IN VIVO S U B - A C U T E P H Y S I O L O G I C A L S T R E S S I N D U C E D BY

S U M I T H I O N ON S O M E A S P E C T S OF O X I D A T I V E

M E T A B O L I S M I N T H E F R E S H W A T E R C R A B

A. B H A G Y A L A K S H M I , P. S R E E N I V A S U L A R E D D Y , and R. R A M A M U R T H I

Department of Zoology, Sri Venkateswara University, Tirupati - 517502, India

(Received September 7, 1983; revised November 30, 1983)

Abstract. Specimens of fresh water crab, Oziotelphusa senex senex were exposed to sub-lethal concentration (25 To of LC5o/48 hr) of Sumithion for 7 days. S umithion was found to inhibit the activity levels of acetylcho- linesterase, succinate dehydrogenase, isocitrate dehydrogenase and pyruvate dehydrogenase, activate lac- tate dehydrogenase and cause accumulation of acetylcholine in the hepatopancreas and muscle. The increase in lactate dehydrogenase and the decrease in succinate, isocitrate and pyruvate dehydrogenases in these tissues indicate the development of anaerobic conditions and is interpreted as a functional adaptation to pesticide induced metabolic stress.

1. Introduction

The organophosphate insecticides are highly effective for the control of mosquitos and agricultural pests in many parts of the world (Muirhead-Thompson, 1971; Duke, 1977). Since these insecticides are non-persistent, they are used repeatedly, and the danger to non-target organisms such as fish, prawns and crabs, the natural enemies of the insect larvae and important members of the food chain, are adversely affected. The tragic incidence of 'Handigodu syndrome' in Karnataka has been attributed to long term consumption of pesticide poisoned crabs and fish by the local population (NIN, 1977). In view of this an elaborate program to evaluate the impact of pesticides on the physiology and biochemistry of several non-target species of aquatic ecosystem has been undertaken. The present paper explains the toxic effects of sublethal concentrations of Sumithion (0-0-dimethyl(-0-3 methyl-4-nitrophenyl) phosphorothioate), which is ex- tensively used locally on certain biologically important enzymes, i.e., acetylcholinester- ase, succinate dehydrogenase, isocitrate dehydrogenase, pyruvate dehydrogenase and lactate dehydrogenase, and acetylcholine levels in hepatopancreas and muscle of the rice field edible crab, Oziotelphusa senex senex. The purpose of this study is to obtain information about the biochemical disturbances in the crab on exposure to Sumithion and to increase our understanding of its mode of action.

2. Materials and Methods

Animals: Healthy, adult, intermolt (Stage C4 according to Sreenivasula Reddy, 1981) male specimens of Oziotelphusa senex senex (weight, 30 + 2g; carapace width, 30 + 4 mm) were collected from local rice fields. They were acclimated to laboratory

Water, Air, and Soil Pollution 23 (1984) 257-262. 0049-6979/84/0233-0257500.90. © 1984 by D. Reidel Publishing Company.

258 A. BHAGYALAKSHMI ET AL.

conditions for 10 days before experimentation. They were fed frog muscle daily, but food was withheld during the experimental period. The properties of tap water were as follows: Temperature 25 + 1 °C; pH 7.3; dissolved oxygen content, 6.2 ppm; hardness 38 ppm of CaCo 3.

Pesticide: Technical grade (96~ W/V) Sumithion (Fenitrothion; 0-0-dimethyl-0 (-3 methyl-4-nitrophenyl) phosphorothioate) obtained from Rallis India Ltd (Bangalore) was used. Sumithion was dissolved in acetone and diluted with tap water so that the final concentration was 0.1 mg L - 1 in 0.001 ~o acetone. Control crabs were also kept in tap water containing the same acetone concentration (0.001~o).

Experimental design: Sixty crabs were exposed for 7 days at 0.1 mg L - 1, 25 ~o of the 48 hr LCso of Sumithion (Bhagyalakshmi and Ramamurthi, 1981). Controls were also set up for comparision. The experimental medium was changed every 24 hr with freshly prepared medium of the same concentration so as to compensate for any change in the concentration of chemical due to degradation or uptake. After 1, 3, and 7 days hepato- pancreas and muscle tissues were dissected over ice, from both control and exposed crabs, placed in crab ringer (Van Harreveld, 1936) and stored in prechilled glass tubes until analyzed.

Enzyme assays: The isolated tissues were minced with scissors and homogenized (2 ~o weight/volume) in cold sucrose solution (0.25 M) at 4 °C in an ice-Jacketed-glass homogenizer with a motor-driven Teflon-coated pestle. The homogenate was centrifug- ed at 1000 x g for 10 min at 4 °C to remove unbroken cells and particulate debris. The obtained cell-free supernatants was utilized as the enzyme source.

Measurements of enzyme activities were performed at 37 °C with appropriate en- zyme and reagent blank using a spectrophotometer. Preliminary experiments establish- ed the optimal conditions for each enzyme with respect to pH, and substrate and cofactor concentration (Bhagyalakshmi, 1981). Optimal as say conditions for individual enzymes (nomenclature, IUB, 1978) were as follows:

acetylcholinesterase, (ACHE) acetylcholin aceylhydrolase EC 3.1.1.8: AChE was assayed (Metcalf, 1957) in a medium of 2 mL which contained: 8 I~moles of acetylcho- line chloride, 100 gmol of phosphate buffer (pH 7.4) and 1 mL of enzyme source.

Succinate dehydrogenase, (SDH) Succinate oxidoreductase, EC 1.3.99.1: SDH was assayed according to Nachlas et al. (1960) using 100 gmol of phosphate buffer (pH 7.4), 40 gmol of sodium succinate, 2 gmol of INT (2-4 Iodophenyl-3 (4 nitrophenyl)-5 phenyl tetrazolium chloride) and 0.5 mL of enzyme source in 2 mL of volume.

Pyruvate dehydrogenase, (PDH) Pyruvate oxidoreductase, EC 1.2.3.3: PDH was assayed (Srikanthan and Krishnamurthy, 1955) in a medium of 2 mL which contained: 100 gmol of phosphate buffer (pH 7.4), 40 gmol of sodium pyruvate, 2 ktmol of INT, 0.1 gmol of NAD (Nicotinamide adenine dinucleotide) and 0.4 mL of enzyme source.

Isocitrate dehydrogenase, (ICDH) Isocitrate oxidoreductase, EC 1.1.1.41: ICDH was assayed by the method of Kornberg and Pricer (1951). The reaction mixture in a final volume of 2 mL contained: 20 lamol of DL-isocitrate, 100 gmol of phosphate buffer (pH 7.4), 4 lamol of INT, 10 lamol of magnesium chloride, 0.2 gmol of ADP, 0.2 gmol of NAD and 0.4 mL of enzyme source.

OXIDATIVE METABOLISM IN THE FRESH WATER CRAB 259

Lactate dehydrogenase, (LDH) L-Lactate: (NAD) oxidoreductase EC 1.1.1.27: LDH was assayed according to Srikanthan and Krishnamurthy (1955) in a medium of 2 mL which contained: 100 gmol of phosphate buffer (pH 7.4), 40 ~tmol of sodium lactate, 2 gmol of INT, 0.1 gmol of NAD and 0.4 mL of enzyme source.

For determination of acetylcholine (ACh) content the method of Augustinsson cited in Glicks (1957) was used. The protein contents in the enzyme source were determined by the Lowry etal. (1951) method, using bovine serum albumin as the standard. Student's t-test was employed to calculate the statistical significance between control and experimental values.

3. Results and Discussion

During the exposure time to a sublethal concentration of Sumithion, observations were made to detect external signs of poisoning in the experimental crabs. The exposure to

T A B L E I

Effect of Sumith ion on some aspects of oxidative metabolism in the hepatopancreas of a fresh water field crab a

Parameter G r o u p Exposure interval (days)

1 3 7

A C h E b Control 1.29 + 0.06 1.27 ± 0.09

Experimental 1.09 _+ 0.08 f 0.92 ± 0.07 f

- 15.5070 - 27.5670

S D H ° Control 148.74 ± 27.12 146.63 ± 21.17

Experimental 118.65 ± 10.41 e 77.56 ± 9.52 f

- 20.24 Yo - 47.11 9o

I C D H ° Control 35.69 ± 7.45 36.07 ± 5.41

Experimental 21.66 ± 4.67 f 17.13 ± 6.11 f

- 39.48 Yo - 52.51 70

P D H ° Control 9.66 _+ 0.54 9.81 ± 0.79

Experimental 6.36 ± 0.31 f 5.01 ± 0.16 f

- 34.1670 - 48.8170

L D H ~ Control 7.31 ± 0.66 7 . 4 9 ± 0.54

Experimental 8.69 ± 0.71 e 9.60 ± 2.51 f

+ 18.88 Yo +28.1770

A C h a Control 1.39 ± 0.01 1.38 _+ 0.02

Experimental 1.52 ± 0.02 e 1.79 ± 0.03 f

+9.3570 +29.7170

1.31 +_ 0.05

0.78 ± 0.08 f

- 40.46 70

152.66 ± 20.09 57.64 _+ 8.64 f

- 62.2870

37.33 ± 6.63

14.29 ± 1.58 f

- 61.71Yo

9.67 + 0.31

4.89 ± 0.21 f

- 52.709°

7.56 ± 0.73

12.12 ± 1.86 f

+ 60.329°

1.34 ± 0.02

2.09 ± 0.08 f

+55.9670

a All values in the table represent the mean _+ SD (N = 10) followed by 70 alteration; b n moles of A C h hydrolyzed/mg protein/h; c n moles of f o r m a z a n formed/mg protein/h; d n moles/g wet tissue; e P < 0.01;

f P < 0.001, student's t-test.

260 A. BHAGYALAKSHMI ET AL.

Sumithion results in sluggish behavior and uncoordinated movements. The crabs after 5 days of Sumithion exposure, showed difficulty in righting themselves (Bhagyalakshmi, 1981). After 7 days, dark frothing fluid was extruded from the mouth, which probably indicates the onset of pathological conditions. Experimental crabs after 5 days displayed inability to feed because of their uncoordinated movements, but there was no mortality up until day 7.

The quantitative changes in ACHE, SDH, ICDH, PDH, and LDH and ACh levels in hepatopancreas and muscle of control and treated crabs after 1, 3, and 7 days exposure to Sumithion are expressed as mean + SD along with percent alteration in Tables I and II.

A significant inhibition in AChE activity was observed in both the tissues after exposure to Sumithion. The maximum ( - 52 ~o) inhibition in AChE activity was observ- ed in muscle after 7 days of exposure and the minimum ( - 15.5~/o) in hepatopancreas after 24 hr of treatment. Coppage (1972) suggested that there is a critical level of enzyme inhibition in the range of 75 to 80~o in cyprinodon, which serves as an index of fish

T A B L E II

Effect o f S u m i t h i o n o n some a spec t s o f ox ida t ive m e t a b o l i s m in the musc l e o f a f resh w a t e r field c r a b a

P a r a m e t e r G r o u p E x p o s u r e in te rva l (days )

1 3 7

A C h E b C o n t r o l 3.42 + 0.30 3.40 + 0.27 3.36 + 0.31 E x p e r i m e n t a l 2 . 4 4 + 0.25 f 2 . 2 0 + 0.24 f 1.61 + 0.11 f

- 2 8 . 6 6 ~ o - 35.29 ~o - 52.08 ~o

S D H ° C o n t r o l 127.55 + 17.01 126.37 + 15.31 133.39 + 11.07

E x p e r i m e n t a l 103.16 + 10.29 e 100.84 + 6.20 f 71.56 + 12.04 f

- 19.12~o - 20.20~o - 46.36~o

I C D H c C o n t r o l 74.94 + 7.99 75.66 + 8.04 73.66 + 5.01

E x p e r i m e n t a l 52.50 + 10.03 f 46 .52 _+ 4.55 f 29.72 ___ 6.55 r

- 29.94 ~o - 38.51 ~o - 59.65 ~o

P D H ° C o n t r o l 4.31 + 0.22 4.66 + 0.21 4.59 + 0.19

E x p e r i m e n t a l 2.96 + 0.18 f 2.04 + 0.19 f 2.01 ___ 0.11 f

- 31.55~o - 56.22~o - 56.29 ~o

L D H ° C o n t r o l 20.67 + 1.07 21.27 + 1.97 20.37 + 1.99

E x p e r i m e n t a l 27.50 + 5.61 e 34.14 + 3.10 e 35.97 _+ 4.18 r

+ 33.04~o + 60.51 ~o + 76.58 ~o

A C h d C o n t r o l 2 . 0 2 + 0.04 2 . 0 4 + 0.06 2.01 _+ 0.07

E x p e r i m e n t a l 2.60 _+ 0.05 f 3.86 + 0.71 f 4.03 + 0.11 f

+ 28.47~/o + 89.03~o + 100.48~o

a All va lues in the t ab le r e p r e s e n t the m e a n + S D (n = 10) fo l lowed b y ~o a l t e ra t ion ;

u n moles o f A C h h y d r o l y z e d / m g p ro t e in /h ; ° n moles o f f o r m a z a n f o r m e d / m g p ro t e in /h ;

a n mo le s /g we t t i ssue;

P < 0.01; f P < 0.001, s tuden t ' s t-test.

OXIDATIVE METABOLISM IN THE F R E S H WATER CRAB 261

poisonign leading to death. In the present study, the inhibition of enzyme activity increased with duration of exposure to sublethal concentration. Similar results were obtained by Coppage and Mathews (1974), Coppage and Braldech (1976), Kabeera- hammad Sahib and Rao (1980), and Rath and Misra (1981).

With AChE inhibition, there was a concurrent increase in ACh content after Sumithion intoxication. The increase in ACh content was progressive with exposure in both the tissues and maximum accumulation was observed in muscle (100~) after 7 days of treatment. The behavioral changes observed in the crab to Sumithion, corre- late with changes in enzyme activity and metabolites. Initially, the crabs showed un- coordinated movements, but, as time progressed, they started to lose equilibrium. This reflects the effects of ACh accumulation at nerve endings, thus disrupting the synaptic transmission of nerve impulses from one neuron to another.

ICDH and SDH, the key citric acid cycle enzymes, were also found to be significantly inhibited by Sumithion. The inhibition of ICDH and SDH increased progressively with exposure time to sublethal concentration. The decrease in ICDH and SDH activity levels suggest that the lowered level of operation of the citric acid cycle at tissue level might in turn be responsible for decreased 02 consumption at whole animal level after Sumithion exposure (Bhagyalakshmi, 1981).

LDH catalyzes the conversion of pyruvic acid into lactic acid under anaerobic conditions and PDH into acetyl Co-A under aerobic conditions. The stimulation of LDH was found to be significant in both the tissues of the crab after 1, 3, and 7 days of exposure to Sumithion. However, the PDH activity significantly decreased. The inhibition of PDH and the stimulation of LDH indicate that pyruvate is not produced via citric acid cycle, but via the lactic acid cycle with the formation of lactic acid in the tissues (Bhagyalakshmi, 1981). Further, the inhibition of PDH, SDH, and ICDH and the stimulation of LDH indicate favoring anaerobic metabolism in Sumithion-stressed crabs. Such an assumption is supported by the findings of Koundinya and Ramamurthi (1978a) and Sastry and Siddiqui (1982) who observed similar elevation in LDH activity and inhibition of SDH and PDH activities in the tissues of fish exposed to Sumithion and Sevin (1-napthyl N-methyl carbamate). In the stressed crabs, various deleterious structural alterations occurred in the gills (author's unpublished data), which affect the respiratory exchange and depleted the 02 uptake (Bhagyalakshmi, 1981), so that anaerobic conditions developed at the tissue level. This view has been supported by a number of workers who found that impaired respiratory function induced by Sumithion (Koundinya and Ramamurthi, 1978b) Methyl parathion (0-0-dimethyl-0-(4-nitrophe- nyl) monothiophosphate) (Nagarathnamma and Ramamurthi, 1982) and Sevin (Koundinya and Ramamurthi, 1981) leads to metabolic depression. Similarly a re- duction in 0 2 consumption in Oziotelphusa after the sublethal and lethal intoxication by Sumithion has been observed by Bhagyalakshmi (1981).

262 A. BHAGYALAKSHMI ET AL.

4. Summary

It can therefore be inferred that the Sumithion stressed crabs met their bio-energetic requirements through anaerobic oxidation as indicated by elevated LDH activity. This was a functional/physiological adaptation to Sumithion intoxication and these enzymes may be used as a good indicator for monitoring the stress condition in crabs.

Acknowledgments

We gratefully acknowledge the CSIR, New Delhi for financial support during the tenure of which the present work was carried out. Thanks are also due to Rallis India Ltd. (Bangalore) for the generous sypply of technical grade sumithion.

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