:MODIFICATION OF AFLATOXIN B,-INUCED CHANGES INCERTAIN MITOCHONDRIAL ENZY~1ES AND LIPIDS BY

MEDROXYPROGESTERONE ACETATE

R. VOHRA, S. MAJUMDAR*, V. MALIK, R. SINGH" AND J. P. NAGPAUL

Department of BiochEmistry.PanJab Univerity. Chano'i[,arh

and*Department of Obstetrics and Gynaecology.

Postgraduate Institute of Medioal Ec'ucation and Research,Chanc'if,arh - 760 072

( Received on septeber 14. 1985 )

Summary: The effect of a single dose of 5 mg/':g body weight of aflalOxin 81 on rat liver mitochondrial

erzymes. ~uccirate dEhydrcgenase (SOH) and Mg++ adenosine triphOsphatase (Mg++ -ATPas::!)

and on certuin lipids we: studif"s at vari0us intervals of time from 3 to 24 hours. A sig~ificant decrease

in the specific activit,! o' SOH was observed after (,.12 .18 and 24 hr treatm3 nt. The l\;lg++ -ATPase ac­

tivity remaired unaffected up to 12 hr but appreciabl,! decreased after.18 and 24 hr of the trealment. The

level of posp/lO!lpids and chOlesterol WE.;e not aitcred c.fter 3. 6 and 12 hr treatment. thereafter (18 and

24 hr) al1 increase waS observld in beth the lipids f;)lIo\',virg the aflatoxin treatment. Medrox,/progestrone

acetate (MFA) did not causs ary alterc.tion in the specific activities of these erz'!mes as well as levels

of che IE:stero I ard phospholipids. The treatmer.l wilh MFA caused Slgr iflcar.t ir.crease in canter.ts of

cytcchr( mes P-4CO. ts ord activities of Ary;rYGwcarbon hydraxylaoe (AHH), UOP-glucuron'!l trans­

ferase (UOP-GT) and NADFH-c'!tochrome C-reductase of hepatic microsomes.

It was observed that preatreatment with medroxy-prcgesterone acetate (MPA) could significantly

mil-imuze the Gepression oaused in mitochondrial SOH and Mg++-ATP2sc activities by aflatoxin Bl.

/

Key words: liver aflatoxin tOXicity

su ccinate dehydrcgcnase

INTRODUCTIION

medroX,/progesterone acetate

ATPase

Af!atoxins. which are hepatocarcinogens produced by Aspergillus, have been shown

to be metabolised by mammalian hapatic mixed function oxidase (MFO) system to anumber of oxidized pt-oducts which are less car-cinogenic and less toxic than the parent

compound (3).

Aflatoxin B1 (AFB 1) has been shown to exert disruptive effect on cellular membraneby altering their structural integrity a~d to elicit toxic effects in mitochondr-ia (24), It has

also been reported that AFB 1 caused decrease in mitochondrial enzymes (11).

COI:le:Sp0r.crr.g author: J.P. Na paul. LectLrer in Biochemistry. Panjab University. Chandigarh - 160014

VOlume ~9

umber 3ModificnliOrl of AFB1 induced changes by M PA 147

Medroxyprogesterone acetate (MPA), a synthetic long acting contraceptive isalso known to induce hapatic drug metabolizing enzymes including M FO system (22)

and thus the drug has got the potentiality to promote the metabolism of xenobiotics. Thre­fore, there is a strong possibility that MPA may alter the bioChemical effects of xenobiotics.

The present study was carried out with a view to find out the time at which maxi­mum toxic effects are exhibited by AFB 1 on mitochonod~ial enzymes and lipids. Further

ex erim9nts were planned to investigate the extent of modification of the toxicity of afla­toxin B1 on these parameters by MPA.

MATERIAL AND METHODS

Female albino rats of Wistar strain weighing 100-125 g were used. The animalswere fed ad libitum rei let diet (Hindustan Lever) and had free access to water. Forexamining the effects of Aflatoxin B. on mitochondrial enzymes. the animols were divided

into two groups; (I) Control (II) Experimental. Each group consist of 30 rats.

The control group received the vehicle (0.1 ml) cimethyl sulfoxide (OMSO) onlyand experimental were administered intraperitoneally a singal dose of AFB1 5 mg/kg, body

weight The animals were sacrificed at various intervels of time 3. 6, 12. 18. and 24 hI'after administration of aflatoxin B1 . A batch of 5 animals were killed in each time intervalunder light anaesthetic ether. The liver was removed quickly. blotted and w~ighed. 10%homogenate of this tissue was prepared in cold 0.25 ~/1 sucrose and mitochondria wasprepared according to the method of Johnson and Laroy (12), using MSE refrigerated.centrifuge.

In order to find out the effect of pretreatment of M PA on the toxicity of AflatoxinBJ on mitochondrial enzymes and lipids, female rats (100-125 g) were divided into twogroups: (I) Vehicle treated. (II) MPA treated. Each group consisted of 40 rats. Theanimals of group (I) were administered 0.1 ml of vehicle on'ly (polysorbate-80. 0.237 mg;

methylparaben, 0.134 mg; propylparaben, 0.0147 mg; polyethylene glycol 4000, 2.852 mgand sodium chlorido 0.8[67 mg per 0.1 ml) every week at 0, 7, 14 and 21 days while

group (II) received MPA i/m 3.5 mg/100 body weight for the same r:eriod. On 22ndday. the animals of group I and II were further divided : (a) Vehicle treated (b) Vehicle

treated + DMSO, (c) Vehicle treated + aflatoxin (d) MPA treated, (e) MPA treatedOMSO (f) MPA treated + Aflatoxin B1 . Animals belongir.g to band e groups wereinjected 0.1 mlof OMSO and c and f were administered intrar:eritoneally 5 mg/kg body weightof aflatoxin B1. The animals were sacrified at 24 hI'S after various treatments Mitochon­drial preparations of the liver were prepared by Johnson and Lardy method (12). Succinicdehydrogenase (SOH) activity was determined by the method of Ernest and Abood (5)

148 Vohra el a I Julv-Sapleml'ar 1985Ind. J. Fhysiol. Pilarmac.

and MgH-Ader,osinetr;r..(-,osphatase (Mg++-ATPase) was assayed by the method of Quigleyand Gotterer (19). Mitochondrial protein was estimated by the procedure of Gornallet at. (7).

The mitochondrial lipids were extracted according to the method of Folch et at.(6). The pllospholipid phosphorus was estimated by Bartlett's method (1) and choles­terol by the method of Zak (25). Hepatic drug-metabolizing enzymes were determined inMPA treated group only and for this purpose. liver was perfused in situ through portalvein with 20-40 m/ cold 015M NaC!. The tissue was homogenised in KCI-Tris-buffer.pH 74 (150 mM KCI/EO Tris-Hcl) and diluted to concentration of 1 g wet weightj4 m!with KCI/Tris-buffer. A small portion of the homogenate was kept for glutathione estima­tion and rest was centrifuged in (Sorvall RC-5B) at 10.000 x g and then at 105,000 x gfor CO min in Beckman ultracentrifuge Model L5-50B.

Glutathione concentration was determined according to the method of Moronet a/ (16) and activity of glutathione-5-transferase assayed following the procedure ofHabig et at. (9). The cOltents of Cytochrome P-450 and bs wore determined. by them,"thod of Omura and Sato (18). The activities of NAOPH-cytochrome C-reductaso. AHH

and UOP-glucuronyl transferase were determined by the m·~thods of Mazel (15). NE:b8rtGelboin (17) and Gorski ano Kasper (8) respectively. Student's t-test was applied forthe statistical' analysis of deta

Chimica/s: Aflatoxin B1 and chemica'is of vehicle WEre purchased tom SiemaChemical Com:)any. USA and MPA was the generous gift hom Upjohn Co.. Belgium

RESULTS

Time depender.t effect of Af/atoxin : The effect of a single dose of Aflatoxin on

the activities of liver mitochondrial SOH. Mg++-ATPasr and levels of phospholipids andcholesterol was studieo at diffel·ont intervals of tim~ to find out the optimum time for m3xi­mum Effect (Fi~. 1). Maximum decrease in SOH and Mg++-ATpase enzyme act;vity was

observe at 2nd hr tlmo> intervals. Similarly thE: membrilne lipid wero altered after 18 hrsbut maxirrum effect was observGd at 24 hr. Thus. 24 hr period was chosen for funher

studies.

Micrcsma/ enzyme induction and AFB toxicity: The effect of M PA on mitochon­drial enzymes and lipids is presented in Table I. The specific activities of SOH and Mg++­ATPase and contents of phospholipid and chclesterol content remained unaff8cted. Asignificant increase in hepatic M Fa enzyme system namely cytochromes P-450. bs. Ary!­hydrocarbon hydroxylase. NAOPH-Cytochrome C-reductase was observed in MPA treatedrats. 1 he activity of UOP-GT was also significantly augmented (Table II).

VOlum 79t,ub ,er 3

Modlical1~n 01 AFR1,ir,duc€d changes by MPA 149

1~0

• I

p<OOj

P<OOl

Ll CON'~Ol

rz;a Su eCI Nit Df: I1YO~O()ENASE

~ "'19'" Al p~,,&

fllJ PH05PHOlI PIG

o (HOl E S 1EROt ••r

lOa,....<I-~...'-'

50'-'...G-

o

Fig.1 : Effect of Aflatoxin 81 on liver mitochondrial SOH and Mg++-ATPase activities and levels of phosphol p ds 3'ldcholestero I. 1. 2. 3,4,5 represent 3.6, 12.18,24 time interva I in hrs at which the animals were Sacrificed aft~r

administration of Aflatoxin BI. Values are expressed as percentage taking control as 100. Each value is amean±S.O. of five individual mitochonarial preparations.

TABLE I ; Effect of Aflatc,xin B. on rat liver mitochondrial SOH, Mg++-ATPase.total chelesterOI and phospholipid in rats pretreated with MPA,

Tr£atment Prctem SOH MfJ++-ATPase Cholesterol Pho<phol;pidz

(mglg of liver) (SF actiVity) (Sp. activ.'ty)

(a) Ve~ir.1<. ·1S5±2.5 48!.i±4.2 0.205±0.u20 °280±O.014 1.1±O32

(b) V"hlclc+OMSO 177±31 481±4.0 O.24±O.014 O.242±O.065 100±O19

(c) Vehicle0.136±O.015"·d 1.48±O.23·d+AflaPxin 1G.2±31 30.6±5.0··d °365±O.035

(d) MPA 187±2.9 528±98 0203±0.C17 °252±O.018 132±O24

(c) MPA+OMSO 178±42 500±82' e o 245±O.019···· °272±O.015··· 1,10±O11'

(I) MPA+Aflaloxin 169±33 478±53·· 0.185±0.O18(:"·1 °34 5±O 26'" 1.22±O185

Value ar mean ±S.O. of 5 individual mitochonGfial preparations.>: = ILmeles of trlphenyltetra~cJlum chluflce (TTC) reouced/~r/mg protem.Y = ILmcles of Pi I'berated/hr/mg proteinZ - mg/g wet tissued = Significant as compared to vehicle+ OMSO grcup.e - Significant as compared to vehicle+aflatoxin.f Sionificant as compared to MPA+OMSO .• = P<:o,05 ; "P<o01 ; ·"P<O.001

50 Vohra et 81. July-September 19B5Ind. J. Physio!. Pharmac.

TABLE II: Effect of MPA 0n hepatic drug-metabolizing enzymes system

Parameter Control MPA tree ted

Cytochrome p-450 (nmol/mg/min) O.S2±O.OS O.99±Ol·

Cytochrome bs (nmol/mg/min) O.50±O.O5 a 66±0.OS··

Arylhydrocarbon hydroxylase (AHH)(p-mol/mg/mm) 225.0±320 362.0±40·O"·

UDP-gucuronyl transferase(nmol/mg/m1n) 36.4±4.5 43.6±6.6

ADPH-Cytochrome C-reductase(n mol/mg/mm) 3S.S±..... 46.2±5.S·

Glutathione «(J.g/g liver) 3.2±O.51 3.74±O6

Glutathione-5-tr~nsfera,en moleCDN B conjugated/min/mg solublfi

71 S±SO.Oprotein 705±tll.0

Values are mean±S,D. of 5 individual observations. Values of Cytochrome P-450. bs. AHH. UDP-glucuronyl trans­ferase and NADPH-Cytochrcme C-rtductase are expressed as per mg microsomal protein.

P<O.05 : ··P<O.Ol : "·P<O.OO1. significant ~s compared to control.

The results of AFB1 treatment (24 hrs) on liver mitochondrial enzymes. SDH andMg++-ATPase as well as on lipids of rats pretreated with MPA for 30 days are shown inTable I. The decrease in spacific activities of SDH and Mg++-ATPase was prevented toa great extent by MPA. but not the AFB 1 induced accumulation of mitochondrial phos­

pholipid and cho'16sterol contents.

DISCUSSION

The results obtained on the depression of SDH activity following AFB 1 <ldminis­tration are in accordance with those of Theron (24) who observed a progressive decreasein staining intensity in tissue sections incubated for demonstration of SDH and ATPaseactivities in animals killed bOTwen 8 to 24 hr. Brown and Abrams (2) had reported thatAFB 1 markedly decreased the activity of certain mitochondrial dehydrogenases in livers.Similar results were observed by Raj and Venkitsubramanian (20) in chicks treated withAFB\. They observed inhibition of SOH activity in chick liver 24 hr after a single doseof AFB\. The results of present study showed that action of the toxin on SDH activity wa5time dependent and maximum decrease in its activity was observed after 24 hr.

There are conflicting reports regarding the effects of aflatoxin B1 on Mg++-ATPasf<activity. Clifford and Rees(4)as well as Ramachandra Pai et al. (21 )observed that aflatoxindid not alter Mg++-ATPase activity. However. Shankaran et al. (23) obc:;elved decreasein Mg++-ATPaie after 2 hI's and then increase after 8 hrs of aflatoxin administration but

VOlume 29Numher 3

Modification of AFB1-induced Changes by MPA 161

in the present study a significant decrease in Mg++-ATPase activity was noted after 18and 24 hr treatment with aflatoxin BJ • The alteration in MgH-ATPase act'ivity might bedue to uncoupling of oxidative phosphorylation by aflatoxin as observed by RamachandraPai et al. (21).

The elevation in mitochondrial phospholipid and cholesterol content might be dueto accumulation of lipids in the liver as effect on lipid metabolism is the primary lesionduri ng aflatoxicosis (10).

There has been great deal of controversy on the role of metabolic activation of AFB J

in the acute toxicity of the compound. It has been reported that there is reduction incytotoxicity after metabolic activition of AFB J (14). But others have contradicted this

view and have reported that the acute toxicity is dependent on the conversion of AFB J

into a toxic metabolite (13). lin the present study aflatoxin toxicity was diminished in MPAtreated rats.

The mechanism of decrease in atlatoxin B J toxicity by MPA could be clue to thestimulatory effect of this drug on hepatic M FO system leading to greater conversion of itto its hydroxylated derivatives which may be less toxic th:1n the pare:lt compound. It is.

therefore. reasonable to conclude that acute mitochondrial membrane dam3ge as seen withtha decrease in anzvm~ activity is due to the native compound which possibily gets conver­

t d to less toxic m~tabolites by microsomal enzymes.

REFERENCES

1. Bartl 0. G.R. PhoEphorus assay in column chromatography. J. BioI. Chem .. 234 : 466·468.1959.

2. Brown. J.M. and L. Abrams. Biochemical studies on afliiloxicosis. Cnderstepoorl. J. Vet. Res.• 32 : 119­146. 1965.

3. Campbell. TC. and J. R Hayes. The role of aflatoxin metabolism in its toxic lesions. Toxieol. Appl. Pharmacal.•35(2) : 199-222. 1975

4. Clifford. J.I. and K.R. Rees. The action of aflatoxin BJ on the rat liver. Biochem J .. 102 : 65-75. 1967.

5. Ernest. K. and L.G. Abood. Colorimetric estimation of succinic dehydrogenase by triphenylletrazolium chlorideSCience. 10 : 144-146. 1949.

6. Folch. J .. M. Less. and G.H.S. Stanley. A simple method for the isolation and purification of total lipid fromChem.• animal tissues. J. BioI. 226 : 497-509.1957.

7. Goma!. AG .. C.S. Barcwill and M.M. David. Spectrophotometaric and turbidimetric methods for measuringproteins. In: Methods in Enzymology (Ed. S.P. Colwick and N.O. Kaplan) New York. Academic Press. 2 :450-451. 1957.

8. GorSki. J.P. and C.D. Kasper. Purification and properties of microsomal UDP. glucuronyltransferase from ratliver. J. BioI. Chem .. 252 : 1336-1343.1977.

9. Habig. W.H .. M.J. Pabst and W.O. Jakoby. The first enzymatic step in mercapturic acid formation. J. BioI.Chem .• 249 : 7130·7139. 1974.

10. Hsi-Tang-Tung. W.E .. D. Donaldson and P.B. Hamilton. Altered lipid transport during aflatoxicosis. TO'l(ieol.App I. Pharmaco I.• 22 : 97 -112. 1972.

11. Jayanth i. Rai. N.. M. Ramachandra Pal and T.A. Vankitasubramanian. MitoChondrial function in aflatoxin toxicityIndian J. Biochem.. Biolphys.. 14: 347-349. 1977.

152 V"nril pI at. uly-S~p!em"er 1985Ind. J. Physiol. Pharm1c

12. Johnson, D. and H. Lardy. Isolation of I,ver llr kidney mitochondria. In: Methods in Enzymology (Eds. S.P.COlowick ar.d N.O. Kaplan. New York. Academic Press, 10 : 94-96, 1967.

13. Lambiotts. M. and N. Thierry. Enhancement or aflatoxin B cytotoxicity in differentiated rat hepat1ma culturesby a prior glucoc'Jrteioid treatment of the monolayer. Biochem. Biophs. Res. Common .. 89 : 933-942.1979

H. Madt--avan• TV. Effects of prednisolons on liver damage in rats induced by aflatoxin. J. Pathol. Bacteriol.. 93 :443-447. 1967.

15. Maze!. P.ln: Fundamentalsofdrug metabolism and drug disposition (Ed. Bert. N. Ladu. H. George. M. Way. E.L.)The Williams and Wilikins Company. Baltimore. 1971. p. 546.

16. Moron. M.S.. JW. Depiarre and B. rviannervik. Levels of glutathione. g!utathions reductase and glutathione-S­transferase activities in rat lung and liver. Biochem. Biophys. Acta .. 582 : 67-78. 1979.

17. Nebert. D.W. and HV. Gelboin. Substrats inducibie microsoma I aryl hydroxylase in mammalian ceil culture.Assay properties of induced enzyme. J. Bioi. Chem .• 243 : 6242-6249. 1968.

1B. Omura. T. and R. Sato, The cllrbon monox;de binding pigment of liver microsomes. J. 8iol. Chem., 239 : 2370­2378. 1964.

19. Quigley. J.P. and G.S. Got!erer, Distribution of (Na+-K+). stimulate ATPase activity in rat intestinal mucosa,Biochem. Biophys. Acta.• 17 : 456-468. 1969.

20. Raj. H.G. and TA. Venkitasubramanian. Carbohydrate metabolism in aflatoxin toxicity. Environmental Physiol..Biochem., 4 : 181-187. 1974.

21. Ramachandra Pai, M., N. Jayanthi Bai and TA. Venkitasubramanian, Effect of aflatoxins on oxidative phosphory­lation by rat liver mitochendria. Chem. Bioi. interactions. 10(2) : 123-131. 1975.

22. Sarani, H.A. T. Jorma. NT. Karki. B.S. Palkonen and A. Sero. Dose dependent effects of medroxyprogp.steroneaceta!e on the hepatoc drug metabolizing enzyme sys'em in rats. Biochem. Pharmacol., 29 : 1155-1159.1980.

23. Shankaran, R., H.G. Raj and T.A. Venkitasubramanian. Effect of afla!Qxin on carbohydrate metabolism inchicken liver. EnzymologllJ, 39 : 371-378. 1970.

24. Theron. J.J. Acute liver injury in ducklings as a result of aflatoxin poisoning. Lab. Invest., 14 : 1586-1592. 196G.

25. Zak. D. Simple rapid microtechniqull for total cholesterol. Am. J. Clin. Path., 27 : 583-592, 1957.