Sub-chronic diclofenac sodium induced alterations of ...

Indian Journal of Experimental Biology

Vol. 49, June 2011, pp. 446-454

Sub-chronic diclofenac sodium induced alterations of alkaline phosphatase

activity in serum and skeletal muscle of mice

Shalini Chouhan & Sushma Sharma*

Department of Biosciences, H P University, Summer Hill, Shimla 171 005, India

Received 29 June 2010; revised 1 March 2011

The present study has been carried on changes in activity of alkaline phosphatase in serum and gastrocnemius muscle

of mice after sub-chronic use of diclofenac. Mice in experimental group received diclofenac (10 mg/kg body wt /day) for 30

days while control group received normal saline. Alkaline phosphatase was assayed in muscle and serum and its activity was

localized histochemically in muscle. Results showed that diclofenac induced changes in specific activity of alkaline

phosphatase at different periods of treatment variably compared to control group. Specific activity of alkaline phosphatase

decreased significantly in gastrocnemius initially (48.74%), increased thereafter (132.96%) and slight decrease (13.97%)

was noticed after 30 days. In serum, the specific activity of alkaline phosphatase decreased slightly after 10 days (18.78%),

increased in the middle of the treatment period (132.04%) as well as showed increase (109.09%) compared to control group

after 30 days stage of investigation. These findings were also confirmed by electrophoretic studies in muscle.

Keywords: Alkaline phosphatase, Diclofenac, Gastrocnemius, Serum, Sub-chronic

Diclofenac residues were found to be the cause for

rapid population decline of Gyps vultures in southern

Asian countries1. The toxic effects of diclofenac were

known however the mechanism of action is still not

fully understood. Nonsteroidal anti-inflammatory

drugs (NSAIDs) continue to be prescribed

as

analgesics for patients with healing fractures even

though these drugs diminish bone formation, healing,

and remodeling2. Diclofenac is a widely used NSAID

for treatment of a variety of inflammatory conditions

such as rheumatoid arthritis, osteoarthritis and

acute muscle aches. Diclofenac inhibits both

cyclooxygenase (COX) isoenzymes, COX-1 and

COX-2, by blocking arachidonate binding resulting in

analgesic, antipyretic and anti-inflammatory

pharmacologic effects3. The enzymes COX-1 and

COX-2 catalyze the conversion of arachidonic acid to

prostaglandin G2 (PGG2), the first step of the

synthesis of prostaglandins and thromboxanes that are

involved in rapid physiological responses. Delayed

fracture healing and no body weight gain was

reported in rats after long term treatment with

diclofenac4. Diclofenac sodium causes a rare but

potentially fatal hepatotoxicity that may be associated

with the formation of reactive metabolites and

subsequent adverse hepatitis effects may arise in

certain individuals5. NS-398, a COX-2 specific

inhibitor has been reported to interfere with the

healing of injured skeletal muscle6. An inhibition

of protein synthesis in rat skeletal muscle with

three different cyclooxygenase inhibitors (aspirin,

indomethacin and meclofenamate) was also reported7.

The elevation in the serum level of alkaline

phosphatase has been used as diagnostic index for

skeletal muscle disorders8. Alkaline phosphatase

showed an association with a number of muscle

diseases including neuromuscular disorders,

myopatheis or other wasting conditions9. The

objective of the study was to investigate diclofenac

induced alterations of alkaline phosphatase activity in

mice skeletal muscle.

Alkaline phosphatase (APase; E.C.3.1.3.1)

represents a group of isozymes that are membrane-

bound glycoproteins which catalyze the hydrolysis of

inorganic and organic monophosphate esters at

alkaline pH in vitro10

. APase is ubiquitous in nature

located in basal membrane of various tissues and also

found in the serum11

. Soluble forms of APase exist in

the serum. Alkaline phosphatases are members of a

rather diverse group of membrane proteins which are

anchored to lipid bilayers in cell membranes by a

phosphatidylinositol-glycan moiety attached to the

——————— *Correspondent author

Telephone: 91-177-2830946

Fax: 91-177-2830495

E-mail: [email protected]; [email protected]

CHOUHAN & SHARMA: DICLOFENAC SODIUM & ALKALINE PHOSPHATASE ACTIVITY

447

carboxy terminus of the protein12

. Serum APase is a

dimer, whereas the membrane-bound forms of APase

are probably tetramers13

. Studies reported earlier

have established that APase does form an important

entity in the muscle cell14

. Patients suffering from

hypophosphatasia, a genetic deficiency in APase

activity, commonly suffer from severe rickets and

osteomalacia15

.

Materials and Methods

Animals―Healthy male mice of Balb-c strain (36)

weighing about 24-27 g were procured from Central

Research Institute (CRI), Kasauli, (Himachal Pradesh),

India. Animals were maintained in hygienic conditions

in a well ventilated room of animal house of Department

of Bio-Sciences of H P University, Shimla with D:L

cycle of (12:12) h and a temperature of 25 ± 2°C.

Animals were provided with commercial feed

(Hindustan Lever Ltd., New Delhi, India) and water ad

libitium. All procedures, including the maintenance of

the animals had the approval of Institutional Animals

Ethics Committee of the University (IAEC approval no:

IAEC/Bio/2009/6-HPU).

Drug administration―Diclofenac sodium was

purchased from Sigma Aldrich Co., USA. Stock

solution was made in distilled water. Further dilutions

were done according to the body weight records of the

animals. All the chemicals used were of analytical

grade. To investigate the changes in the specific

activity of APase in control and experimental group,

mice (18) received diclofenac intramuscularly at

the dose rate of 10 mg/kg body wt. /day for 10, 20 and

30 days while 18 of the control group received normal

saline. The drug dose chosen was higher than the

recommended therapeutic dose (8 mg/kg) for mice.

Mice were sacrificed by cervical dislocation, the next

day at the end of the experiment. Gastrocnemius

muscle was excised and serum was collected at each

stage of the investigation.

Histological study―Bouin fixed gasrocnemii were

embedded in paraffin wax after dehydrating in

ascending grades of alcohol and clearing in xylene.

Further, 7-8 µm thin sections were processed for

Haematoxylin-Eosin staining. Lastly, after clearing in

xylene, sections were mounted in DPX and

photographed.

Histochemical localization―APase was localized

histochemically in cryostat cut sections of the

gastrocnemius muscle by Gomori’s calcium cobalt

sulphide method16-18

with slight modifications.

Briefly, tissues were fixed overnight in neutral

calcium formol and next day, processed for

histochemical staining. The cryostat cut sections were

preincubated in 100 mM Tris-maleate buffer

containing 1% MgCl2 overnight at room temperature.

Thereafter, the preincubated sections were incubated

in the substrate solution (pH 8.9) containing sodium

β-glycerophosphate for half an h at 37ºC and preceded

according to Gomori’s method16

. These were quickly

dehydrated in ascending grades of alcohol and

mounted in Canada balsam and photographed.

Biochemical assay―The muscle homogenate was

prepared in ice cold distilled water which was

centrifuged at 1664 × g for 20 minutes at 4°C. The

supernatant was used for the enzyme assay. Serum was

collected and centrifuged at 2599 × g for 15 min at 4°C.

Muscle and serum APase was assayed by the method of

Weil and Russel19

. Breifly, sodium β-glycerophosphate

was used as substrate. The homogenates were incubated

with the substrate solution (pH 8.9) for one h at 37°C.

The reaction was stopped by 10% TCA. Finally, the

solution was centrifuged at 936 × g at 4ºC and the

supernatant was collected for determination of enzyme

activity. The color intensity was read at 650 nm in a

Hitachi 150-ophrey Double Beam Spectrophotometer.

The amount of phosphate released was plotted against

known concentrations of KH2PO4. Protein content was

estimated according to method of Lowry et al.20

. The

specific activity of alkaline phosphatase was calculated.

Homogenization of the tissue―Muscle homogenate

was prepared according to the method previously

described21-22

with slight modifications. Tissue was

homogenized in 5 volumes of Tris buffer, 10 mM, pH

7.4 containing 1% triton X-100, 2 mM MgCl2 and

0.025 mM ZnCl2. Homogenate was centrifuged at

15,000 × g for 20 min. The pellet was rehomogenized,

centrifuged at 15,000 × g for another 20 min.

Supernatants were combined and again centrifuged at

20,000 × g for 30 min. All the steps in this procedure

were carried out at 4°C.

Electrophoresis—It was conducted according to the

method of Epstein et al.23

with some modifications.

Supernatants were used for electrophoretic studies.

Proteins were estimated according to Lowry et al.20

.

The enzyme was resolved on 7% separating and 5%

stacking gel. Equal amount of protein was loaded in

each lane. The enzyme was localized using activity

staining technique earlier described by some

workers24

with certain modifications. Gels were

analyzed densitometrically.

INDIAN J EXP BIOL, JUNE 2011

448

Statistical analyses―Values are represented as

mean ± SEM. Results were analysed using Student’s

‘t’ test.

Results

Toxic effects of diclofenac treatment were

observed in mice gastrocnemius in terms of altered morphological and biochemical levels. The histopathological changes pointed towards massive tissue damage. Diclofenac treatment caused numerous pathological changes during different stages of investigation as compared to the control group.

Lightmicrographs of control mice gastrocnemius depicted normal fascicles surrounded by perimysium encasing group of myofibers having relatively uniform pattern. Individual muscle fibers showed endomysium around them, each one having normal peripheral arrangement of the nuclei (Fig. 1A and

1B). Diclofenac treatment for 10 days on the other hand resulted in structural disorganization in terms of degenerating fibers, atrophied fibers and inflammatory cell infiltration (Fig. 1C). After 20 days of drug treatment, merging of fibers was noticed which had irregular limiting membranes, many

fibrolysing as well as abnormally hypertophied fibers were also observed. Previous peripheral arrangement of the nuclei delineated which occupied central position now; (Fig. 1D). More disorganized structure of the muscle was observed after 30 days of diclofenac treatment. Chains of darkly stained nuclei

were noticed in interfibrillar regions. Muscle fibers lost proper shape and uniformity due to loss of perimysial and endomysial boundaries, many atrophied fibers were noticed to be without nuclei, degeneration continued as well as merged fibers were also noticed (Fig. 1E and 1F).

With regard to histochemical studies, the

micrographs of gastrocnemius muscle exhibited

darker staining with intense APase activity around the

sarcolemmal regions in the control group. Further,

section from control group showed normal fibers with

intact boundaries having a uniform pattern (Fig. 2A);

whereas the intensity of enzyme positive areas was

found to be lighter in the section from treated group

after 10 days of treatment. Degenerating fibers and

merging sarcolemma were also noticed. The enzyme

activity was found to be more pronounced around the

smaller fibers as compared to the hypertrophied ones

where enzyme activity seemed to be less (Fig. 2B).

After 20 days of treatment with diclofenac, the

increased number of hypertrophied as well as

degenerating fibers with degeneration foci were

observed (Fig. 2C). Also lesser enzyme activity

around the merging sarcolemma was seen as

compared to control. After 30 days, weaker staining

for APase was observed; many fibers were found

without sarcolemma being merged with each other

along with somewhat larger and intact fibers showing

more enzyme activity in comparison to other

degenerating fibers. However, some atrophic fibers

also had darker staining for APase. Fibrolysis was

also noticed along with lesser enzyme positive areas

(Fig. 2D).

These histochemical findings were also confirmed from biochemical assay where APase activity in gastrocnemius decreased significantly as compared to control after first stage of investigation. Activity of APase in gastrocnemius showed a decrease from 0.0143 ± 0.0014 µM Pi/µg of protein/h to 0.0073 ± 0.0003 µM Pi/µg of protein/h after 10 days of treatment. Significant increase was observed in alkaline phosphatase activity after 20 days of treatment from 0.00364 ± 0.00016 µM Pi/µg of protein/h to 0.00484 ± 0.00024 µM Pi/µg of protein/h. After 30 days of treatment, slight decrease in APase activity [0.0022 ± 0.0001 µM Pi/µg of protein/h to 0.0019 ± 0.00014 µM Pi/µg of protein/h; was found (Fig. 3)].

The biochemical findings were further supported

by electrophoretic study (Fig. 4). An image analysis

of gels showed that in treated group, alkaline

phosphatase activity decreased significantly from

61.62 ± 1.227 to 38.37% ± 1.227 after 10 days of

treatment, while at the second stage of investigation,

Fig. 1―(A)-Lightmicrograph of control mice gastrocnemius showing normal fascicles (NF) and individual muscle fibers with peripheral

arrangement of the nuclei (NU); H & E × 200; (B)-T.S. gastrocnemius of control mouse showing perimysium (PM) around individual

fiber bundle, Endomysium (EnM) surrounds muscle fiber; peripherally placed nuclei (NU) are clearly visible (H & E; × 400); (C)- T.S. of

gastrocnemius of diclofenac treated mice for 10 days showing loss of cellular architecture (←), atrophied fiber (AF), blood vessel ( BV)

is also seen (H & E × 400); (D)-Histopathological changes like irregular limiting membrane, exhibiting merged adjacent fibers (MF),

centrally placed nuclei (*), fibrolysed fibers (F Fb) and hypertrophied fibers (HF) observed in gastrocnemius after 20 days diclofenac

treatment; chains of nuclei is noticed; H & E × 400; (E)-Diclofenac treatment for 30 days resulted in aggravated structural disorganization

in terms of fiber degeneration (*), atrophied fibers showing loss of nuclei (AF) and infiltration of inflammatory cells (•) in mice

gastrocnemius; H & E × 200; (F)-Photomicrograph from 30 days diclofenac treated gastrocnemius muscle exhibiting degenerating fibers

(DF), atrophied fiber (AF), merged fibers (MF); clumped inflammatory cells also visible H & E × 400.


449


450

noticeable increase in the enzyme activity (45% ±

1.041 to 55% ± 1.041; as compared to control

group was seen. In the similar trend as that of first

stage of investigation, appreciable decline in the

enzyme activity after 30 days of drug

administration (57.74 ± 1.171 to 42.25% ± 1.171)

was noticed (Fig. 5).

With regard to the biochemical analysis of APase

in serum, insignificant decrease in the serum APase

activity was noticed after 10 days treatment from

0.0458 ± 0062 µM Pi/µg of protein/h to 0.0372 ±

0.0022 µM Pi/µg of protein/h which amplified after

20 days from 0.0181 ± 0.0012 µM Pi/µg of protein/h

to 0.0239 ± 0.001 µM Pi/µg of protein/h and again

showed a significant hike after 30 days from 0.0308

± 0.0011 µM Pi/µg of protein/h to 0.0336 ± 0.0011

µM Pi/µg of protein/h (Fig. 6).

Discussion

Many nonsteroidal anti-inflammatory drugs have

been reported to cause alterations in different tissues.

Diclofenac associated hepatitis and histopathological

lesions in liver and kidney have been reported in

earlier studies25-26

. The present investigation revealed

remarkable alterations at morphological as well as

biochemical levels in mice gastrocnemii. A number of

deviations in the muscle under investigation were

noticed in terms of degenerating sarcolemma,

fibrolysis, atrophying fibers, abnormally

hypertrophied fibers, merging of fibers and delineated

arrangement of nuclei etc. which seemed to be

irreversible during this stretch of study. With

fibrolysis taking place in the fiber, tension generated

within the fiber is altered, thus, causing bilateral

compression which results in reduction of sarcoplasm

Fig. 2―(A)-T.S. Gastrocnemius control: Intact fibers (IC) showing intense APase activity around sarcolemma shown with (↑); (B)-T.S.

of Gasrocnemius of diclofenac treated group for 10 days exhibiting intense APase activity (↑), atrophied cells (↑↑), degenerating cells

with noticeable degenerating Foci (↕); (C)-Section of gastrocnemius from diclofenac treated group for 20 days showing overall lesser

enzyme activity around the fibers; higher enzyme activity(↑) around intact cells (IC), reduced activity around degenerating cells ( ↓), few

merged fibers(*) also seen; (D)-Section of gastrocnemius from diclofenac treated group for 30 days revealing lighter staining for APase,

merging fibers(↓), fibrolysed fibers ( ↓↓ ) , APase activity positive around intact cells(↑).


451

and the myofibrils27

leading to atrophy. COX-2

activity is essential for efficient repair after muscle

injury6 as well as recovery from atrophy

28. NS-398, a

specific COX-2 inhibitor was documented to inhibit

the proliferation and differentiation of myogenic cells

in vitro6. Furthermore, it decreased the regeneration of

injured muscle by delaying the maturation of

regenerating myofibers, promoted fibrosis by

upregulating transforming growth factor (TGF-β1)

expression in mice gastrocnemius. It is speculative

that the observed hisopathological changes in

gastrocnemius might be due to diclofenac

administration which is also a partial COX-2

inhibitor. Rows of nuclei in interfibrillar region and

their displacement from peripheral positions to centre

of the muscle fiber were indicative of some

alterations. Such changes in the nuclear morphology

are apparently on account of loss of nucleoplasmic

substances29

. The administration of different classes

of COX inhibitors like aspirin, nimesulide and

celecoxib when used in animal studies at the clinically

safer doses have also caused alterations in the

biochemical and biophysical state of the intestinal

brush border membrane30

while diclofenac was

reported to have negative effect on colon anastomotic

healing in rats31

. Tubular epithelial cell degeneration

and necrosis in kidney and acute hepatitis has been

earlier reported in rats on administration of the high

dose of diclofenac32

that may be reversible or

irreversible.

The present study revealed that diclofenac induces

remarkable changes in specific activity of alkaline

phosphatase in serum and mice muscle as well.

Alkaline phosphatase has been categorized as cell

activity biomarker33

as well as hydrolytic enzyme

functioning at alkaline pH optima by a number of

workers earlier34

. Biochemically, it was found that

specific activity of APase in gastrocnemius decreased

to 48.74% and increased to 132.96% after 10 and 20

Fig. 3―APase activity in mice gastrocnemius in µM Pi /µg of

protein / h [***P <0.01,**P<0.02, *P<0.05; n=6].

Fig. 4―APase activity, analyzed by native PAGE in mice

gastrocnemius: D10 control group (lane 1), D10 diclofenac treated

group (lane 2), Lane 3 ,4 depicting enzyme activity in control

group and diclofenac treated group for 20 days (D20 group)

respectively, enzyme activity in control and drug treated group for

30 days (lane 5 and 6).

Fig. 5―Alkaline phosphatase activity (%) in mice gastrocnemius

[**P<0.001; *P<0.01; n=7 each].

Fig. 6―APase activity in serum of mice in µM Pi /µg of protein /

h [**P<0.001, *P<0.01; n=6].


452

days respectively. These altered patterns of the

enzyme activity were further confirmed by

densitometric analysis of gels where APase activity

declined (37.73%) which augmented to 122.2% after

20 days drug administration. The lytic process and

stimulation of alkaline phosphatase has been

documented in skeletal muscle by some workers35

earlier. In another study, enhanced fibrolysis was

documented with parallel rise in APase activity in

sciatectomized chick gastrocnemii36

.

In the treated spleen, a significant increase in the activity of APase was observed in the foamy cells and macrophages as well after use of diclofenac in rabbit

37. After 30 days of treatment, activity of APase

decreased to 13.97% in gastrocnemius in biochemical assay while elctrophoretic analysis also recorded parallel results (26.83% diminution). In previous studies with aspirin and nimuslide on rat intestine, the increase in the alkaline phosphatase enzyme activity was indicative of intestinal dystrophy

30. The decline

observed in APase activity during initial stage could be due to the cytoprotective role of non-steroidals to some extent. However, when the drug was continued for another 10 days, enhanced APase activity was noticed which further diminished after 30 days (13.97% diminution). From these varied trends of

APase activity, it is hypothesized that by lowering APase, diclofenac has played role of anti-inflammation protecting the tissue while in 2

nd stage

of the study, trend of enzyme reversal may be due to an adverse effect of the drug. Higher levels of alkaline phosphatase activity in diseased human muscle has

been associated with lytic role of enzyme and also leading to proliferation of non-contractile connective tissues

9. It is noteworthy that the role of alkaline

phosphatase varies differently in different tissues. APase has been proposed to be a function related marker in the renal proximal epithelia

38 having

protective effect on proximal tubule damage of the kidney

39. However, after aspirin treatment, 80%

decrease in APase activity in rat heart was documented to play cardiovascular protective role by preventing calcification of the system

40.

Advanced age and long-term physical exercise

cause changes in the activity of APase in rat muscle41

.

The nonspecific increase in the activity of alkaline

phosphatase by diclofenac exposure may be a result

of the incorporation of the drug in the place of zinc

atoms leading to an increase in the activity of this

enzyme as APase being a metaenzyme37

. In serum,

APase specific activity decreased to 18.78% and

showed 132.04% increase after 10 and 20 days and

again after 30 days of diclofenac treatment, increase

(109.09%) was noticed. Previous studies have shown

that cataflam (potassium diclofenac) significantly

increased the activity of alkaline phosphatase in

rat serum42

which is indicative of skeletal and

muscular disorders8.

The trends of APase in gastrocnemius and serum

in the present investigation exhibited unexpected similar decline in the enzyme activity after 10 days of drug treatment and hypothesized that decrease in the serum APase could be accounted for its hike in other tissues. However, increase in the serum APase during the last two stages definitely hints towards

some deleterious effects of the drug on different tissues including gastrocnemius for which many morphological evidences have been proposed. Increased transmembrane transport of diclofenac has been suggested where APase is involved in the absorption and transportation across these

membranes43

. The involvement of alkaline phosphatase in active transport

44 was reported by a

number of workers. Histochemical studies on gastrocnemius muscle

revealed lesser APase activity around sarcolemma initially. It was noticed that atrophied (narrow) fibers

showed greater activity as compared to the normal fibers after 10 days of treatment. It has been reported earlier that increased fiber alkaline phosphatase positivity is correlated with an increased incidence of degeneration and regenerative changes, fibrosis and atrophy in skeletal muscles

45-46. An association

of APase with pathological conditions has also been reported for skeletal muscle of chicks and rats, in which striated muscles revealed increased activity in pathological conditions such as denervation

47.

Declined levels of APase later on suggested that

the system may show adaptive responses towards

otherwise cytotoxic effects of the drug. However, the

level of serum APase was found to be higher in last

two stages of investigation suggesting some kind of

deleterious effects caused due to diclofenac

treatment. An increase in the activity of serum

alkaline phosphatase after aspirin treatment was

reported earlier and this increase was linked with the

hepatotoxic effects caused by aspirin48

. Although,

there remains a question of future research due to

limitation of this study as to look for other possible

reasons of raised levels of serum APase taking other

vital organs such as liver, kidney, bone and intestine

etc. into consideration. It is concluded from


453

the present study that altered APase activity after

sub-chronic diclofenac treatment can cause

histopathological and physiological changes in

skeletal muscle and may affect other tissues as well.

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