Indian Journal of Marine Sciences Vol. 28, March 1999, pp. 45-49

Cr (VI) induced changes in the activity of few ion dependent A TPases in three vital organs of mudskipper Periophthalmus dipes (Pisces: Gobidae)

Jayesh Thaker, Jignasa Chhaya, Renu Mittal, Sheeba Nuzhae & Rahul Kundu2

Department of Biosciences, Saurashtra University, Rajkot 360 005, Gujarat, India

Received 7 October 1997, revised 21 September 1998

Experiments were carried out to assess the dose and duration dependent influence of Cr(Vl) to few ion dependent A TPases in liver, brain and muscle of a coastal euryhaline teleost Periophthalmus dipes. Fishes were exposed to three sublethal concentrations (5, 10 and 15 mg/I) of Cr(VI), prepared by using K2CrO. with normal seawater, for three exposure duration (2, 4 and 6 days). In the present report, effects of Cr(VI) toxicity on total ATPase, (Na+,K+)-ATPase, (Ca++)-ATPase, (Mg++)-ATPase, (Ca++,HCOJ -)-ATPase and (Mg++,HCOJ-)-ATPase in liver, brain and muscle were evaluated. Though results indicated a general dose and duration-dependent inhibitory trend, exposure duration was predominant over dose in the inhibition of the enzyme activity.

Chromium compounds are considered highly carc'inogenic and can produce potential hazards at a very low concentration I . The principle source of chromium in the aquatic environment is from discharge of industries using large amount of chromate and dichromates. Toxicity of potassium chromate on some parameters on some fresh water fishes were earlier reported2• Heavy metals depress ion transport in several important osmoregulatory systems of teleosts leading to structural damage to various organs like gills, brain and kidney3. Lakshmi et al.4 reported the effect of mercury toxicity on six ion dependent ATPase in the gill ' s tissue of B. dentatus. Therefore, in the present communication an attempt has been made to evaluate the dose and duration dependent effects of Cr(VI) as K2Cr04

on the A TPases in liver, brain and muscle of a coastal amphibious fish the mudskipper, Periophthalmus dipes Bleeker (Pisces:Gobidae).

Materials and Methods A total of 275 numbers of a coastal teleost, commonly

known as mudskipper, Periophthalmus dipes, were selected for the present investigation. Other details regarding fish are same as reported earlier5. Analytical grade of K2Cr04

(E. Merck) was .dissolved in normal seawater to a desired stock solution. Different concentrations of this toxicant were prepared with seawater (salinity 35.14 ppt). Other details regarding toxicant are as reported earliers. On the basis of LC50 value (47 .84 mg/l) three different sublethal concentrations were prepared (5, 10 and 15 mg/l i.e., 0.096

1 Department of Biochemistry, Allahabad University, Allahabad - 211002, India. 2For correspondence

M, 0.192 M and 0.288 M respectively) with seawater having salinity5 of 35.14 ppt. At least 10 fishes were put in each concentration and exposed for three different durations (2 , 4 and 6 days). Separate control groups were also maintained along with each treatment. The toxic media were renewed every 12 h. Animals were sacrificed after scheduled exposure and selected tissues were quickly dissected out and placed 111 chilled sucrose - EDTA­imidazole buffer (PH 7.1) to remove excess blood and adhering tissues .

Pieces of liver and myotomal muscle were taken from approximately same portion of the tissue from all killed specimens, these samples were also washed and blotted. The brain was removed from the skull, washed and blotted. Each tissue sample from all the killed animals was combined together and 200 mg of each tissue was taken for enzyme extraction. The tissue preparation, enzyme estimation and other assays were carried out by the methods described earlier4•6.7. The activities of (Na+-K+)-ATPase, (Ca++)- ATPase, (Mg++)- ATPase, (Ca++, HC03-)-ATPase and (Mg++, HCOJ-)- ATPase were estimated4 . All experiments were repeated at least thrice and obtained data were subjected to various statistical analyses8.

Results and Discussion Results of the present investigations showed the

behavioural manifestations upon exposure to the toxicant. As soon as the fishes were introduced to the toxic media, they started jumping and tried to come out of the media. After this unsucccssful attempt fishes showcd erratic swimming for sometime. Then the fishes settled down. It

46 INDIAN 1. MAR SCI., VOL. 28, MARCH 1999

was observed that fishes became sluggish as the time elapsed. Generally, in maximum time duration (6 d) specimens became absolutely sluggish with slight change in their body colour (bluish gray to yellowish gray).

In liver, the specific activity of most of the enzymes showed dose and duration dependent inhibition. The activity of total and (Na+, K+)-ATPase are effected mostly. In these cases a gradual inhibition was observed. The activity of (Mg++, HC03-)-ATPase showed initial stimulation in lower dose (Smg/l). In all other cases less inhibition was observed. However, the activity of all enzymes studied showed maximum inhibition in fishes exposed to highest dose for longest duration (Table I). Varied changes in the activity of different enzymes in the whole brain were evident from the present study. Most of the enzymes showed inhibition. However, in few cases slight stimulation in the enzyme activity, after an initial fall in lower concentration, was also evident (Table 2). Marked changes in the activity of different enzymes in the myotomal muscle tissue was evident. The activity of total ATPase and (Na; , K;)- A TPase fell sharply in fish exposed to S mg/I and 10 mg/I concentrations, but stimulated in the highest dose. However, inhibition of varied magnitude in the activity of other ATPases was observed (Table 3).

In the present study a general inhibitory trend was observed in all the ion dependent A TPases, in all the tissues studied of Cr(VI) intoxicated fishes . However, slight irregularities were observed in some cases on exposure to Cr(VI) . (Na ' K;)- ATPase is one of the major enzymes responsible for movement of Na+ and K+ ions across membrane and in the proper functioning of the Na+, K+ pump. It is apparent that inhibition of (Na+, K+)- ATPase by Cr(VI) may cause alteration in the ion transport processes in the cell membrane and impair metabolite uptake and transfer in the liver tissue9 McCarty & Houston l O reported significant deviations in plasma sodium and chloride levels as well as ti ssue sodium and water content on exposure to sublethal concentrations of heavy metals. Strick et al. II observed a decrease in the Na+ concentrations of the blood of rainbow trout intoxicated with lethal doses of chromium. This drastic inhibition of (Na+, K+)-A TPase may disrupt the active Na; reabsorption which explains for the Na+ loses observed in the cells9. Brain is always in a state of constant electrical activity and most of the effector activity taking place is initiated from the brain. Chromate ions I are also known to inhibit electrical potential across the membrane. The resulting impairment of neurotransmitter release and transport could be responsible for the paralytic symptoms observed in this study. The drastic inhibition in the activity of muscular (Na+, K+)-ATPase in the present investigation may have resulted from the binding of these metals with the enzyme protein which usually denatures them l 2 The inhibited (Na+, K+)- ATPase activity may cause a blockade of Na+, K+ coupled pump. Since this pump is

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Table 2 - Mean spec ific activity ( 11 mol Pi mg ' Protein h ') o f six e nzy mes in the brain tissue of the mudskippers exposed to varying concentrations ofCr(VI) for 2,4 and 6 days respectively

I 'v'a lucs rcp rcscnlmean ± standard deviation o f tripli cate data]

En/ymcs

Tota l . \ TPasc

Na- K' ATP asc

Ca ­

ATPase

Mg'­

ATPase

Ca~

HCO, ' ATPase Mg~

HCO,' ATPase

2 da ys

" l> 3 17 ±

26.32 291.88

± 25.73 17 .2 ±

2.72 16.83

± 3.56 14 .44

± \.52

18.08 ±

2.05

Contro l

~ days

.. 55 .55

± 24 .3 1

291 .02

± 30.29 17.69

± 5.96 10.46

± 2 .11 11.77

± 2.29

18.54

± 3.25

6 days

.... 8 .11

± 24 .92

291.63

± 3 1.63 17 .58

± 3.32 14 .67

± 2.52 17.49

± 3.84

18.86

± 3.63

2 days

390.48

± 25 .2 1 126 .58

± 10.7 1 1606

± 6.36 15. 10

± 302

11 .65

± 1.69

16.46

± 1.92

5 mg ! I 4 da ys

380 .50

± 22.62 11 0 .74

± 18.32 6.98

± 1.29 4 .21

± 0 .92 6 .01

± 0.93

6.67

± 1.12

6 days

244 .12

± 22.2 8

213.48 ±

22 .63 17.32

± 3.91 12 .76

± 2 .6 1 17 .49

± 3.35

22 .86

± 4 .21

2 days

458 .29

± 29.92

2R 1.73

± 18.72 7.91

± 1.92

13 .5 1

± 2.9 1 12 .04

± 202 13 .09

± 1.63

10 mg I I 4 days

371.98

± 21.62

21 704

± 17.32 6 .52

± 1.15 7.86

± 1.63 8.12

± 1.29 9.69

± 1.92

6 days

360.59

± 25 .25 187.54

± 15.62 10.84

± 2.59 11 .65

± 1.93

11 .08

± 2.72

1090

± 3.21

2 days

3256

± 22 .63 99.77

± 13 .73 12 .14

± 3.32 16 .09

± 3.69 \1 .17

± 1.67

12.23

± 1.51

15 mg I I

4 days

273.75

± 23 .62

270.66

± 24 .95 16 .76

± 2 .69 18.61

± 3 .81 15 .09

± 3 .31 18.49

± 3.63

6 days

270.09

± 19.57 98 .52

± 6 .65 10.59

± 2 .63 11 .46

± 1.95 8.89

± 0 .81 11.71

± 1.92

Tab le 3- Mean specific act ivity (11 mol Pi mg " Prote in h " ) of six enzymes in the musc le tissue of the mudskippers exposed to varying concentrations ofCr(VI) for 2, 4 and 6 days respectively

[Values represent mean ± standard deviation of triplicate data]

Enzymes

Total ATPase

Na- K ' ATP ase

Ca­

ATPase

Mg­

ATPase

Ca ­HCO, '

ATPase Mg~

HCO, ' ATPase

2 days

119.01

± 21.63 40.05

± 10 .25 20.98

± 3.37

26.58

± 8.52 14 .23

± 3.31

20.83

± 9.91

Control

4 da ys

11604 ±

19.23 40 .69

± 9.96

20 .24

± 3.45

21. 98 ±

8.48 13 .7 1

± 2.96 18.25

± 9 .27

6 days

112 .86

± 18.69 35 .00

± 8.59

16.38

± 2.96 19.52

± 7.73 I 1. 87

± 2.23 17. 12

1:

6 .58

2 days

57 .63 ±

10.25 327

± 9.82 4 .36

± 0 .78 6.02

± 1.2 1 5.87

± 1.12 6 .0 1

± 1.16

5 mg I I 4 days

91.74

± 15.6 1 30 .10

± 5.42 3.3 1

± 1.12 347

± \.59 4 07

± 1.26 4 .12

± 1.76

6 days

40 .00

± 10.22 23 .10

± 7.21 4 .69

± 1.1 1 4 .52

± 1.92 3.87

± 1.72 6 .12

± 1.72

2 days

34.53

± 9 .82

20.00

± 2.72 4 .00

± 1.22 1.89

± 0 .35 5.00

± 1.91 5.00

± 1.2 1

10 mg / l

4 days

113 .89

± 18.62 79. 12

± 11 .23 3.97

± 1.69 2.48

± 0 .97 4 .67

± 1.37 6 .13

± \.59

6 days

53 .23 ±

6. 59 18 .85

± 3.32

1.61 ±

0 .51 1.29 ±

0.21 2.07

± 1.69 3.46

± 1.86

2 days

105.45

± 10.63 21.43

± 3.31 2 .74

± 0 .68 2.04

± 0 .84 2.31

± 1.37 3.80

± 1.83

15 mg / l 4 days

124.89

± 20.21 72.85

± 12.96 2.81

± 0 .91 15 .13

± 5.31 4 .39

± 1.52 4 . i2

± 1.81

6 days

47 .85

± 6 .15 18 .60

± 3 .62 2 .44

± 093 2 .64

± 0 .29 6.41

± 2.62 2 .44

± 0 .59

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48 INDIAN J. MAR. SCI., VOL. 28, MARCH 1999

responsible for the action potential the conductivity of muscle may be significantly reduced making the whole muscular tissue nonfunctional6. Inhibition in the activity of (Ca .... )-ATPase and (Mg++)-ATPase in liver may be due to the accumulation of large amount of Cr (VI) in the liver cells which combine with the enzyme protein thus altering its activity. Similar inhibitions in the activity of the (Ca O-+)-ATPase and (Mg++)-ATPase in the liver of fish exposed to HgCl2 have been reported13. In the present investigation, inhibition of these two vital enzymes clearly

indicates a blockade in the transport of these ions. Mg++ together with Ca++ is essential for neurotransmittory fu nction especially reflex action. Therefore, the reduction of Mg++ might cause delay in transmission at the neural junction7. Results of the present investigation showed duration dependent inhibition in the activity of (Ca++)-A TPase and (Mg++)-A TPase in the muscular tissue of intoxicated fishes . This inhibition probably reduced the uptake and transport of Ca++ and Mg++ which are essential for the contractile property of the muscle . It is also possiblel 4 that this imposed stress impairs functions of sarcoplasmic reticulum and the transport and storage of Ca ..... Inhibition in the activity of the anion dependent A TPases clearly revealed the disrupted transport of anions through the membrane which eventually might have caused other functional disorders in the liver. However, the slight stimulation in the enzyme activities observed in some cases

could be due to the initial effort made by the animals to adapt to the imposed stress. The role of bicarbonate ions in the central nervous system (CNS) is not very clear at this point. However, they are believed to be associated with axoplasmic flow and normal maintenance of nervous tissue. Heavy metals are reported to cause irregularities in their release and uptake of neurotransmitters in the brain which may be due to the inhibition of HC03- ATPases l5 . The observed neurobehavioural disturbances in the mudskippers may also be due to this impaired neurotransmission. However, sudden enhanceme~t in the activity of (Ca++, HCO) -)- A TPase in Cr(VI) exposed fishes was probably due to necrosis in the membl \ne where the entry of anions was not controlled by the active transport l6. The drastic inhibition of (Na" K' )- ATPase created a blockade in the transport of Na' and K' and hence, to equilibrate the electrical potential considerable amounts of negatively charged ions entered the cell which may explain the stimulation in the enzyme responsible .

Results of the statistical analysis of the activity of the enzymes studied revealed a predominant exposure duration dependent effect (Table 4) . The overall results of Cr(VI) intoxication revealed a drastic impairment in the cellular active transport of different vital ions and metabolites in liver, brain and muscle tissues. The hampered uptake and transport of important ions such as Ca++ and Mg++ may cause failure of the contractile property of the muscle. The

Table 4- F' ratIOs derived from the two way nested ANOV A test of the specific activity of various enzymes studied between different treatments and exposure duration s. Calcu lated "F" values are given in the Table. "Degrees of Freedom" (dt) are (a) among doses = 3, 8

and (b) among durations within doses = 8, 24. Critical values of F for these df are also given

Liver

Among doses Among durat ions within doses

Brain

Among doses Among durations within doses

Muscle

Among doses Among durations within doses

Critical values of "F" at various df :

Na-K ATPase

3.96 340.15

0.72 27 .62

0.77 63.42

Probability

P = 0.05 P = 0.01 p = 0.001

Ca ATPase

1.34 87.34

0.9i 57.48

1.81 76.79

F [3,8] F [8,24]

4.07 2.36 7.59 3.36 15.83 4.99

Enzymes

Mg ATPase

0.92 148.08

0.77 52.45

1.24 152.07

Ca-HCO) ATPase

1.36 12767

0.67 41.36

2.78 71.76

Mg-HCOJ

ATPase

2.39 373.44

1.71 60.16

4.38 106 .92

THAKER et af. : Cr fNDUCED CHANGES fN P. dipes 49

observed depolarization was probably due to increased intracellular Na+ resulting from inhibition of membrane (Na+K+)-A TPase activity. This leads to rise in the intracellular Ca++ as is evident from the stimulation in the activity of (Ca++)-A TPase. This ultimately effected the contractility of muscular tissue 17. The fatal conseqqences of the blockade in the ion pumps and metabolite transport may be coupled with the behavioural observations where a failure in muscular mobility followed by paralysis was evident.

Acknowledgement Authors are thankful to Dr. Harish Dave, Deputy

Director of Fisheries, Govt. of Gujarat, India for technical assistance. This work was fmancially supported by Ministry of Environment & Forests, Govt. of India, Grant No. 19/107/91-RE of 18-11-92.

References I Venko V, J Hyg Epidemiol Microbiol Immunol (Prague) 29

(1985) 37. 2 Patel R & Saxena A B , Indian J Zool, II (1983) 43 . 3 Renfro J L, Schmidt-Nielson B, Miller D, Benos D & Allen

J, in Pollution and physiology of marine organisms, edited by F J Vernberg & W B Vern berg (Academic Press, New York) 1973,101.

4 Lakshmi R, Kundu R & Mansuri A P, Acta Hydrochim Hydrobiol, 18 (1990) 605 .

5 Thaker J, Chhaya J, Nuzhat S, Mittal R, Mansuri A P & Kundu R, Toxicology, 112 (1996) 237.

6 Kundu R, Lakshmi R & Mansuri A P, Proc Acad Environ Bioi, I ( 1992) I.

7 Kundu R, Lakshmi R & Mansuri A P, Bull Environ Contam Toxicol , 55 (1995) 723 .

8 Sokal R R & Rohlf F J, Biometry, (W H Freeman and Company, San Francisco) 1969, pp 260.

9 Gilles R, in Mechanisms of osmoregulation in animals, edited by R. Gilles (Wiley, New York) 1979, 113.

10 McCarty L S & Houston A H, J Fish Bioi, 9 (1976), II . II Strik J J TWA, De longh H H, Van Rijn Van Alkemade J

W A & Wuite T P, in Sublethal effects of toxic chemical on aquatic animals, edited by J H Koeman & J J TWA Strik (Elsevier, New York) 1975, 179.

12 Sahana S S, Jana S S, Choudhuri N A & Choudhury D K, Physiol Bohemoslovaca, 35 (1986), 81.

13 Verma S R, Jain M & Tonk I P, Toxicol Lett (Arnst), 16 (1983), 305 .

14 Chhaya J, Thaker J, Mittal R, Nuzhat S, Mansuri A P & Kundu R, Indian J Mar Sci, 26 (1997) 186.

15 Hseih K, Sue Y, Yau-Huai W, Cheih-Fu C & Ru-Dong W, Toxico!. , 24 (1986) 153.

16 Needham D M, in The Structure and fun ction of muscle, edited by G H Bourne, (Academic Press, New York) 1973, 363.

17 Chen Z, Coan C, Fielding L & Cassafer G, J Bioi Chem , 266 (1991), 12386.