Changes in hemolymph glucose, hepatopancreas glycogen, total carbohydrates, phosphorylase and amino transferases of sumithion-stressed freshwater rice-field crab (Oziotelphusa senex senex)

277

Toxicology Letters, 18 (1983) 277-284 Elsevier

CHANGES IN HEMOLYMPH GLUCOSE, HEPATOPANCREAS GLYCOGEN, TOTAL CARBOHYDRATES, PHOSPHORYLASE AND AMINO TRANSFERASES OF SUMITHION-STRESSED FRESHWATER RICE-FIELD CRAB (OZIOTELPHUSA SENEX SENEX) (Crustacean;

pesticide; glucogenesis)

A. BHAGYALAKSHMI,

P. SREENIVASULA

REDDY*

and R. RAMAMURTHI

Department of Zoology, Sri Venkateswara University, Tirupati - 517502 (A.P.) (India) (Received

March

(Revision

received

(Accepted

18th, 1983) April

May l&h,

19th, 1983)

1983)

SUMMARY Adult crabs were exposed to 3 concentrations Hemolymph

glucose

demonstrating the controls

Significant

and elevations

when compared influences

of sumithion

in each group

with a control

group

over a 20-day period.

over the course of the experiment.

The group

the most acute and sustained hyperglycemia (2 ppm sumithion) was then analyzed, for changes in hepatopancreas glycogen, total carbohydrates, phosphorylase

aminotransferases. bohydrates

levels were monitored

(P
of phosphorylase

with controls.

of hyperglycemic

These changes hormone,

depressions

in hepatopancreas

and transaminases are discussed

which is released

were observed

glycogen

and

total

in sumithion-stressed

with respect to gluconeogenesis into hemolymph

of stressed

as also and carcrabs

and possible

crab.

INTRODUCTION

Due to indiscriminate and widespread use of pesticides in agricultural and public health operations, many non-target species, some of them important members of food chain, are adversely affected. The tragic incidence of ‘Handigodu syndrome’ of Karnataka has been suggested to be long-term consumption of pesticide-exposed crabs and fish by the local population [l]. In view of this, an elaborate programme to evaluate the impact of pesticides on the physiology and biochemistry of some selected ‘non-target’ animals of the rice-field ecosystem has been undertaken. * To

whom

Abbreviations:

0378-4274/83/$

correspondence AAT,

should

aspartate

be addressed.

aminotransferase;

03.00 0 Elsevier

Science Publishers

AlAT,

B.V.

alanine

aminotransferase.

278

of

Previous studies by our group have shown sumithion (10 hg) to crabs induces

acetylcholinesterase [2]. This

report

activity discusses

that administration of a single dose a significant inhibition in the of nerve mass after 1, 3, 7, 14 and 24 h of intoxication

some

of the

biochemical

changes

in the

crab

after

sumithion intoxication. Crabs were exposed to 3 concentrations of sumithion over 20 days and the group demonstrating the most acute and sustained hyperglycemia was analyzed as also the controls for changes in hepatopancreas glycogen, total carbohydrates, phosphorylase and aminotransferases. MATERIALS

AND

METHODS

Animals Healthy freshwater rice-field crabs (Oziotelphusa senex senex) were collected from local rice fields. 300 crabs with a body weight of 30 -t 2 g (mean * SD) were placed in glass aquaria and allowed to acclimatize for 2 weeks. Ambient temperature was maintained at 26 + 2°C and relative humidity 85 rt 5% under a 12:12 (06.00 to 18.00; 18.00 to 06.00 h) light-dark regimen. The crabs were fed an ad lib. diet of frog muscle daily. The medium in which they were placed was changed after each 24 h.

Pesticide Technical grade (96% w/v) sumithion (fenitrothion; 0,0-dimethyl-O-(3 methyl-4-nitrophenyl) phosphorothioate) obtained from Rallis India Ltd (Bangalore) was used. Sumithion was first dissolved in ethanol and diluted with tap water so that the final concentration was 0.5, 1 and 2 ppm containing 0.001% ethanol. The control crabs were kept in tap water containing the same ethanol concentration (0.001%) as in tap water containing sumithion.

Experimental

design

Following the acclimatization of sumithion. A fourth group group at various time intervals

period, the crabs were exposed to 0.5, 1 and 2 ppm served as control. Crabs were sampled from each up to 20 days of exposure. As mortalities occurred

at the highest sumithion concentration, only crabs which were normal from all external appearances were sampled. To minimize circadian rhythm in the metabolic status of the crabs all sampling was done between 09.00 and 11 .OO h. Crabs were taken individually and hemolymph was taken from the arthrodial membrane using a syringe. The hepatopancreas was excised and frozen. Hemolymph glucose level was determined in all the crabs 1, 3, 5, 7, 15, and 20 days after exposure. Hepatopancreas glycogen, total carbohydrates, phosphorylase and aminotransferases were determined in each crab from the 2 ppm and the control group, 1, 3 and 5 days after exposure.

219

Hemolymph

glucose determination

A 1.O ml of the hemolymph was quickly deproteinized with barium hydroxide (1.5 ml) and zinc sulphate (1.5 ml). After thorough shaking, the precipitate mixture was centrifuged twice to obtain a clear supernatant which was drawn off and stored at 0°C. Glucose determinations were carried out by a calorimetric method [3]. Hepatopancreas

glycogen and TCHO determination

Tissue homogenates of 1% (w/v) were made in 10% trichloroacetic acid solution. Total carbohydrate level was estimated in trichloroacetic acid supernatant and glycogen in the ethanolic precipitate of trichloroacetic acid supernatants by the method of Carroll et al. [4]. A standard sample containing a known quantity of analar glucose solution was run with the experimental samples. The level of the contents was expressed as mg/g wet weight of tissue. Assay of phosphorylase

The activities of phosphorylase ‘a’ (active) and ‘ab’ (total) were estimated in the direction of glycogen synthesis by calorimetric determination of inorganic phosphate released from glucose l-phosphate. The assay has been described by Cori et al. [5]. Inorganic phosphate determinations were made by the method of Fiske and Subbarow [6].

-

1.0

PPm PPm

[)---o

2.0

PPm

I

I

I

I

I

3

5

7

15

Fig. 1. The mean hemolymph of sumithion

ppm sumithion

glucose

Y

levels (+ SD.) Significant

days 1, 3, 5 (P
and day 15 (P~0.01);

levels in hemolymph

I

20

S

for up to 20 days.

groups,

3, 5, 7 (PccO.001) Glucose

0.5

ad

1

DA

trations

CONTROL

-

of controls

for fresh-water differences

and day 7 (P
for 2.0 ppm sumithion throughout

crab exposed

from control

the experiment,

to different

1.0 ppm sumithion group,

days

concen-

levels are as follows: group,

0.5

days 1,

1, 3 and 5 (P
and on day 15 and 20, the 0.5 ppm

group and on day 20, the 1 .O ppm group were not significantly altered. determined in the 2.0 ppm group after day 5. N = 8 for all the groups.

Hemolymph

glucose

were not

280

Assay of aminotransferases The activity

of alanine

and aspartate

aminotransferases

was measured

by the

method of Reitman and Frankel [7]. The reaction mixture in a total volume ml contained: 100 pmol of phosphate buffer at pH 7.4; 20 pmol of L-aspartic (substrate

for AAT)

or 50 pmol of DL-alanine

(substrate

for AIAT);

of 1 acid

2 pmol of L-

ketoglutarate and 0.2 ml of tissue supernatant (5% w/v) prepared in ice-cold 0.25 M sucrose solution as enzyme source. The levels of oxaloacetate or pyruvate formed at the end of incubation (37°C) were measured on Bausch and Lomb calorimeter at 545 nm.

Protein determination In the enzyme source protein content was estimated al. [B] using bovine serum albumin as standard.

by the method

of Lowry et

Data analyses For statistical

analyses

of the data,

Student’s

t-test have been used throughout.

RESULTS

No mortalities occurred at either of the two lower sumithion concentrations or in the control group during the experiment. At the highest sumithion concentration (2 ppm) mortalities commenced after 24 h and continued until day 5. The crabs in this group were depleted by day 5 as a result of sampling and mortalities.

Hemolymph

glucose

The mean hemolymph

glucose levels for each group are shown in Fig. 1. Control

crabs showed a mean value of 8.83 + 0.09 mg of glucose/100 ml of hemolymph. Hemolymph glucose level was significantly (P< 0.001) higher than the control levels in all 3 experimental groups on day 1. The higher the sumithion concentration, the greater will be the hemolymph glucose level. The peak values were obtained at 3 days after exposure to 0.5 and 1 ppm of sumithion and at 5 days after exposure to 2 ppm. The glucose levels showed a gradual decrease from day 3 and the control value was approached at day 15 in crabs exposed to 0.5 ppm and at day 20 in crabs exposed

to 1 ppm.

Hepatopancreas

glycogen and total carbohydrates

Hepatopancreas glycogen and total carbohydrates for the control crabs and those in the highest sumithion concentration are shown in Fig. 2. In the sumithion-stressed crab, glycogen and total carbohydrate levels in hepatopancreas were significantly Glycogen and total carbohydrate levels of (P
281

Hepatopancreas phosphorylase Changes in hepatopancreas phosphorylase activity after sumithion intoxication are shown in Table I. Sumithion-exposure prompted significant elevation in the hepatopancreas phosphorylase activity. Both phosphorylase ‘a’ and ‘ab’ were augmented. The increase in phosphorylase ‘a’ is coupled with the increase in the ratio of phosphorylase a/ah. This indicates the interconversion of inactive phosphorylase to active phosphorylase and explains the decrease in glycogen content after sumithion-intoxication. Hepatopancreas aminotransferases The quantitative activities of AAT and AlAT in hepatopancreas of control and sumithion-exposed crabs after 1, 3 and 5 days are shown in Fig. 3. AAT and AlAT was significantly (P
The results of this study have shown that exposure to lower concentrations

2

-

CONTROL

-

2.0ppm

r

1.4

4

I

I

I

1

3

5

F

P

I

260

4-

I

I

I

1

3

5

DAYS

of

DAYS

glycogen

and total

carbohydrates

levels

(k SD.)

Fig. 2. The mean

hepatopancreas

sumithion-exposed sumithion-exposed

(2.0 ppm) freshwater crabs over 5 days. Glycogen and total carbohydrate levels in the crabs as compared with control crabs are significantly (PC 0.001) lower on days 1,

for control

and

3 and 5. N = 8 for all the groups. Fig. 3. The mean (+ S.D.) hepatopancreatic exposed

(2.0 ppm) freshwater

as compared

with the controls

aminotransferase

crabs over 5 days. Aminotransferase are significantly

(P
activity

levels for control

and sumithion-

levels in the sumithion-exposed

crabs

higher on days 1, 3 and 5. N = 8 in all groups.

282

sumithion

(0.5 and

hemolymph subsequently simply

1 ppm) cause a transitory

hyperglycemia.

The varied

levels of

glucose only occur during the first few days of sumithion exposure and approach control levels for the rest of the exposure time. This may

be due

to the accIimatization

of crabs

to the

toxicant.

At the

higher

sumithion concentration (2 ppm) (but still below LGo concentration), there is a more pronounced and sustained hyperglycemia. This is accompanied by a depletion of glycogen and total carbohydrate levels in hepatopancreas and a significantly higher Ievel of phosphorylase activity and the observed carbohydrate imbalances is, however, not unexpected. Elevation of phosphorylase activity in the h~patopancreas of the crab, whereby glycogenolysis results in hyperglycemia. Crabs stressed by pesticides such as sumithion exhibit gill damage and epithelial mucus accumulation which may lead to hypoxia (author’s unpublished data). There is evidence that this hypoxia in turn is a stimulus to increase circulating glucose levels as an anaerobic energy source. This elevation of hemolymph glucose levels during hypoxia has been attributed to an hyperglycemic agent [9]. Recent studies with crabs have demonstrated repetitive discharges of neurons and hyperglycemia in DDT-intoxicated crabs [lo]. Fingerman et al. [lo] suggested that the discharged material is hyperglycemic hormone. In view of previous findings, it is conceivable in the present case, that sumithion could cause discharge of hyperglycemic hormone from the neurosecretory cells that synthesize it, in a similar way. Earlier, Hohnke and Scheer [I 1] suggested that the primary role of the so-called ‘crustacean hyperglycemic hormone’ is not to elevate hemolymph sugar level, but to elevate intracellular glucose, through the degradation of glycogen by activating the enzyme phosphorylase. This glucose leak into hemolymph causes hyperglycemia [ 12, 131. Further, this suggestion is supported by the fact that in eyestaIk-ablated crabs, injec-

TABLE

I

VARIATIONS

IN HEPATOPANCREAS

SENEX SENEX AFTER Concentration

Enzyme

exposed

Control

2 ppm

EXPOSURE

PHOSPHORYLASE

ACTIVITY

OF OZIOTELPHUS~4

TO 2 ppm OF SUMITHION Autopsy

interval

(days)

_5

1

2

a

2.7 rt 0.1

2.7 + 0.1

2.7 + 0.1

ab

4.9 + 0.4

4.9 i

0.5

4.1 + 0.2

a % ab a

55 3.7 t 1.1*

4.0 t

0.9*

57 4.3 +- 1.0*

ab

1’ 40) 5.6 i 1.0**

(+ 48) 6.1 I 0.7*

(+ 57) 6.9 + 1.0*

a oio ab

55

(+ 15) 67

(+ 24) 66

c+ 45)

(+

(19)

(+

22)

62 0

Values are mean (pm01 of Pilmg protein/h) rt S.D. of eight individ~lals. VaIues in parentheses represent % change over control. Significantly different from control level at *p
283

tion of sumithion [I4] and BHC 1151did not increase the hemolymph glucose level and supports the hypothesis that the sinus gland in the eyestalks of this crab is the main release site for hyperglycemic hormone [ 161. Depletion of glycogen and total carbohydrates was significant (see Fig. 2) in the tissues of crab after exposure to sumithion [17]. Hence it is likely that, to meet the energy demands, the sumithion-exposed crabs depend on other sources for survival Since free amino acid content increases in the tissues of the sumithion-exposed crab [ 171this serves as a precursor for aminotransferases by converting the strategic compounds like cr-ketoglutarate, pyruvate, oxaloacetate, glutamate, alanine and aspartate for gluconeogenesis to meet the energy demands during the sumithion-imposed stress conditions, the same might be envisaged of the role of aminotransferases during sumithion intoxication of the crab. ACKNOWLEDGEMENTS

One of the authors (PSR) gratefully acknowledges financial support by the Council of Scientific and Industrial Research, New Delhi. We are also grateful to Rallis India Ltd. (Bangalore) for the supply of technical grade (96% w/v) sumithion. REFERENCES 1 Nationaf Institute of Nutrition, Annual report (Indian Council of Medical research publications), 1977, p. 184. 2 A. Bhagyalakshmi and R. Ramamurthi, Recovery of acetylcholinesterase activity from Fenitrothioninduced inhibition in the fresh water field crab (Oziotelphusu senex senex), Bull. Environ. Contam. Toxicol., 24 (1980) 866-869. 3 Technicon Corporation, Technicon Autoanalyzer Methodology, 1965, Bulletin N-26 I/II. 4 N.V. Carroll, R.W. Longiey and J.H. Roe, Glycogen determination in liver and muscle by use of anthrone reagent, J. Biol. Chem., 32 (1965) 583-593. 5 G.T. Cori, B. Illingworth and P.J. Keller, in S.P. Coiowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. 1, Academic Press, New York, 1955, pp. 200-204. 6 C.H. Fiske and Y. Subbarow, The calorimetric determination of phosphorus, J. Biol. Chem., 66 (1925) 375. ‘7 S. Reitman and S. Frankel, A coiorimetric method for the determination of serum glutamic oxaloacetate and glutamic pyruvic transa~nases, Am. J. Clin. Pathol., 28 (1957) 56-63. 8 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 9 D. Porte and R.P. Robertson, Control of Insulin secretion by catecholamines, stress and the sympathetic nervous system, Fed. Proc. Fed. Am. Sot. Exp. Biol., 32 (1973) 1792-1796. 10 M. Fingerman, M.M. Hanumante, U.D. Deshpande and R. Nagabhushanam, Increase in the total reducing substances in the hemolymph of fresh water crab, ~~ryre~~~ff~u guerini, produced by a pesticide (DDT) and an indole-alkylamine (serotonin), Experientia, 37 (1981) 178-179. 1I L. Hohnke and B.T. Scheer, Carbohydrate metabolism in crustaceans. in M. Florkin and B.T. Scheer (Eds.), Vol. V, Academic Press, New York, 1970, pp. 147-164. 12 R. Keller and E.M. Andrew, The site of action of the crustacean hyperglycemic hormone, Gen. Comp. Endocrinol., 20 (1973) 572-578.

284

13 M. Telford,

Blood glucose

hyperglycemia

in crayfish,

III. The sources

of Cambarus robustus, Comp.

14 A. Bhagyalakshmi,

P. Sreenivasula

duced hyperglycemia

Reddy,

in the freshwater

Biochem.

of glucose Physiol.,

V. Chandrasekharam

field crab,

and role of the eyestalk

factor

in

51 (1975) 69973. and R. Ramamurthi,

Oziotelphusa senexsenex, Toxicol.

Sumithion Lett..

in-

12 (1982)

91-94. 15 P. Sreenivasula

Reddy,

S.B. Ramesh

Babu and R. Ramamurthi,

field crab (Oziotelphusa senex senex) produced

by a pesticide

Hyperglycemia (BHC),

in the fresh water

Z. Naturforsch.,

37c (1982)

545-546. 16 P. Sreenivasula

Reddy,

Neuro-endocrine

Oziotelphusa senex senex. Ph.D. 17 A. Bhagyalakshmi, Fabricius

in relation

Physiological to pesticide

control

dissertation, studies impact,

of molt and metabolism S.V. University,

on the fresh Ph.D.

Thesis,

water

‘Tirupati field crab

S.V. University,

in the fresh water field crab, (A.P.)

(India),

1981.

Oziotelphusa senex senex Tirupati,

(India),

1981.