Indian Journal of Experimental Biology Vol. 41, April 2003, pp. 336-340

Toxicity of PCB 1232 on mitochondria of fish Arius cae latus (Valenciennes)

Dravyam Selvarani * & C Rajamanickam

Department of Biochemistry, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India

Received 19 February 2002, revised 24 January 2003

Mitochondrial proteins and phospholipids were estimated and SOH, Na+ - K+ - ATPase and Mg2+ - ATPase activities were analysed in the gill, liver and heart tissues of PCB 1232 (sublethal doses) treated fish A. cae/alUs. Protein and phos­pholipids were found to be decreased significantly and SOH, Na+ - K+ - ATPase, Mg2+ - ATPase and other enzyme systems displayed an inverse relationship with PCB dosage. Statistical analysis was carried out to indicate the relationship between sublethal doses of varying concentration and the activities of the enzyme systems involved in energy metabolism. The stud­ies indicated impairment in mitochondrial functions .

Organochlorines have been found to uncouple mito­chondrial oxidative phosphorylation and the respira­tory chain. Ela et al. I presumed organochlorine like DDT to be an inhibitor of oxidative phosphorylation rather than an uncoupler. Uncouplers enhance ATPase acti vity by causing the break down of A TP2.

Na+-K+-ATPase and Mg2+-ATPase are both mem­brane bound3 and maintain ionic balance by active transport. They have been found to be affected by or­ganochlorines4

,5 . DDT has also been reported to in­hibit SOH activitl, which is an indication of im­paired oxidative metabolic cycle. Cytochrome c oxi­dase, being the terminal enzyme of the electron trans­port system was found to be the site of organochlorine action7

.

Polychlorinated biphenyl (PCB)-an organochlo­rine is an universal priority toxic pollutantS, which is transported through the food chain and biomagnified at higher tropic levels9

• Hence in the present study the effects of PCB 1232 on the key enzymes of mito­chondria of the gills, liver and heart of an estuarine fish Arius caelatus have been analysed.

Materials and Methods Fish Arius caelatus weighing 95-100 g were col­

lected along the coast of Pazhayakayal near Tuticorin (Longitude East 79° of Greenwich and Latitude 8° North) and maintained in three separate netted enclo­sures in the estuary itself. The fishes were fed with beef. Five sets of experiments were performed. Each

* Address for correspondence: Department of Biology, Pope's College, Sawyerpuram 628 251, India. Phone: 0461 - 2327759

set consisted of 3 fishes . The first set which served as the control received ip injection of I ml corn oil. The fish in the second, third, fourth and fifth sets were given, 0 .6, 2, 4 and 6 mg PCB/kg respectively in I ml corn oil. PCB 1232 was purchased from NSI Envi­ronmental Solutions, North Carolina, USA. After 5 days of injection, the gills, liver and heart were re­moved and processed for isolation of mitochondria. All proceedings were carried out at 0°_5°C.

Isolation of mitochondria from gills-Mitochondria from the gills were separated 10. The gill bars were scraped off without any cartilage and after washing with 0.15 M KCl solution, were homogenised in 0.25 M sucrose containing 0.002 M Tris-HCI, pH 7.6 and 0.002 Methylene diamine tetra acetic acid (EDTA). The homogenate was centrifuged at 600 g for 10 min to remove nuclei and cell debris. The supernatant was again centrifuged at 10,000 g for 10 min to obtain mitochondrial pellet. The pellet was washed thrice and finally suspended in the same medium.

Isolation of mitochondria from heart and liver­Isolation of mitochondria from heart and liver was carried out ll . The organs were blotted free of blood, weighed and washed several times in ice-cold 0.25 M sucrose solution containing ImM EDTA, 20mM tris­HCI (PH 7.2) and 0.2% bovine serum albumen (BSA, pH 7.2-SET buffer). The tissues were sliced into small pieces. The whole liver was homogenised, while only the most metabolically active chambers of the heart, such as, the atrium and ventricle were used. The homogenisation was performed in 10 volume (w/v) ice-cold SET buffer. Nuclei and cell debris were removed and mitochondria were isolated from the homogenate by differential centrifugation.

SELVARANI & RAJAMANICKAM : TOXICITY OF PCB 1232 ON MITOCHONDRIA OF FISH ARIUS CAELATUS 337

Protein was estimated by the method of Lowry et al l2

. The phospholipids were extracted by. the method of Bligh and Dyerl3 and estimated by the method of Fiske and SUbbarow l4.

ATPase was assayed following the procedure of Tzagoloffl5 with minor modifications. The reaction mixture contained a total volume of 1ml, 50mM Tris HCI (PH 8.5), 4 mM ATP, 5 mM MgS04 and the mi­tochondrial protein. Ouabain (2 mM was added when estimating Mg2+-ATPase activity. The reaction was initiated by adding ATP and stopped by the addition of 1ml of 10% TCA and kept at 10°C for 15 min. The supernatant was centrifuged at 10000 rpm for 10 min and assayed for inorganic phosphate by the method of Fiske and SUbbarow '4 . Na+-K+-ATPase activity was calculated as the difference between inorganic phos­phate liberated in presence or absence of 2 mM ouabain. Quabain insensitive ATPase was considered Mg2+-ATPase. SDH I6 and cytochrome c oxidase '7

were assayed. The statistical analyses followed in the present

study are (l) standard deviation, (2) students t test, (3) simple linear regression and correlation, (4) one-way analysis of variation, (5) two-way analysis of varia­tion, and (6) Student Newman-Keul's test.

Results and Discussion Irrespective of the tissues, injection of the lowest

PCB dose stimulated protein synthesis (Table 1). This may be an adjustment to detoxify the toxicant. Further increase in PCB dose gradually suppressed protein synthesis especially in gill and liver mitochondria. As a result the protein content of the fish that received the highest PCB dose was less than that of the control. According to Syverson lS the protein synthesis is in­hibited by toxicants. The present study reveals that a part of the protein content may be utilized to counter­act the pesticide stress as evidenced by the decline of

protein content and another part may be transformed into nitrogenous wastes like ammonia as evidenced by the elevation of ammonia excretion l9. This may also indicate rapid utilization of protein or that dietary pro­tein consumed by the fish is not stored. The decline of protein is suggestive of intensive proteolysis support­ing the hypothesis of Kabeer20. Moreover PCB treated fish would have obtained the extra energy from body tissue protein.

Injection of 0.6 mg PCB/kg body weight dose in­creased the phospholipid content which decreased significantly with further increase to 2, 4 and 6 mg/kg body weight (Table 2). At the highest dose the mito­chondrial phospholipid content decreased from the level of sham treated fish .

Data on SDH activity (Table 3) point out that the lower two PCB doses significantly decreased the ac­tivity and with the higher doses there was no signifi­cant decline of the enzyme activity. Similar results were obtained when Channa striatus was subjected to DDT6 . Sakthivel et al. 2I observed decreased SOH activity in Cyprinus carpio in response to textile dye stuff effluents and stated that it was a indication of depression of cellular metabolism and that the meta­bolic pathway may have turned anaerobic to meet the increased energy demands during stress caused by the pollutant. Joyce et al. 22 in their study using malathion on Clarias batrachus remarked that decreased SOH activity indicated decreased funnelling of intermedi­ates into the citric acid cycle.

Like SDH, Na+-K+-ATPase (Table 4) and Mg2+_ ATPase (Table 5) activities decreased with increasing PCB dose. Influence of ATPase in fish by PCB is var­ied. Koch et.al. 5 have reported Na+-K+ -ATPase inhi­bition in kidney, liver and brain of Pimephales prome­las while Folmar et.al. 23 did not observe any change in Oncorhynchus kistuch. Similarly organochlorine DDT decreased Na+-K+-ATPase activity of intestinal

Table 1 - Mitochondrial protein content (mg/g tissue) of gill, liver and heart of A. caelatus 5 days after injection of PCB 1232

[Values are mean ± SD of 3 estimations. Figures in parentheses are % increase (+) or decrease (-) over controll

Sample PCB dose (mg/kg bod~ weight) LSD value 0.0 0.6 2 4 6

Gill 4.633 ± O.073c 5.247 ± 0.085d 4 .938 ± O. I03cd 3.870 ± 0.773b 3.220 ± 0.257" 0.3328

(+13 .26) (+6.58) ( -16.47) ( -30.5)

Liver 11.286 ± 0.178b 12.925 ± 0.192c 11.335 ± 0.173b 9.318 ± 0.400' 9.400 ± 0.176' 0 .5352

(+ 14.52) (+0.43) (-17 .44) (-16.71)

Heart 4 .870 ± 0.271" 6.695 ± O.346b 6.273 ± 0. 149b 6.160 ± 0.244b 5.267 ± 0.261"

0.5661 (-37.47) (+28.81) (+ 26.49) (+8.15)

Note: Values in a row superscribed by different alphabets are significantly different at P < 0.05 (SNK Test)

338 INDIAN J EXP BIOL, APRIL 2003

Table 2 - Mitochondrial phospholipid content (nmol Pi/mg protein) of gill, liver and hear of A. cae/atus 5 days after injection of PCB 1232

[Values are mean ± SD of 3 estimations. Figures in parentheses are % increase (+) or decrease (-) over control]

Sample PCB dose (mg/kg body weight) 0.0 0.6 2 4 6

Gill B 0.418 ± 0.022 0.541 ± 0.042 0.512 ± 0.028 0.434 ± 0.014 0.455 ± 0.017

(+29.43) (+22.49) (+18.66) (+8 .85)

Liver A 0.239 ± 0.015 0.332 ± 0.Q28 0.283 ± 0.019 0.222 ± 0.014 0.227 ± 0.017

(+38.91) (+18.41) (-25 .52) (-5 .02)

0.408 ± 0.037 0.480 ± 0.637 0.469 ± 0.016 0.460 ± 0.Dl5 0.462 ± 0.043

Heart B (+17.65) (+14.95) (+12.75) (+13.24) a c b a A

Two way SNK test - LSD at 0.05 level: Between doses = 0.0301 ; Between tissues = 0.0233 Different lower case alphabets under different columns in the last row indicate significant difference between the respec­tive doses irrespective of the tissues. Different upper case alphabets against tissues indicate significant difference between the tissues.

Table 3 - Mitochondrial SDH activity (nmol DCPIP reduced/min/mg protein) of gill. liver and heart of A. Cae/atus 5 days after inject~on of PCB 1232

[Values are mean ± SD of 3 estimations. Figures in parentheses are % increase (+) or decrease (-) over control]

Sample PCB dose (m~kg bod:t weight) 0.0 0.6 2 4 6

Gill A 0.261 ± 0.001 0.215 ± 0.005 0.210 ± 0.016 0.208 ± 0.002 0.202 ± 0.006

(-17.63) (-19.64) (-20.31) (-22.61)

Liver C 0.983 ± 0.061 0.763 ± 0.042 0.509 ± 0.001 0.501 ± 0.220 0.497 ± 0.031

( -23.58) (-48.22) (-49.03) (-49.44)

0.822 ± 0.035 0.653 ± 0.056 0.430 ± 0.003 0.381 ± 0.014 0.335 ± 0.073

Heart B (-20.56) (-27.13) (-53.65) (-56.08) c b a a A

Two way SNK test - LSD at 0.05 level: Between doses = 0.0399; Between tissues = 0.0309. Different lower case alphabets under the columns in the last row indicate significant difference between the respective doses irrespective of the tissues. Different upper case alphabets against tissues indicate significant difference between the tissues.

Table 4-Activity of Na+ - K+ -ATPase (Ilmol Pi liberated/min/mg protein) of gill, liver and heart of A. cae/alus 5 days after injection of PCB 1232

[Values are mean ± SD of 3 estimations. Figures in parentheses are % increase (+) or decrease (-) over control]

Sample PCB dose (mg/kg bod:t weight) 0.0 0.6 2 4 6

Gill C 0.105 ± 0.006 0.095 ± 0.032 0.055 ± 0.062 0.049 ± 0.002 0.046 ± 0.007

( -9.52) (-47 .62) (-47.62) (-56.19)

Liver A 0.021 ± 0.003 0.112 ± 0.004 0.010 ± 0.004 0.009 ± 0.00 1 0.009 ± 0.003

(+43.33) (-52.38) (-57.14) (-57 .14)

0.095 ± 0.005 0.062 ± 0.002 0.043 ± 0.002 0.040 ± 0.002 0.033 ± 0.002

Heart B (-34.74) (-54.74) (-47 .37) (-65.26) c b a a A

Two way SNK test - LSD at 0.05 level: Between doses = 0.0113; Between tissues = 0.0087. Different lower case alphabets under different columns in the last row indicate significant difference between the respec­ti ve doses irrespecti ve of the tissues. Different upper case alphabets against tissues indicate significant difference between the tissues .

SELVARANI & RAJAMANICKAM: TOXICITY OF PCB 1232 ON MITOCHONDRIA OF FISH ARlUS CAELATUS 339

Table 5 - Mitochondrial Mg2+-ATPase (flmol Pi liberated / min / mg protein) of gill. liver and heart of A. cae/alliS 5 days after injection of PCB 1232.

[Values are mean ± SD of 3 estimations. Figures in parentheses are % increase (+) or decrease (-) over control]

Sample PCB dose (mg/kg body weight) 0.0 0.6 2 4 6

Gill B 0.161 ±0.010 0.079 ± 0.001 0.077 ± 0.001 0.176 ± 0.003 0.076 ± 0.005 (-50.93) (-52.17) ( +9.32) ( -52.8)

Liver A 0.043 ± 0.002 0.029 ± 0.003 0.021 ± 0.002 0.020 ± 0.002 0.018 ± 0.002 (-32.56) (-51.16) ( -53.49) (-58.14)

0. 128 ± 0.007 0.109 ± 0.007 0.094 ± 0.001 0.088 ± 0.003 0.085 ± 0.003

Heart C (-14.84) ( -26.56) (-31.25) ( -33.59) c b a a A

Two way SNK test - LSD at 0.05 level: Between doses = 0.0053; Between ti ssues = 0.0041 . Different lower case alphabets under different columns in the last row indicate significant difference between the respec­tive doses irrespective of the tissues. Different upper case alphabets against tissues indicate significant difference between the tissues.

Table 6- Mitochondrial cytochrome c oxidase activity (flmol cytochrome c reduced/min protein) of gill. liver and heart of A. cae/alliS 5 days after injection of PCB 1232.

[Values are mean ± SD of 3 estimations. Figures in parentheses are % increase (+) or decrease (-) over control]

Sample PCB dose (mg/kg body weight) 0.0 0.6 2 4 6

Gill A 0.159 ± 0.060 0. 139 ± 0.027 0.102 ± 0.002 0.099 ± 0.005 0.083 ± 0.002

(-12.58) (-35 .9) (-37.74) (-47.8)

Liver B 0.313 ± 0.027 0.26 1 ± 0.027 0.226 ± 0.02 0.217 ± 0.014 0.213 ± 0.020

(-16.61) (-27.8) (-30.67) (-31.95)

0.666 ± 0.020 0.617 ± 0.040 0.435 ± 0.004 0.404 ± 0.013 0.363 ± 0.021

Heart C (-7.36) (-34.69) (-39.34) ( -45.5) d c b ab A

Two way SNK test - LSD at 0.05 level: Between doses = 0.0211; Between ti ssues = 0.0164. Different lower case alphabets under different columns in the last row indicate significant difference between the respec­tive doses irrespective of the tissues. Different upper case alphabets against tissues indicate significant difference between the tissues.

epithelium of Anguilla rostrata24 and brain Mg2+_ ATPase activity of Channa striatui5

. In Channa gachua 0.0022 mg/l endosulfan enhanced Na+-K+­ATPase activity in the liver while 0.0056 mg/l en­hanced Mg2+-ATPase activit/6

. Therefore it can be concluded that organochlorines seriously interfere with SOH, Na+-K+-ATPase and Mg2+-ATPase and enzymes of oxidative phosphorylation.

As SOH and A TPases, cytochrome C oxidase of sham treated A. caelatus displayed highest activity (Table 6). Injection of 0.6 and 2.0 mg/kg dose resulted in marked decrease in the enzyme activity. Cytochrome C oxidase like phospholipid is embedded in the inner mitochondrial membrane27. Hence the inhibition by the highest PCB dose can be related to the depletion of phospholipid. Surprisingly the phos­pholipid content of A. caelatus subjected to 0.6 mg PCB/kg increased while the cytochrome C oxidase activity reduced. Exposure of channel catfish28 to

stressor like hydrogen sulphide has been reported to cause similar reduction in the activity of cytochrome C oxidase. Hence cytochrome C oxidase inhibition is a response to stress.

The present study reveals that eventhough PCB 1232 has only 32% chlorine it is also hazardous as other PCBs with more chlorine content.

Acknowledgement One of the authors (OS) acknowledges the finan­

cial assistance from UGC, New Delhi through a Teacher Fellowship.

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