1991 BP Nazareth Etal

8/12/2019 1991 BP Nazareth Etal

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Biochmical Pharmacology, Vol.42,No. 4,pp.931-936,991. O -2952/913.00 0.00PrintednGreat ritain. @ 1991.ergamonress plc

EFFECT OF PARACETAMOL ON MITOCHONDRIALMEMBRANE FUNCTION IN RAT LIVER SLICES

WILHERMINA M. A. NAZARETH, JASWINDER K. SETHI and ANDRE E. M. MCLEAN*Laboratory of Toxicology, Department of Clinical Pharmacology, University College London,5 University Street, London WClE 655, U.K.

(Received 29 October 1990; accepted 1 May 1991)Abstract-The effect of paracetamol on mitochondrial function was studied using rat liver slices.Changes in the potential of the mitochondrial and plasma membrane were monitored using [3H]-triphenylmethylphosphonium (TPMP+) and [4CJthiocyanate (SCN-) probes, respectively. Liver sliceswere exposed to 10 mM paracetamol for various time periods (O-360 min) after loading with TPMP+.The release of TPMP+ which correlates with a decrease in the mitochondrial membrane potentialbecame significant after 30 min incubation with 10 mM paracetamol. The change in the mitochondrialmembrane potential was shown to be independent of cytochrome P450 activity. No significant changein plasma membrane potential was observed, until the release of lactate dehydrogenase (LDH) hadbegun, 4 hr after exposure, reflecting the ultimate stages of cell injury by paracetamol. These resultssuggest that paracetamol elicits a direct effect on the mitochondrial function before cell injury developsand adds further evidence to the role of mitochondria in paracetamol toxicity.

Paracetamol (N-acetyl-p-aminophenol, acetamino-phen) in overdosage leads to liver damage in manand many species of animals [l-4]. Paracetamollevels in the peripheral blood can reach aconcentration of more than 3 mM for 4 hr after anoverdose of 20 g or more [l]. The portal blood willpresumably reach near saturation, especially in thefirst few hours. The initial phases of paracetamolmetabolism involve the formation of glucuronideand sulphate conjugates, which are safe excretableproducts [5]. A small fraction of the paracetamol isalso oxidized in the cytochrome P450 system to form areactive metabolite, N-acetyl-p-benzoquinoneimine(NAPQIT) which combines with glutathione.However, as the glutathione levels fall, furtherreactive metabolite attacks cell components whichleads to final cell destruction [6,7].The mechanism of the late stages of cell toxicityremains unestablished, but several ideas have beenproposed. They involve covalent binding of thereactive metabolite to essential hepatic proteins[8,9], oxidation of macromolecules especially cationpumping ATPases [lo] and alteration in the electrontransport chain of the cell [ll]. Histochemical andultrastructural studies have also shown that the latestages of liver necrosis are associated with structuraldamage to subcellular components including themitochondria [12,131. Although the exact mechanismis not known, there is general agreement that failureof intracellular calcium ion regulation is a commonlate and perhaps irreversible final stage in the processof cell necrosis [14]. Mitochondria have beenimplicated as being a potentially sensitive target for

* Author to whom correspondence should be addressed.t Abbreviations: NAPQI, N-acetyl-p-benzoquinone-imine; TPMP+ triphenylmethylphosphonium; SCN- thio-cyanate; GSH, gkathibne; L-D-H, lactate dehydrogenase;CCCP, carbonylcyanide m-chlorophenylhydrazone; SKF525A, diethylaminoethyl 2,2_diphenylvalerate.

damage and their integrity can be followed by amitochondrial membrane probe, such as TPMP+(triphenylmethylphosphonium) [15,16].TPMP is a penetrating cation which accumulatespredominately in the mitochondrial matrix becausethis has a high negative potential relative to thecytoplasm [151.Loss of potential causes the release of TPMP+from the organelles and subsequently from the cells.Using a similar approach, the plasma membranepotential can be monitored by measuring thedistribution of a permeant anion SCN, which isexcluded from cells maintaining an intact potential[15,16]. A fall in the plasma membrane potentialcan therefore be followed by an increase in theuptake of SCN- by the cell.The aim of this study was to investigate theinvolvement of the plasma and mitochondrialmembrane potential in paracetamol toxicity usingliver slices as the in-v i t ro model.The model developed in this laboratory allowsinvestigation of the two phases, paracetamolexposure and subsequent development of cell injuryin paracetamol toxicity [11,17]. Phenobarbitalpretreated rats were used because they show a greatsensitivity to paracetamol poisoning.In many respects, liver slices behave in a similarfashion to the liver in the whole animal. The slicecells are not permeable to molecules such as EDTA,which are excluded in uivo but which enter freelyinto suspended hepatocytes. Slices maintain theinter-cell adhesion and communication in contrastto isolated hepatocytes which have their surfacesstripped by exposure to proteases and environmentalchanges which force major change in morphologyand activity. Liver slices from phenobarbitalpretreated rats show a marked loss of potassium,gain in water content and leakage of soluble enzymessuch as lactate dehydrogenase in the 4 hr following2 hr of exposure to paracetamol. The slices from rats

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932 W. M. A. NAZARETH J. K. SETHI and A. E. M. MCLEANTable 1. Effect of Valinomycin and CCCP on the intracellular retention levels of the membranepotential indicators, TPMP+ and SCN- in rat liver slices

AdditionsControl medium (Low K+)Control+CCCP (80 PM)+Valinomycin (10 ,ug/mL)High K+ mediumControl+Valinomycin (10 pg/mL)

Intracellular marker[HITPMP [JC]SCN-(% retention (% retentiondem/mg relative to dem/mg relative toslice wt control) slice wt control)

4933 t 266 100 862 t 68 1001297 - 51* 26 879 * 76 1021485 -+ 64* 30 940 r 81 1094341 * 174 88 1402 r 73* 1621365 * 48* 27 1630 - 84* 198

Liver slices were incubated as described in Materials and Methods and exposed to the inhibitorsfor 30 min. Three flasks were used per condition in each experiment. Results are means 2 SD forthree separate experiments. Low K medium (ordinary Ringer medium, 7 mM K+), High K+medium (Ringer medium containing all potassium salts instead of sodium salts, 150 mM K)* Indicates significant difference from control values (P < 0.05).

not given phenobarbital are far less sensitive toparacetamol and can be used as controls in whichparacetamol exposure does not lead to cell injury.U.K.). SKF-525A was a gift from Smith Kline andFrench (Welwyn Garden City, U.K.).

MATERIALSAND METHODSMeasurement of changes i n mi t ochondri al membranepotential

AnimalsMale Wistar rats (OLAC, Bicester, U.K.)weighing 120-150 g were fed stock pellets (SDS,Witham, U.K.) and given sodium phenobarbitoneas a solution containing 1 mg/mL as a sole sourceof drinking water for at least 5 days, where increasedcytochrome P450 levels were required [18]. Ratswere killed by exsanguination under fentan(0.105 mg/kg, i.m.) and diazepam (2.5 mgr

1 citratekg, i.p.)anaesthesia (Janssen, Wantage, U.K.). The liverwas rapidly removed and the liver slices of 0.3 mmthickness or less were cut by hand on a Stadie-Riggsstage with a long razor blade (A. H. Thomas Co.,Philadelphia, PA, U.S.A.).

Changes in the mitochondrial potential wereassessed by determining the intracellular [3H]TPMP+remaining in the slice after treatment [19]. A knownmitochondrial uncoupler CCCP and valinomycin aK+ ionophore were used to check the validity of thesystem [15,20]. High K+ medium was prepared byreplacing all the sodium salts of the Ringer mediumwith potassium salts [15].

Slices weighing about lOO--120mg were put into25 mL Erlenmeyer flasks containing 4 mL of Ringersolution with the following composition: 125 mMNaCl, 6 mM KCl, 1.2 mM MgS04, 1 mM NaH2P04,1 mM CaClz, 10mM glucose and 15 mM Hepesbuffer, pH 7.4 at 37 as described previously [17].The slices were put into the Ringer solution at roomtemperature and the experiment started by placingthe flasks into an incubator bath at 37 under oxygenwith shaking (90 strokes/min).

Method I. The slices were initially loaded withTPMP+ by incubating in Ringer medium containing0.25 &i/mL [3H]TPMP+ for 45 min. These loadedslices were then transferred to fresh Ringer mediumcontaining, where appropriate, 10 mM paracetamol.In experiments where the time course was greaterthan 2 hr, the slices were taken out of the flasks andre-incubated in fresh Ringer medium. At the end ofthe incubations, the slices were digested in 1 MNaOH 2 mL at 37 for 2 hr. The intracellular [3H]-TPMP+ was measured in 200 PL aliquots neutralizedwith 100 PL of glacial acetic acid by liquid scintillationcounting (LSC).

Chemicals

The quantity of cell protein was determined bythe method of Lowry et al. [21], using crystallinebovine serum albumin (fraction V) as standard.Control and paracetamol treated slices were takenat each time point, and radioactivity (dpm/mgprotein) compared.Paracetamol, carbonyl cyanide m-chlorophenyl- Method II. In some experiments the slices werehydrazone (CCCP), valinomycin, cysteine and po- loaded using 0.25 or 0.05 ,nCi/mL [3H]TPMP+ withtassium [t4C]thiocyanate (3.9 mCi/mmol) were pur- 0.125 ,uM cold carrier TPMP+. The concentration ofchased from the Sigma Chemical Co. (Poole, U.K.). the [3H]TPMP+ in the incubation medium and in[methyl-3H]Methyltriphenylphosphonium iodide the slices was measured and used to determine the(52.8 Ci/mmol) was purchased from Du-Pont Ltd, intracellular to extracellular distribution ratio i.e.New England Nuclear Products Division (Stevenage, (dpm/pL slice water)/(dpm/pL incubation medium).U.K.). All other reagents were of analytical grade Cell water content was assumed to be five times theand were bought from Sigma or BDH Ltd (Poole, protein content. The results could equally have been


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Paracetamol and mitochondrial membrane 93325

504 I0 30 80 90 120 150Time min)

Fig. 1. Loss of [3H]TPMP+ from slices during 10mMparacetamol exposure (O-120 min) relative to control slicestaken at each time point. Each point represents the mean+ SD of at least three experiments performed in triplicate.* P < 0.05 as compared to control groups.

expressed as the ratio of dpm/mg protein dividedby dpm/mg incubation fluid.Measurement of changes i n pl asma membranepotential

Changes in the plasma membrane potential weremonitored by the increase in the uptake of [Cl-SCN- by the slice. For verification of the procedurethe slices were incubated with 10 pg/mL valinomycinin low or high K+ medium for 30 min, before additionof [14C]SCN-.Using 10 mM paracetamol the slices wereincubated for 2 hr. After exposure, the slices weretransferred into fresh Ringer medium containing0.5 ,uCi/mL [4C]SCN- and allowed to incubate forvarious time periods. At the end of the incubation,the slices were digested as described previously andan aliquot measured by LSC.

Measurement of enzyme leakage (LD H) andglutathione

Injury was assessed by measuring leakage oflactate dehydrogenase from the slice into theincubation medium. Lactate dehydrogenase activity(LDH) released was expressed as a percentage ofthe amount of enzyme activity originally present inthe flask, based on the original slice weight andLDH assays on homogenates of liver slices sampledbefore incubation [17,221.Glutathione (as acid soluble SH) was measuredby the method of Ellman improved by Beutler et al .1231.Stat ist i cal analysis

Significance of the differences were determinedusing Students t-test with a P < 0.05 being taken asindicating significant difference.

RESULTSIntracellular levels of indicators of membranepotential

The suitability of TPMP+ and SCN- as indicatorsof changes in membrane potentials for suspensionsand monolayer cultures of hepatocytes has been

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/~8--_O------ -_o Control I

03 l I2 4 c 8Time IL-)

Fig. 2. LDH leakage in the second period of incubation(2-6 hr) following exposure to 10 mM paracetamol for 2 hr.Each point represents the mean - SD of at least threeexperimentsperformedintriplicate. * P < 0.05ascomparedto control groups.

previously demonstrated [15,16, 191. Since sliceswere used in this study, we verified the applicabilityof the probes under these conditions. Uptake ofTPMP+ was essentially complete after 30 min whenthe ratio of more than 30 : 1, intracellular toextracellular, had been reached. Further incubationtimes did not lead to increased uptake andtetraphenylboron did not affect uptake.The slices were treated with known uncouplers atconcentrations stated in Table 1. After 30 min ofincubation with an excess of the mitochondriauncoupler CCCP more than 70% of the accumulatedTPMP+ was released. Valinomycin also caused a70% drop in TPMP+. Incubation of liver slices in ahigh K+ medium caused a relatively small decreasein the intracellular level of the mitochondrial marker(12%). Non-specific binding to the slice wasdemonstrated by initially freezing the slice for anhour before undertaking the experiment. Less than4% of the marker was detectable in the intracellularfraction (data not shown). The effects of valinomycinremained similar in the high K+ medium, indicatingthat in our model using slices the release of TPMP+mainly reflects a loss of mitochondrial membranepotential as has been previously shown forhepatocytes [15, 16, 191.The accumulation of SCN- under the samecondition was also measured. In a high K+ medium,the intracellular level of SCN- increased, inagreement with the expected depolarization of theplasma membrane. A further rise was observedwhen valinomycin was added, probably reflecting ahigher uptake of K+ by the permeable high K+slices. This data indicates that the SCN- proberesponds readily to a change in the plasma membraneand is also suitable in our model.Exposure of loaded slices to 10 mM paracetamolled to a consistent decrease (28%) in TPMP*retention from 30 min of exposure onwards (Fig. 1).Slices exposed to 10mM paracetamol for 2 hrfollowed by removal of paracetamol gave the sameresult (data not shown). For the first 3 hr ofincubation there was no significant evidence of cellinjury in the form of increased enzyme leakage


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934 W. M. A. NAZARETH,J. K. SETHI and A. E. M. MCLEAN

Time hr)Fig. 3. Effect of paracetamol on the intracellularaccumulation of [T]SCN- following 10 mM paracetamolexposure (2 hr). Slices were transferred to fresh mediumcontaining [r4C]SCN- and accumulation monitored relativeto control slices taken at each time point. Each pointrepresents the mean f SD of at least three experimentsperformed in triplicate. * P < 0.05 as compared to the

control.

(LDH). Both control and paracetamol treated slicesshowed the usual slight loss of enzyme activity tothe medium. Only after 4 hr did major signs of cellinjury appear Fig. 2) as has been described previouslyfor enzyme leakage and potassium loss [17]. Atconcentrations below 10 mM paracetamol thedecrease in mitochondrial membrane potential wasreduced proportionally to dose, above 10mMparacetamol there was no extra effect (data notshown).Figure 1 shows an early time course where changesin mitochondrial membrane potential were significantafter 30 min, after this the change was not timedependent.

Figure 3 shows relative change in the plasmamembrane monitored by an increase in the uptakeof [i4C]SCN- by the slice. Significant changes onlyoccurred after 5 hr, by which time massive enzymeleakage was taking place. The slight deficit at anearlier time point was not significant.Addition of 0.5 mM cysteine or 30 PM SKF 525Ato the system did not prevent the reducedmitochondrial membrane potential (Fig. 4). Whenslices from an uninduced rat were used changes inthe membrane potential were still detectable (Tables2 and 3).The addition of carrier TPMP+ to the loadingconditions and expression of the results as a ratio ofintra- to extracellular concentrations (Method II)led to a more stable and robust system. This alsoshowed the drop in TPMP+ retention whenparacetamol was added to the system, using slicesfrom control rats not pre-induced with phenobarbital(Table 3).

DISCUSSIONOur present results demonstrate that paracetamolelicits a direct and early change in the membranepotential of the mitochondria. Studies with cadmium,mercuric chloride and cyanide using hepatocyteshave all demonstrated that early mitochondriadepolarization is an initial injury which is followedby ATP depletion and cell death [19,20,24].Alteration in mitochondrial function was moni-tored through the detection of changes in themitochondria membrane potential. The penetrating

cation TPMP has been shown to be a suitableindicator in studies using hepatocytes in suspensionand in monolayer cultures [15,16,19]. In this studywe have demonstrated that TPMP is also suitablefor liver slices, more than 70% of TPMP+ retention

Co11lrol Paracet. SKF 525A Paracet. Cysl. ParacetSKF 525A cyst.Fig. 4. Effect of (30 PM) SKF 525A and (0.5 mM) cysteine added together with 10 mM paracetamolfor 30 min. Loss of intracellular f)HlTPMP+ determined as described in Materials and Methods. Eachbar represents the mean f SD of at least three experiments performed in triplicate. * P < 0.05 ascompared to control groups.


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Paracetamol and mitochondrial membraneTable 2. Measurement of intracellular [3H]TPMPC following incubation with andwithout 10 mM paracetamol for 30 min using slices from an induced and uninducedrat

dpm/pg slice protein % ChangeControl Paracetamol relative to controlInduced 35.8 f 2.2 24.1 * 2.4 -31.0*Uninduced 41.7 2 2.1 32.3 2 2.7 -22s

Each value represents the mean of two separate experiments each performed in

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quadruplicate.* P < 0.05 as compared to control.

Table 3. Effect of 10 mM Paracetamol on the distributionof [3H]TPMP+ in liver slices from control ratsDistribution ratio after

t3H]TPMP+ concn incubationfor loading Control Paracetamol0.125 PM0.05 @i/mL 118.4 f 5.1 57.3 2 14.3*0.125 PM0.25 &i/mL 130.3 + 2.8 64.1 + 13.3*

Slices were pre-loaded with 0.05 or 0.25 pCi/mL [HI-TPMP+ in the presence of 0.125 PM cold carrier for 1 hr.They were then incubated with and without paracetamolfor a further 30min in fresh medium without addedTPMP+, under the standard conditions as described inMaterials and Methods. The distribution ratio equals theamount of [3H]TPMPf per unit volume of intracellularwater (i.e. per mg protein X 5)/extracellular concentration.The values represent the means (+SD) of two separateexperiments each performed in triplicate.* P < 0.05 as compared to control.

by the slice was dependent upon the mitochondrialmembrane potential (Table 1). We have observedthat the first significant efllux of the marker wasafter 30 min exposure to 10 mM paracetamolindicating that within this short period, paracetamolinduced a collapse of the mitochondrial membranepotential (Tables 2 and 3). It is established that themitochondrial membrane potential depends uponthe H+ gradient generated by the electron-transportchain [20]. A collapse in the electrochemical gradientmay occur via an alteration in the activity of therespiratory chain or in the citric acid cycle whichsupplies NADH.In order to investigate the possible consequencesof mitochondrial attack and to establish that thechange in mitochondrial membrane potential wasnot due to plasma membrane potential change wemonitored changes in the plasma membranepotential. After verification of the use of thepenetrating probe SCN- with slices, the effect ofparacetamol on the uptake of SCN- was evaluated.No significant alteration in SCN- penetration wasobserved until 5 hr after exposure, at which timethere is a massive release of LDH from the slice.Increase of GSH concentration by the addition ofcysteine did not alter TPMP+ release indicating thatthe early effect was not GSH dependent.

SKF 525A a known cytochrome P450 inhibitorwhen added to the system did not diminish the effectof paracetamol on the mitochondrial membranepotential. This was confirmed by using an uninducedrat for the preparation of the slices. Rats which arenot treated with any inducer are highly resistant toparacetamol toxicity n v vo or i n v tro [26],since the background cytochrome P450 activity isinsufficient to elicit damage readily in this species[17]. This suggests that the formation of the activemetabolite NAPQI is not necessary to cause changein mitochondrial membrane potential. Equallythe observation that the paracetamol causes amitochondrial defect in liver slices from uninducedrats shows that the mitochondrial defect on its ownis not sufficient to cause cell death. It is notable thatfurther incubation with paracetamol and progressiontowards cell injury does not increase the mito-chondrial lesion. Direct cytotoxic effects of par-acetamol have previously been reported using V79Chinese hamster cells [25,27].This study therefore suggests that early par-acetamol damage involves mitochondria. A declinein ATP levels can be critical, a negessary, but notsufficient event for the development of cell damage[28-311. Low ATP could lead to reduced calciumATPase activity and thus a failure to repair anyinjury caused by reactive metabolites, such as anincrease in intracellular calcium, so leading to finalcell death.Acknowledgements-This work was partially supported bygrants from the Sir Halley Stewart and Welcome Trusts.

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