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Histoenzymological study on the toxicity of copper sulphate in the

digestive glands of Lymnaea luteola

Sarvesh Mathur and Ashok Kumar Gupta*

College of Fisheries, Department of Aquaculture, Maharana Pratap University of

Agriculture and Technology, Udaipur - 313 001, India

(Received: August 30, 2005 ; Revised received: March 26, 2006 ; Re-revised received: August 18, 2006 ; Accepted: August 28, 2006)

Abstract: During 24 and 48 hr of exposure, the digestive glands of Lymnaea treated with a lethal concentration of 0.038 mgl-1 CuSO4 revealed intense activity

of acid phosphatase in perilobular margin. On the other hand, same area of the gland showed moderate activity of ATPase during 24 and 48 hr of exposure.

However, alkaline phosphatase showed average activity in perialveolar region and perilobular margin during 24 and 48, and 72 hr of exposure respectively. The

changes in the activity of these enzymes were nonsignificant in alveolar margin and perialveolar region of the gland. It is interesting to note moderate activity

of acid phosphatase in perialveolar region during 24 hr of exposure.

Key words: Acid phosphatase, Adenosine triphosphatase, Alkaline phosphatase, Copper sulphate, Digestive gland, Lymnaea luteolaPDF of full length paper is available with author ( *drashokgupta9712@rediffmail.com)

Introduction

Enzymatic malfunctioning can cause a rapid breakdown of

cell components that are otherwise quite stable leading to death of an

organism. Many toxic chemicals are known to affect the permeability

of cell membranes, disturb energy metabolism and cell functions by

releasing hydrolases due to increased fragility of lysosomal

membranes. Chemically induced alternations in enzymatic activities,

can be used to monitor the functional alterations in the intoxicated

tissues of an organism (Zonek et al., 1966; Jackim et al., 1970;

Hinton et al., 1973). Alkaline phosphatase, acid phosphatase and

adenosine triphosphatase (ATPase) are the hydrolytic enzymes

which release phosphoric acid from different substrates. The

susceptibility of these enzymes to toxic challenge is well established

in the vertebrates (Jackim et al., 1970; Hinton et al., 1973; Sastry

and Agrawal, 1977; Srivastava and Pandey, 1982; Gupta and

Rajbanshi, 1985; Watson and Benson, 1987; Benders et al., 1994;

Li et al., 1996, 1998; De Boeck et al., 2001; David et al., 2003) and

to some extent in the invertebrates (Banna, 1979; Satyaparameshwar

et al., 2006; Lodhi et al., 2006). Histoenzymological studies on the

tissues of snails exposed to molluscicide toxicity are scanty. Therefore,

an attempt was made to investigate the effect on hydrolytic enzymes,

viz., acid phosphatase, alkaline phosphatase and ATPse in digestive

glands of normal and molluscicide (copper sulphate) exposed

Lymnaea luteola.

Materials and Methods

Live specimens of Lymnaea were collected from the Kamal

Talai pond, Udaipur and acclimatized to laboratory conditions for a

period of ten days on lettuce diet. The digestive glands of control and

treated snails were excised by sacrificing living specimens from static

bioassay (APHA, 2005) used for copper sulphate (CuSO4.5H

2O)

as toxicity (0.038 mgl-1) at different time intervals of 24, 48 and 72

hours. The tissues were immediately washed in a jet of saline (1%

NaCl solution) and fixed in 10% chilled neutral formaline for varying

length of time. The fixed frozen sections were cut at 10 µ and then

processed for histochemical localization for following enzymatic

reactions.

1. Acid phosphatase: Lead nitrate method (Gomori, 1941).

2. Alkaline phosphatase: Calcium cobalt method (Gomori and

Takamatsu, 1939).

3. ATPase: Calcium method (Padykula and Herman, 1955a, b).

For the above enzymatic reactions proper controls were

run simultaneously.

Results and Discussion

A comparative account of the distribution and activities of

three hydrolytic enzymes viz., acid phosphatase, alkaline

phosphatase and ATPase in control and copper sulphate exposed

digestive glands of Lymnaea, at different time intervals are

summarised in Table 1. The enzymatic activities were assessed

visually and from the figures (Plate 1, Fig. 1-6 for acid phosphatase

and Plate II, Fig. 1-6 for alkaline and ATPase) at high magnification.

The three hydrolytic enzymes studied are known to be involved in

the metabolic processes of cells. An increased activity of acid

phosphatase in the perilobular margin of the digestive gland after 24

hr appears to be due to the cellular breakdown and release of acid

phosphatase from the ruptured lysosomes. Acid phosphatase is a

lysosomal enzyme and plays role in the synthesis of cell organelles

(Novikoff, 1960; Thakar and Tewari, 1967; Bresnick and Schwartz,

1968). Stress factors are known to affect some hydrolases, the release

of which from their membrane binding sites lead to the autolysis of the

cell (Moore and Halton, 1973; Moore and Stebbing, 1976; Establier

et al., 1984). Increased activity of acid phosphatase has also been

reported in the manganese exposed rabbit testes (Zonek et al.,

1966) and mercury exposed fish kidney (Sastry and Agrawal, 1977).

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202

Table - 1: Activities of acid phosphatase, alkaline phosphatase and ATPase in different regions of digestive glands of L. luteola, exposed to lethal concentration

of copper sulphate

Concentration of CuSO4 (mgl-1)

Digestive gland

Alveolar margin Perialveolar reigon Perilobular margin

(AM) (PAR) (PLM)

0.00 mgl-1 CuSO4 (control)

Alkaline phosphatase + + ++

Acid phosphatase - + + +

ATPase - + +

Duration 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr

0.038 mgl-1 CuSO4

Alkaline phosphatase + - - + ± + ++ +++ +

Acid phosphatase + + - + + - +++ +++ +++

ATPase + - - + + + ++ ++ +

+++ Intense; ++ Moderate; + Average; + Dull; - Nil

Plate - 1: Photomicrographs showing histochemical localization of acid phosphatase in digestive gland of L. luteola exposed to lethal concentration of

copper sulphate (0.038 mgl-1)

Fig. 1,2: Localization of acid phosphatase in three regions of digestive gland from control groups. X 280 and X 700

Fig. 3,4: Sections exhibit dull activity of acid phosphatase in alveolar margin (AM) and perialveolar region (PAR) after 24 and 48 hr exposure of copper sulphate X 280

Fig. 5,6: After 72 hr exposure of copper sulphate the digestive gland exhibits negative activity of acid phosphatase in alveolar and intense in perilobular margin

(PLM). X 280 and X 700

Sarvesh Mathur and Ashok Kumar Gupta

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203Toxicity of copper sulphate in the digestive glands of Lymnaea

It is interesting to note that acid phosphatase activity was inhibited in

the alveolar margin and perialveolar region of digestive gland in

copper sulphate treated Lymnaea. Inhibition of acid phosphatase

activity was also observed in kidney and liver of the channel catfish,

lctalurus punctatus after 24 hr of mercury treatment (Hinton et al.,

1973) and in different tissues of Heteropneustes, Ophiocephalus

and Lebistes exposed to sublethal dose of mercury, lead and zinc

(Sastry and Gupta, 1979; Sastry et al., 1981; Gupta and Rajbanshi,

1985; Sharma and Sharma, 2005). This decrease in activity has

been attributed to the necrosis of affected cells or the direct binding of

metals to the enzyme proteins associated with lysosomal damage.

Alkaline phosphatase is a brush border enzyme involved in

transphosphorylation reaction and transport of mediated membranes

(Goldfischer et al., 1964). The activity of alkaline phosphatase

generally decreased on exposure to CuSO4 in the digestive gland

of Lymnaea. Zinc treated developing Lebistes reticulatus also

showed moderate activity of alkaline phosphatase in hepatocyte as

compared to strong activity in untreated (control) liver (Sharma and

Sharma, 2005). Reduction in alkaline phosphatase activity of digestive

gland was probably due to the transphosphorylation reaction, which

is adversely affected by the impairment of mitochondrial function

(Zalme et al., 1976) or as a sequal of the reduction in chemical

Plate - 2: Photomicrographs showing histochemical localization of alkaline phosphatase and ATPase in digestive

gland of L. luteola exposed to lethal concentration of copper sulphate (0.038 mgl-1)

Fig. 1:Localization of alkaline phosphatase activity in three regions of digestive gland from control group. X 312

Fig. 2:Digestive gland exhibits dull activity of alkaline phosphatase in alveolar margin (AM), average in perialveolar region (PAR) and moderate in perilobular

margin (PLM) after 24 hr in lethal concentration of copper sulphate (0.038 mg/l). X 787

Fig. 3:After 48 hr treatment of CuSO4, digestive gland shows nil activity of alkaline phosphatase in alveolar margin (AM), dull in perialveolar region (PAR) and

intense in perilobular margin (PLM). X 312

Fig. 4:The section exhibits localization of adenosine triphosphatase in digestive gland from control group. X 787

Fig. 5:After 24 hr exposure of CuSO4 ATPase showing dull activity in alveolar margin (AM), average in perialveolar margin (PAM) and moderate in perilobular

region (PLR). X 787

Fig. 6:After 48 hr exposure digestive gland showing nil activity of ATPase in alveolar margin, average in perialveolar region and moderate to intense in

perilobular margin. X 787

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204

perception and ion uptake on one hand, and control of water

absorption on the other (Banna, 1979).

ATPase is an important hydrolytic enzyme which has

significant role in energy metabolism, transphosphorylation of

substrate, maintenance of Na/K balance and exchange of ions and

nutrients through the plasma membrane (Skou, 1965; Booij, 1966).

In the present study, increase in ATPase activity in the digestive

gland is probably reflective of the organisms effort in escalating its

energy metabolism to cope with the toxic stress of CuSO4 and to

survive. Copper and other positively toxic metals are known to

cause a variety of cellular and enzymological alterations regulating

in death of organism (Jackim et al., 1970; Establier et al., 1984; De

Boeck et al., 2001).

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Sarvesh Mathur and Ashok Kumar Gupta