhibited moderate activity while zymogenic and argentaffin cells exhibited dull activity. After copper nitrate treatment the strong positive activity became moderate and moderate activity became dull, zymogenic and argentaffin cells showed negative activity. But after cadmium nitrate treatment the activity became poor in tunica externa (serosa), tunica muscularis and tunica submucosa, tunica mucosa layers and gastric glands and cells showed nil activity. Details are given in table-2.

Acid Phosphatase – (Phosphomonoesterase- II):

Stomach- Strong positive reaction was noticed in tunica externa (serosa) and a moderate reaction was observed in tunica muscularis, tunica sub mucosa, tunica mucosa layers, mucous cells, while dull activity was noticed in oxyntic and zymogenic cells. Argentaffin cells showed nil activity. The effect of copper nitrate gave moderate reaction in serosa and tunica muscularis, dull activity in tunica mucosa and gastric glands gave negative reaction. While cadmium nitrate treatment exhibited an adverse or nil activity in tunica submucosa, tunica mucosa, gastric glands and cells, but showed dull activity in tunica externa, (serosa) and tunica muscularis. Details are given in table-3.

Intestine: The intestine of Rattus-rattus albino showed a positive reaction for acid phosphatase in tunica externa (serosa), muscularis, submucosa, mucosa and mucous cells, but showed dull activity in oxyntic (parietal), zymogenic (chief) and argentaffin cells. Copper nitrate gave a dull activity in all the layers except oxyntic cells, zymogenic cells and argentaffin cells which showed nil activity. Cadmium nitrate showed a poor and feeble reaction in tunica externa (serosa) tunica muscularis tunica submucosa layers while rest of the layers and glands did not respond to any type of activity. Details are given in table-4.

Table 3: Distribution of Acid Phosphatase in the stomach of Rattus-rattus albino after treatment of copper nitrate and cadmium nitrate

Treatment	(Serosa ) Tunica Externa	Tunica Muscularis	Tunica submucosa	Tunica mucosa			Glands/ Cells
				Muscularis mucosa	Lamina Propria	Mucosal epithelium	Mucous cells	Oxyntic cells (Parietal)	Zymogenic (Chief)	Argentaffin cells
Control	++	+	+	+	+	+	+	+	+	-
Copper nitrate	+	+	+	+	+	+	+	-	-	-
Cadmium Nitrate	+	+	-	-	-	-	-	-	-	-

++ = Strong activity, + = Dull activity, + = moderate activity,-= Nil activity

Table – 4: Distribution of Acid Phosphatase in the intestine of Rattus-rattus albino after treatment of copper nitrate and cadmium nitrate

Treatment	(Serosa ) Tunica Externa	Tunica Muscularis	Tunica submucosa	Tunica mucosa			Glands/ Cells
				Muscularis mucosa	Lamina Propria	Mucosal epithelium	Mucous cells	Oxyntic cells (Parietal)	Zymogenic (Chief)	Argentaffin cells
Control	+	+	+	+	+	+	+	+	+	+
Copper nitrate	+	+	+	+	+	+	+	-	-	-
Cadmium Nitrate	+	+	+	-	-	-	-	-	-	-

++ = Strong activity, + = Dull activity, + = moderate activity, - = Nil activity

Alkaline phosphatase is present in every oragan but its physiological role in different localization is still uncertain. In order to establish the exact localization of functional metabolic changes of enzymes, histochemical methods were employed. In stomach both copper and cadmium inhibited alkaline phosphatase. This reflected the damage to plasma membrane. Reaction for alkaline phosphatase in the intestine of copper and cadmium fed rats also reduced alkaline phosphatase activity, stomach also exhibited lysosomal damage. Loss in acid phosphatase activity from the stomach and intestine of copper and cadmium fed rats exhibits lysosomal damage. Copper is known to form inclusion bodies that alter the lysosomal structure and function. Inhibition in the activity of acid phosphatase in the target tissues after heavy metals was noticed. Rapid loss of lysosomes from atrophine cells may be another reason for the diminished activity of acid phosphatase. One more reason in the fall of enzyme activity is due to a reflection of slow metabolism of nerve cells which are in the process of atrophy.

Acknowledgement:

The authors are thankful to Dr. Shakti Bharadwaj, Head department of Environment toxicology and Dr. Wilson Masih for providing constant support in this work.

References:

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Received on 14.12.2009

Accepted on 12.02.2010

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Research J. Science and Tech. 2(3): May –June. 2010: 57-59