Indian Journal of Experimental Biology Vol. 41 , A ugust 2003, pp. 814-820

Inhibition of membrane Na+-K+ ATPase of the brain , liver and RBC in rats administered di(2-ethyl hexyl) phthalate (DEHP) a plastici zer used

to polyvinyl chloride (PVC) blood storage bags

C R Dhanya, A R Indu, K V Deepadevi & P A Kurup*

Department of Bio logical Sciences, R&D. Terumo Penpol L imited, Tri vandrum-6950 14, Indi a

Received 16 Seplelllher 2002: revised II March 2003

Significant amounts o f di(2-ethy lhexy l) phthalate (DEHP) leach out into blood stored in DEHP pl as ti cized polyvi ny l chloride (PVC) bags resul ting in the ex posure of recipients of blood transfusion to thi s compound. The aim of thi s study was to find out whether DEHP at these low leve ls has any effect on the acti vi ty of membrane Na+-K+ ATPase. since a decrease in thi s enzyme acti vit y has bcen reported to take place in a number of di sorders like neurodegenerati ve and p,ychiatri c disorders. coronary artery disease and stroke, syndrome- X , tumours etc. DEHP was adm inistered (ip) at a low dose of 750 Ilg/ l OO g body we ight to rats and the acti vit y of membrane a+-K+ ATPase in li ver, brain and RBC was est imated. Histopathology of bruin. activity of HMG CoA reductase (a major rate limiting enzyme in the isoprenoid path way of which digox in, the phys iolog ica l inhibitor of Na+- K+ ATPase is a product), intracellular concentrati on of Ca~+ and Mg~+ in RBC (wh ich is altered as a result of inhi bit ion of Na+-K+ ATPase) were also studied. (In the light of the observati on of increase of int racellular Ca2+ load and intracellu lar depletion of Mg2+ when Na+- K+ ATPase is inhibited). Histopathology of brain revea led areas of degenerati on in the ra ts admi nistered DEHP. There was signi fi cant inhibition of membrane Na+-K+ ATPase in brain , l iver and RBC. Intracellular Ca2+ increased in the RBC while in tracellular M g2+ decreased. However acti vity of hepatic HMG CoA reductase decreased. Activi ty of Na+- K+ ATPase and HMG CoA reductase, however returned to normal levels w ithin 7 days of stopping admin istra ti on of DEHP. T he inhi bition of membrane Na+-K+ ATPase activi ty by DEHP may indica te the possibi l ity of predispos ing recipients of transfusion of blood or hemodialys is to the various disorders mentioned above. However since thi s effect is reversed when DEHP administrati on is stopped, i t may not be a seri ous problem in the case of a few transfusion; but in pat ients receiving repeated blood transfusion as in thalassemia patients or pati ents undergoing hemodialysis. possib ility of thi s ri sk has to be considered. T his inhibiti on is a direct effec t of DEHP or its metabo lites, since acti vi ty of HMG CoA reductase, (an enzyme which ca talyses a major rate li miting step in the isoprenoid path way by which digoxin, the ph ys iolog ica l inh ibi tor of Na+- K+ ATPase is synthesized) showed a decrease.

K eywords : DEHP. HMG CoA reductase. In tracell ular Ca!+ and M g!+ concentration, M embrane Na+-K+ ATPase, Poly vinyl chloride

DEHP [d i(2-ethylhexy l)phthalatel , a widely used pl asticizer in blood storage bags, leaches out in appreciable amounts into blood (about 10- 15 mg1l 00ml ) 1.2 resulting in ex posure of rec ipients of blood transfusion to thi s compound3

-s. Vari ous reports

indicate the tox icity of DEHP, particul arl y in li ver and reproducti ve organs6

- t:1 bu t all these stud ies used large doses (up to 2g or more/kg body weight) and oral route of administration, which are not relevant to the intravenous admini stration during blood transfusion or the low amounts of DEHP present in blood. We had earlier reported that admini stration of DEHP at low doses (corresponding to transfu sion of 2,4,6 and 10 units of blood) to rats did not produce any hi stopathological changes in the li ver or testes or any

*Correspondent author : E-mail : tp lrd @asianeti ndia.com Phone: (047 1) 337355 Fax : (047 1) 72 I 5 19

signi ficant alterati on in many of the biochemical parameters studied like y-glutamyl transferase, alanine aminotransferase, CPK, LDH , alkaline phosphatase ~­glucuronidase of the li ver, plasma and testes, serum al! ll tmin , acid phosphatase of the testes etc l

. There was however decrease in concentrati on of vitamin E in the liver and testes whi ch however returned to normal within 72 hr to 7 days 14. Decrease in the concentrati on of vitamin E in the blood stored in DEHP plasticized PVC bags was also observed by

14 us .

We have now can'ied out further detai led studies on the effect of DEHP and in vestigated the changes in the membrane Na+-K+ ATPase of the brain , li ver and RBCs, since a decrease in thi s enzy me acti vity is known to be involved in the pathogenesis of a number of disorders like neurological di sorders, hypertension, diabetes, coronary artery di sease and stroke, tumors

DHANYA el at.: INHIBITION OF MEMBRANE Na+-K+ ATPase BY DEHP 8 15

etc. 15.22. Na+-K+ ATPase is a membrane bound

enzy me invo lved in the transport of various cations and the steroidal g lycos ide digox in, now known to be ynthesized by the mammalian hypothalamus23 is the

physiological inhibitor of this enzy me24. It is also

known that an inhibition of thi s enzyme results in a depletion of intracellul ar Mg2+ and increase in intracellular Ca2+ load25 . The change in the intracellular levels of calcium and magnesium is believed to be the major factor in the pathogenesis of the various disorders mentioned above. If DEHP at the level present in blood stored in DEHP plastici sed PVC bags causes any inhibition of Na+-K+ ATPase, then the possi bility of exposing a recipient of blood transfusion to a ri sk for the above-mentioned disorders, has to be reckoned with.

We therefore studied the changes in the activity of this enzy me in the membrane of brain, liver and RBCs in rats admini stered DEHP at a dose of 750 Ilgfkg body weight which corresponds to success ive transfusion of 10 units of blood in a recipient. The intrace1 lular Mg2+ and Ca2+ concentration was also studied in the RBCs. Since the physiological inhibitor of Na+-K+ ATPase is digoxin, which is known to be synthesized by the hypothalamus, from acety l CoA by the isoprenoid pathway 15, the acti vity of a key regulatory enzyme, viz. HMG CoA reductase of this pathway was also studied to ascertain whether DEHP acts via influencing this pathway. Concentration of cholesterol in the serum, which is also sy nthesized by this pathway, was also studied .

Necessary ethical clearance of this work was obtained by the ethical committee of the Institute.

Materials and Methods Female, albino rats (Wistar strain , average body

weight 150 g) were used for this study. The rats were grouped randomly into two groups with 16 rats in each group.

I. Group I 2. Group 2

control rats experimental rats

The experimental rats were admjni stered DEHP at a dose of 750llgfkg body weight. Emulsion of DEHP was prepared in 2.2% glycerol containing 1.2% egg yolk lecithin by sonication under sterile conditions and was administered intraperitonially as described earlier l4

. The control rats received the same volume of vehicle. The animals were caged individually in polypropylene cages and maintained on normal laboratory feed (Sai feed) at temperature 28° ± 1°C. Food and water were available to the rats ad libitum.

Administration of DEHP was made on alternate days. A total of 7 injections were given and at the end of 14 days, 8 rats in each group were deprived of food overnight and blood was removed to citrated tubes for preparation of RBCs. They were then stunned by a blow at the back of neck and killed by decapitation. Brain and li ver tissues were removed to ice-cold containers for various estimations.

The injection of DEHP was stopped in remaining 8 rats in group II at the end of 14 days and these rats along with control rats continued on their diet for another 7 days. They were then deprived of food overnight, and killed as above. Blood, liver and brain were collected for various estimations as before. This was done to ascertain whether the effect produced by DEHP was reversible or not, when DE HP administration was stopped.

A nalytical Procedures Estimation of membrane Na+ -K" ATPase actlvlty

(EC 3.6.1.37)- Preparation of RBC membrane: RBCs were separated from citrated blood by centrifugation at 1300 rpm in a Sorvall super Tl centrifuge (USA) using rotor SL-50T for 15min at 4°C. The erythrocyte fraction after removal of buffy coat and plasma was washed thrice by centrifugation and resuspended in 0 .9% NaCI solution (3000 rpm for 10 min at 4°C). The cells were then lysed with di stilled water and the cell membranes washed twice with 1 mM tris-HCI buffer (PH 7.4) containing 1 mM EDT A at 4°C followed by washing with 1 mM tris­HCI buffer containing 10 mM EDT A and finally thrice with 2 mM Tris-HCl buffer (PH 7.4). The RBC membrane was finally suspended in 0.0625M Tri s­HCI buffer pH 7.4. The membrane bound enzyme was released by freezing the suspension and thawing it and repeating the process26

. This suspension was used for the assay of Na+-K+ ATPase.

Preparation of the membrane from brain and li ver ti ssues: Ti ssues were homogenized under ice-cold conditions in 0.25 M sucrose and centrifuged at 3500 rpm for 10 min at 4°C. The residue containing cell debris and unbroken cells was washed with normal saline twice and the rest of the procedure was the same as described for RBC.

Assay of Na+-K+ ATPase activity: The following reaction mjxture was used. Reagents : (1) NaCI-0.25 M , MgS04-O.00138 M and KCI-

0.0125 M-O.4 m1 (2) Na-ATP -0.00 125 M-O.4 m1

8 16 INDIAN J EXP BIOL, AUGUST 2003

(3) Tris-HCl buffer - 0.0625 M (pH 7.4), containing 3.l 2x 10.4 M EDTA-0. 16ml

Two sets o f tubes, o ne test and the o ther control , containing the above reagents in a tota l vo lume of 0.96ml , were preincubated at 37°C for 5 min and 0.2ml of the enzy me preparation (20-25 mg/ml prote in in the case of li ver membrane, 0 .8-lmg/ml prote in in the case of brain membrane and 4-5 mg/ml prote in in RBC membrane) was then added to the test. The tubes were incubated at 37°C for I hr and were then transferred to powdered ice and ice-co ld 12% TCA ( I ml ) was added to both followed by 0.2 ml enzy me preparation to the control. The ATP remaining in the test (after the action of the enzy me) and in the control (enzy me inacti vated) was then measured by using kit purchased from Sigma chemica l. (USA) Concentra­tion of the protein in the enzy me preparation was estimated after di ssolving the TCA prec ipitate in 0 .0 1 NaOH. Lowry ' s procedure was used for

. ·· ?7 protell1 es tllnatlon- . Analysis of intracellular Ca2+ and Ml+ in the

RBC - For the analys is of intracellul ar Ca2+ and Mg2+ in the RBC, the ce ll s were lysed with millipore deionised spectroscopy grade water and depro te ini sed with perchl ori c ac id (fina l concentratio n-4%). The supernatant was neutra li zed with KOH and kept at 4°C overn ight. The precipitated potass ium perchl orate was removed by centrifugati o n and the supernatant was used for estimatio n o f Ca2+ and Mg2+ using commercial kits (Ci+-Bayers and Mg2+-Randox).

Activity of HMG CoA reductase (EC 1.1.1.34)­Activity of HMG CoA reductase in the liver was studi ed by the method of Rao and Ramakri shnan by measuring the ratio of HMG CoA to mevalonate28 . A low ratio indicates higher activity. Choleste rol in serum was estimated by us ing kit ava ilable from Randox.

For histopatholog ical examination of the brain , the ti ssue was transferred to buffered 10% formalin and the sections were sta ined with haematoxylin and eos ll1 .

Statistical analysis was carried out using ' ANOYA, 29. SPSS statistical programme was used and the results were evaluated using analysis of variance utilizing the ' t ' test. The results were presented as the mean ± SD. Differences among the means o f the groups were assessed using the Duncan Proceedure to determine which mean values were significantly different at P<O.OI and P between 0.01 and 0.05 .

Results

Histopatholog ical evaluation - Hi stopatho logica l examination of the bra in revealed areas of degenerati on (Fig . I ). Results of hi stopatho logial examination of live r was reported before' . No gross changes were observed in the li ver.

Activity of Na+ -IC ATPase in the membrane of RBe, liver and brain cells (Table J) - There was significant inhibition of the enzyme activity in the membrane of RBC, liver and bra in in the rats admini stered DEHP, when compared to control rats. The effect was reversed and near normal va lue of enzyme activity was observed at the end of 7 days afte r withdrawal of DEHP.

. . II I C 2+ d M 2+ . ConcentratlOn of lntrace u ar a an g 111

the RBC (Table 2)- Sig nificant decrease in the Mg2+ in RBC was observed in the rats admini stered DEHP

Fig. I - Histopathology of brain from rats receiving 750 JLgl l OO g body weight by successive administration. Sections stained with H&E. 200 x. [A -Control rats, B - Experimental rats, i-Normal neuronal cells, ... - Neuronal degeneration

DHANYA el al.: INHIB ITION OF MEMBRANE Na+- K+ ATPase BY DEHP 817

when compared to contro l rats. This decrease was reversed after 7 days when the injec ti on of OEHP was stopped, indicating that the effect is reversible.

On the other hand intracellular Ca2+ concentration in the RBC showed significant increase, which too returned to near normal leve ls after 7 days of withdrawal of OEHP admini stration .

Activity of hepatic HMG eoA reductase and concentration of serum cholesterol (Table 3)­Activity of hepatic HMG CoA reductase and concentrat ion of serum cholesterol showed significant decrease in the li ver of the rats admini stered OEHP, when compared to control rats. Both these also returned to very near normal levels when the admini stration of DEHP was stopped .

Discussion The observation that administration of OEHP on

alternate days at low level s (corresponding to the successive transfusi on of about 10 units of blood in a recipient) causes degenerative changes in the brain is

important. Prev ious studies carried out in thi s laboratory however showed no gross histopatho­log ical changes in liver or testes at these levels l

.

The results obtai ned indicate that administration of DEHP at low levels caused significant inhjbition of membrane Na+-K+ ATPase in the brai n, li ver and RBC. Thi s is an important observation in view of the reports of a significant decrease in this enzyme activity in the RBC membrane in many neurological and psychiatric disorders {epilepsy , Parkinson's d isease (PO), schi zophrenia, manic-depress i ve psychosi s (MOP), CNS g lioma, multiple sclerosis (MS), sub acute scleros is pan encepha litis (SSPE) } 15.17.30, CAD (coronary artery di sease) and stroke, syndrome-X and tumors). Inhibition of thi s enzyme activity was also observed by us in the heart in isoproterenol induced myocardial infarction in rats (unpublished report) and in the brai n in quino linic ac id induced neurodegeneration in rats31

.

Thus the present observation may suggest that transfu sion of blood stored in OEHP pl asticized PVC

Table I - Effect of admini stration of DEHP to rats on the acti vity of Membrane Na+-K+ ATPase in the brain. li ver and RBC

[Values. expressed as tLmole of ATP hydrolysed/hr/mg protein , are mean ± SD of 8 rats in each group J

Group Activity of membrane Na+ K+ ATPase at the end of Acti vity of membrane Na+- K+ ATPase after 7 days of administration of DEHP stopping DEHP

Brain Li ver RBC Brain Liver RBC

Control rats 6.75±0.981 0.461±0.019 0.442 ± 0.054 6.92±0.4 11 0.432 ± 0.0 12 0.452 ± 0.099

Test rats 3.24 ± 1.511 " 0.258 ± 0.064' 0.274 ± 0. 1 04" 6.5 1 ±0.295 0.425 ± 0.0 19 0.396±0. 121

P values:"< 0.0 1, bbetween 0.0 1 and 0.05 compared to corresponding control s

Table 2 - Effect of admi ni stration of DEBP to rats on the concentration of intracellular Ca2• and Mg2+

[Values. expressed as mg in RBC from 100 ml blood, are mean ± SD of 8 rat s in each group]

Group Conc. of intracellu lar Ca2+ & Mg2+ at the end Conc. of intracellular Ca2+ & Mg2+ after 7

of administrat ion of DEHP da:ts of stopping DEHP

Ca l + Mg2+ Ca l + Mg2+

Control rats 0.67 ±0.04 3.634 ± 0.186 0.68±0.04 3.7 1 ±0.75

Test rats 0.826 ± 0.05" 3.097±0.37b 0.72±0.043 3.47±0.42

P values: "< 0.0 I, bbetween 0.0 I and 0.05 compared to corresponding controls.

Table 3 - Effect of admini strati on of DEHP to rats on the Activity of HMG CoA reductase and concentration of serum cholesterol

[Values, are mean ± SD of 8 rats in each group]

Groups

Control rats

Test rats

Activity of HMG CoA Activity of HMG CoA reduct ase at the end of reductase after 7 days of

admini stration of DEHP stopping DEHP HMG CoAlMevalonate ratio*

1.893 ± 0.085

2.38 ± O. 107"

1.910 ± 0.077

J. 931 ± 0.086

*Higher the ratio: lower the acti vity

Con. of serum cholesterol at Con. of serum cholesterol the end of adm ini stration of after 7 days of stopping

DEHP DEHP

74.4± 2.59

70.5±2.25b

mg/dl serum

74.58 ±2.57

75.04±2.62

P values : '< 0.0 I, bbetween 0.0 I and 0.05 compared to corresponding controls.

818 INDI AN J EXP BIOL, AUGUST 2003

blood bags can expose a rec ipient to the risk o f any of the various disorders mentio ned above. However the fact that thi s inhibition of Na+-K+ ATPase activity is reversed when DEHP is withdrawn appears to be a saving factor. But with frequent or repeated transfusions of blood sto red in DEBP plasti c ized PVC bags as in the case of tha lassemia patients or patients undergoing hemodialys is, thi s ri sk facto r has to be considered. In fac t, in the recent National seminar on thalassemia he ld in ew Delhi (281h April 200 1), Phys ici ans treating tha lassemi a patients reported neurological, card io log ical and other complications in those getting repeated transfusion o f blood stored in DEHP plasti c ized PVC bags.

Membrane bound a + -K+ ATPase is an important enzy me utili z ing the energy of ATP hydrolysis for transport of severa l cati ons. It i known that an inhibition of thi s enzy me produces an increase in intracellular Ca2+ and a decrease in intracellul ar Mg2+25. Wang e l 0 1.

32 reported e levation of intrace llul ar Ca2+ from 125 nM to 408 nM when Na+­K+ ATPase activity was inhibited. Yuan Wei et al. 33

also recently confirmed that inhibitio n of Na+­K+ ATPase with strophanthidin increased intrace llular Ca l

+ to a very hi gh level. Inhibition of membrane Na+- K+ ATPase results in

increased re lease of Ca2+ from endopl asmic reti culum stores and increased entry of Ca2+ via the vo ltage gated calc ium chan nel. Inc reased intrace llular calc ium disp laces magnesium fro m binding sites and magnesium being free ly permeable, leaks into se rum . There is defec tive reabsorption of magnes ium from the renal tubules because of increased tubular cell ca lc ium I9.25

. Similary increased intesti na l epithe li a l ca lcium inhibits magnes ium absorpti on from the gut. Th us inhibiti on of membrane Na+-K+ ATPase can lead to increased entry of calcium into the cell and produce cellul ar magnesium depl eti on. The present observat ion o f decrease in intrace llul ar Mg2+ and increase in intracellular Ca2+ in the RBCs in rats administered DEBP ag rees w ith thi s. A direct consequence of inhibition of Na+-K+ ATPase is :ncrease in concentratio n of intrace llul ar Na+ and d..:pletion of K+. Increase in intrace llular Na+ in tum ra ises intrace llular concentration of Ca2+ by stimulating Na+/Ca2+ exchanger. There is thus d isru ption of ion homeostasis .

Increased intracellular Ca2+ can trigger apoptos i ~ or cell death by several mechanism. It can activate ci+ dependant calc ineurin signal transduction pathway which can produce T cell activation resulting in the

secretion of TNF a lpha (Tumour Necrosis Factor a lpha) among other things34 TNF alpha binds to its receptor and in tum activates the caspase cascade, espec ia lly downstream caspase-9 and produce apoptos is35

. Increased intracellul ar calc ium can open the mitochondri a l PT (permeability transition) pore causing co ll apse of the proton g rad ient across the inner membrane and uncoupling of the respiratory chain36 Thi s causes the rupture of the outer membrane of mitochondria resulting in the re lease of AIF (apoptos is inducing factor) and cy to C (cy tochro me­C) to the cytoplasma. Thi s results in acti va tion of caspasc-9, which produces cell death . [n thi s connec tion X iao et 0 1. 37 observed from their studies o n cultured cortical neurons that inhibiti o n o f a+ K+ ATPase resulted In neuronal death invo lving concurrent apopto ti c and necrotic components, mediated by intracellular depletion o f K+ and accumul ation of Ca2+and a+ respecti ve ly. Di sruptio n of io n homoeost.1sis was considered a major factor by them. We have earli er mentioned di ruption of ion homoeostasis as a consequence of Na+ K+ ATPase inhibition . Apoptosis has been implicated in neuronal degeneration36

. Mg2+ deficiency , re lated A TP synthase defect, amo ng other things can also contribute to mitocho ndri a l dys fun ct io n resulting in incomple te reductio n of O 2 and increased product io n of free radical s. Increased Ca2+ level can a lso acti vate NOS (nitric ox ide synthase) causing increased production of NO which combines with superox ide radi cal to form peroxynitrite io n promoting lipi d perox idati o n38

. Free rad ical damage has been implicated in the neuronal degeneratio n39

.

Inc reased intracellular Ca2+ a lso acti vates the signal transducti on mechani sm in volved III NM DA transmiss io n which results in inc reased g lutamaterg ic exci to tox icit y, important in neuronal degenerati ve di sorders4o, ~ I .

The inhibition of Na+-K+ ATPase may be caused directl y by DE HP or its important metabo lite MEHP or indirectly by its stimulatio n of the isoprenoid pathway of which digox in , an inh ibitor of thi s enzy me, is one of the products. In most . of the di seases particularl y epilepsy, PO, schizophrenia, MS , SSPE, CAD and stroke, syndrome-X, an increase in serum digox in has been reported 15. 17.30. The fact that

HMG CoA reductase acti vity , the major rate limiting step in the isoprenoid pathway, is decreased in rats administered DEBP may argue aga inst the poss ibility that DEBP acts via stimul ating the endogenous synthesis of digox in . The observed effect on

DHANYA et 01.: INHIBITION OF MEMBRANE Na+- K+ ATPase BY DEHP 819

membrane Na+-K+ ATPase may therefore be a direct effect of DEHP or its metabolites, particularly MEHP. [0 thi s connection, we had reported earlier that significant amount of DEHP is observed in the RBC membrane in blood stored in DEHP plasticized PVC bags42

. This indicates that DEHP gets incorporated in to the cell membrane. We had further reported changes in the protein, glycosaminoglycans(GAG), carbohydrate components of glycoprotei ns, cholestero l, phospholipids and vitamin E in the RBC membrane in the presence of DEHP42. The protein : GAG ratio, protei n: carbohydrate rat io, cholesterol: phospholipid ratio as well as protein : lipid ratio, all showed significant increase in the RBC membrane in the presence of DEI-IP42. It is clear from these observations that incorporation of DEHP into the RBC membrane results in significant alteration of the integrity of the membrane. This may possibly be responsible for the inhibition of the Na+-K+ ATPase, which is a membrane, bound enzyme. Further work is III progress to elucidate the mechanism of this inhibition.

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