Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Chem.-Biol. Interactions, 47 (1983) 249--263 Elsevier Scientific Publishers Ireland Ltd.

249

PARACETAMOL-STIMULATED LIPID PEROXIDATION IN ISOLATED R A T AND MOUSE HEPATOCYTES

EMANUELE ALBANO, GIUSEPPE POLI, ELENA CHIARPOTTO, F I O R E L L A BIASI and MARIO UMBERTO DIANZANI

lstituto di Patologia Generale dell'Universitd, Corso Raffaello 30, 10125, Torino (Italy) (Received April 15th, 1983 ) (Revision received June 7th, 1983) (Accepted June 15th, 1983)

SUMMARY

Treatment o f isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to p r o m o t e lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the c y t o c h r o m e P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production. Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack. In isolated mouse liver cells paracetamol does not require preliminary c y t o c h r o m e P 4 5 0 induction to stimulate MDA formation, even at concentrations ineffective in rat cells. However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not p r o m o t e MDA formation either in the rat or in the mouse hepatocytes. This effect m a y be due to the ability o f paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems. Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA Abbreviations: DM S O , dimethylsulphoxide; DPPD, N,N'-diphenyl-p-phenylenediamine; DNTB, 5,5'-dithiobis-(2-nitrobenzoicacid); EGTA, ethyleneglycol-bis-(~-amino-ethylether)N,N'-tetraaceticacid; GSH, reduced glutathione; HEPES, N-2-hydroxy-ethylpiperazine-N'-2-ethane sulfonic acid; LDH, lactate dehydrogenase; M D A , malonaldehyde; TCA, trichloroaceticacid. 0009-2797/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

250 formation is inhibited by high doses of the drug or by addition of antioxidants such as a-tocopherol and diphenylphenylenediamine (DPPD). Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.

Key words: Paracetamol -- Isolated hepatocytes -- Lipid peroxidation -Glutathione depletion

INTRODUCTION

Stimulation of lipid peroxidation has been postulated as one of the mechanisms by which chemical compounds can cause cell damage [1]. Among the protective reactions against such oxidative attacks, GSH plays a major role as cofactor for the enzyme glutathione peroxidase (EC 1.11.1.9) in reducing hydrogen peroxide and organic peroxides [ 2]. Furthermore GSH interacts, in reactions catalyzed b y glutathione transferases, with several electrophilic intermediates, which might bind to cell macromolecules or initiate lipid peroxidation [3]. A wide range of chemicals have been shown to lower, to various extents, hepatocyte GSH levels and, in most cases, to stimulate lipid peroxidation and cause cell damage both in vivo and in vitro [4--8]. Depletion of hepatic GSH has also been observed following overdoses of the widely used analgesic drug, paracetamol. This effect is a consequence of paracetamol activation by the cytochrome P-450-dependent microsomal mixed oxidase system, to a reactive metabolite which is then inactivated by conjugation with GSH, to be finally excreted as mercapturic acid derivative [9]. Upon exhaustion of the greater part of the liver GSH content, the reactive intermediates become covalently bound to cell protein leading in this way to a loss of cell functions and to hepatocyte necrosis [9]. Moreover, the lowering of GSH is a condition allowing liver cells to undergo lipid peroxidation as already mentioned. Wendel et al. [10] observed that paracetamol treatment of starved mice initiates lipid peroxidation as measured in vivo by ethane expiration or by MDA formation in liver homogenate.~rhey also reported that such peroxidative stimulation is related to a decrease in the activity of selenium-dependent glutathione peroxidase, as a consequence of the lowering of GSH levels [11]. MDA formation as a result of paracetamol treatment has also been detected in rat liver homogenate [ 12 ] as well as in post-mitochondrial supernatant [ 13 ] ; in both cases the appearance o f MDA was demonstrated to be dependent on a previous depletion in GSH content exerted by the drug activation.

251 Isolated rat and mouse hepatocytes have been shown by Molddus [14] to be a suitable experimental model for in vitro studying paracetamol metabolism and its toxicological consequences. We are here reporting on the use of isolated rat and mouse liver cells to investigate the conditions in which paracetamol induces lipid peroxidation and to elucidate the role that such oxidative reactions might have in causing liver necrosis as a result of the drug intoxication. MATERIALS AND METHODS

Male Wistar rats (200--300 g body wt.) and male Swiss mice (30--40 g body wt.) were purchased from Nossan (Correzzana, Milano, Italy) and were fed ad libitum on a semisynthetic diet devoid of any antioxidant (Piccioni, Brescia, Italy). Paracetamol metabolism was stimulated in the rats by pretreatment with 3-methylcholanthrene (20 mg/kg i.p. in light liquid paraffin) 72 h and 48 h before hepatocyte preparation, or with 0.1 g% phenobarbital in the drinking water for at least 5 days until 24 h before cell isolation. All chemicals were of reagent grade and were obtained from the following sources: collagenase type I (EC 3.4.4.19), soya bean lipoxygenase (EC 1.13. 12.11), ethyleneglycol-bis-(~-amino~thylether)N,N'-tetraaceticacid (EGTA), N-2-hydroxy-ethylpiperazine-N'-2-ethane sulfonic acid (HEPES), 5,5°
252 lation of lipid peroxidation by soya bean lipoxygenase was assayed by incubating 30 min at 37°C 15 000 U/ml of the enzyme with the mierosomal suspension, as previously mentioned, or with linoleic acid (0.5 mg/ml in 0.1 M Tris buffer (pH 7.4) containing 0.05% deoxycholate); in the latter experiments 10 gM hematin was added to enhance the Fe-mediated breakdown of lipid peroxides [18]. The incubation was stopped by adding an equal vol,.une of 10% trichloroacetic acid (TCA) and MDA production was evaluated as hereafter reported. Intracellular GSH content was routinely estimated by the method of El]man with the modification of Sedlack and Lindsay [19]; no difference was found in the estimation of total GSH levels with this method and with the enzymatic assay according to Owen and Belcher [20]. GSH measurements were performed as follows: aliquots (0.5 ml) of the hepatoeyte suspension (2 X l 0 6 cells) were centrifuged at 80 × g for 5 min; the supernatant was kept for the estimation of lactate dehydrogenase release, while the cell pellet was shaken with 0.5 ml of 5% TCA, and following protein precipitation by centrifugation (10 min at 3000 rev./min), 0.4 ml of the protein free supernatant was used for the eolorimetrie reaction. Cell structural integrity was evaluated by monitoring LDH (EC 1.1.1.25) release in the eeU-free incubation medium [ 15], obtained as above mentioned. Lipid peroxidation was measured determining MDA in the hepatocyte suspension [15] and the diene conjugation absorbance band on total cell phospholipids, according to the procedure described by Casini and Farber [8]. Covalent binding of activated paracetamol metabolites to rat and mouse hepatocytes was determined as follows: [14C]paracetamol (50 pCi; radiochemical purity exceeding 98%), was solubilized in the incubation medium containing unlabeUed paracetamol, in order to obtain a 25 mM solution with 106 dpm/mmol.Various amounts of this solution were added to hepatocyte suspension, as previously mentioned, and the cells were incubated for up to 180 min at 37°C. Every 60 min 2 ml of the suspension were withdrawn, the cell separated by centrifugation (5 min at 80 × g) and the pellet resuspended in 5 ml 5% TCA. The protein was precipitated and processed as described by Massey and Racz [21]. Protein bound radioactivity was measured using a Packard Tri-Carb 460 C liquid scintillation counter. RESULTS

Despite the relative resistance of rats to paracetamol poisoning, their pretreatment with cytochrome P-450 inducers has been demonstrated to greatly enhance the activation of the drug [9,22]. Preliminary experiments showed that the same dose of paracetamol was approximately twice as effective in reducing GSH in hepatocytes obtained from 3-methylcholanthrene-induced rats than in those obtained from phenobarbital-induced animals. In the latter group no increase in MDA formation was detected throughout the incubation period.

253 On the other hand, mice do not need c y t o c h r o m e P-450 induction and are known to be highly susceptible to paracetamol-produced liver injury [9]. Thus the effect of paracetamol poisoning on the occurrence o f lipid peroxidation was investigated using hepatocytes isolated from rats pretreated with 3-methylcholanthrene or from untreated mice. Isolated liver cell suspensions (4 × 106 cells/ml, final concentration) were incubated up to 180 min at 37°C in the presence of paracetamol (0.5--5.0 mM), and cell GSH levels, MDA formation and LDH release were monitored. In 3-methylcholanthrene-induced rat hepatocytes paracetamol dosing caused a time and dose~lependent depletion of cellular GSH (Fig. la). In the same cell suspension, MDA accumulation became evident after the second hour from paracetamol addition (Fig. l b ) . Such a lag period is probably related to the time necessary to lower cell GSH content below 20--30% of the normal values, the level considered critical for the detection o f peroxidative events caused by GSH-depleting agents [4]. MDA formation reached a maximum in isolated hepatocytes receiving 1 mM paracetamol, whereas it was practically absent in cell suspensions incubated with 5 mM of the drug, even if the hepatocyte GSH content was very low (Figs. l a and lb). Different authors reported that paracetamol addition to isolated hepatocytes was able to inhibit lipid peroxidation [ 5,7 ], b u t at the present no reliable explanation for such an effect is available. In order to further investigate this point, paracetamol, in concentrations ranging between 0.5 and 10 mM, was added to rat liver microsomes, stimulated to peroxidize by 30 rain incubation in the presence of 0.5 mM NADPH plus 0.1 mM FeC13 or 1 mM CC14, or b y treatment with soya bean lipoxygenase (15 000 U/ml o f microsomal suspension). In all the experimental conditions a dose-related reduction of MDA formation was apparent, with a maximum (approx. 65--70%) at paracetamol concentration of 5 mM (Fig. 2). A comparable effect was also observed in the case of lipoxygenase~atalyzed peroxidation of linoleic acid (Fig. 2, dotted line). It follows that paracetamol is interfering to the same extent with lipid peroxidation caused by both free radical attack to unsaturated fatty acids and enzymatic lipoperoxide formation mediated by soya bean lipoxygenase. Thus it is likely that the so~called propagation phase of lipid peroxidation [19] might be affected b y paracetamol acting as an antioxidant. Hepatocytes isolated from 3-methylcholanthrene-induced rats were also investigated for the presence of conjugated diene absorbtion band. Figure 3 shows that a slight increase in the absorbance at 230--233 nm was detectable in the phospholipids extracted from hepatocytes dosed with 1 mM paracetamol, in contrast with those from ceils receiving 5 mM of the drug. In any case, the stimulation of diene absorbance was less evident than MDA accumulation. A similar lack of correlation between MDA production and diene conjugation has also been observed b y Poli et al. in isolated hepatocytes

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Fig. 1. T i m e course o f (a) p a r a c e t a m o l - i n d u c e d G S H - d e p l e t i o n , (b) M D A f o r m a t i o n and (c) LDH release in isolated h e p a t o c y t e s f r o m 3 - m e t h y l c h o l a n t h r e n e - p r e t r e a t e d rats. P a r a c e t a m o l c o n c e n t r a t i o n s w e r e as follows: o, n o n e ; e, 1 mM; =, 2.5 raM; , , 5 mM. LDH release is expressed as percentage o f t h e t o t a l e n z y m e c o n t e n t as previously described [ 15 ]. Each p o i n t r e p r e s e n t s t h e m e a n o f 5- 6 e x p e r i m e n t s _+ S.D.

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Fig. 2. Effect of paracetamol on MDA formation in peroxidizing rat liver microsomes and linoleic acid suspension. The rrricrosomal suspension (15--20 mg protein/ml) was incubated 30 rain at 37°C with a NADPH-regenerating system [17] and 0.1 mM FeC13 (~) or 1 mM CC14 (#) as DMSO solution. Alternatively lipid peroxidation was stimulated by soya bean lipoxygenase (15 000 U/ml) addition to a similar microsomal suspension (~,, continuous line), or purified linoleic acid suspension (% dotted line), incubated 30 rain at

37°C. Suitable amounts of a 25-raM paracetamol solution were added to the incubation mixtures (1 ml final volume) to obtain the concentrations reported in the figure. treated with CC14 and ADP-Fe 3÷ and has been related t o a different generation o f aldehydic end products by the various peroxidizing agents [23]. The influence o f factors, such as t he stimulation of t he drug metabolism and the lowering o f cell GSH c o n t e n t , known t o enhance paracetamol t o x i c i t y , were investigated in relation t o t he occurrence o f lipid peroxidation. According to Mold~us and Gergely [24] addition of 100 mM acetone t o isolated h e p a t o c y t e suspensions greatly increased the rate of paracetamol activation by the c y t o c h r o m e P-450-dependent system and potentiated the drug action on both GSH depletion (P < 0.05) and MDA form at i on (P < 0.001) (Fig. 4). Moreover acetone induced a substantial increase in the prooxidant effect o f low doses (0.5 mM) of paracetamol per se uneffective in p r o m o t i n g MDA accumulation. The in vivo lowering hepatic GSH by various means has been shown t o aggravate liver lesions and to stimulate ethane expiration in course o f paracetamol intoxication [10,11]. In agreement with these findings the previous lowering o f h e p a t o c y t e GSH c o n t e n t n o t only induced a massive MDA formation, but also reduced the lag phase in the appearance of lipid peroxidation {Table I). Under these conditions 0.5 mM paracetamol, which

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WAVELENGTH ( n m ) Fig. 3. C o n j u g a t e d d i e n e a b s o r b a n c e o f p h o s p h o l i p i d s e x t r a c t e d (a) f r o m 3 - m e t h y l c h o l a n t h r e n e - i n d u c e d r a t h e p a t o c y t e s a n d (b) f r o m u n t r e a t e d m o u s e h e p a t o c y t e s , i n c u b a t e d respectively for 180 a n d 120 rain at 37°C in t h e p r e s e n c e of: (1) n o p a r a c e t a m o l ; (2) 1 m M p a r a c e t a m o l ; (3) 5 m M p a r a c e t a m o l . P h o s p h o l i p i d s were purified a c c o r d i n g t o Casini e t al. [ 8 ] a n d dissolved in c y c l o h e x a n e at t h e c o n c e n t r a t i o n o f 2 m g / m l . T h e inserts r e p r e s e n t t h e differential U V s p e c t r a o f c o n t r o l cells (1) vs. 1 m M p a r a c e t a m o l t r e a t e d h e p a t o c y t e s (2).

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Fig. 4. E f f e c t o f a c e t o n e a d d i t i o n o n (a) p a r a c e t a m o l - m e d i a t e d G S H d e p l e t i o n and (b) M D A p r o d u c t i o n in isolated h e p a t o c y t e s f r o m 3 - m e t h y l c h o l a n t h r e n e - i n d u c e d rats. O p e n s y m b o l s r e p r e s e n t t h e cells i n c u b a t e d w i t h o u t a c e t o n e , while t h e filled s y m b o l s t h o s e receiving 1 0 0 m M a c e t o n e . P a r a c e t a m o l c o n c e n t r a t i o n s were as follows: o, • n o n e ; 4, • 0.5 m M ; D, • 1 mM. E a c h p o i n t r e p r e s e n t s t h e m e a n o f 3--4 e x p e r i m e n t s -+ S.D.

257 TABLE I PARACETAMOL-INDUCED LIPID PEROXIDATION IN ISOLATED HEPATOCYTES HAVING LOW GSH CONTENT Isolated hepatocytes were prepared from 3-methylcholanthrene induced rats avoiding the procedures suggested by Vifia et al. [16] to preserve cell GSH content. Values represent the means of 3 different cell preparations +_S.D. Treatment

Incubation 60 min

None Paracetamol (0.5 raM) Paracetamol (1.0 raM)

120 rain

GSH (nmol/106 cells)

MDA (nmol/10' cells)

GSH (nmol/106 cells)

MDA (nmol/106 cells )

24.6 +_1.3 15.2 +_1.0 6.8 +_0.7

1.03 + 0.11 1.12 +_0.08 0.88 +_0.15

22.8 +_1.6 7.2 +_0.8 4.3 +_1.3

2.27 4-_0.23 6.16 +_0.48 4.35 +_0.36

did n o t s h o w p r o ~ x i d a n t effects in h e p a t o c y t e s with physiological G S H c o n t e n t , was able t o initiate lipid p e r o x i d a t i o n (Table I). Isolated m o u s e h e p a t o c y t e s are k n o w n t o readily activate p a r a c e t a m o l w i t h o u t requiring p r e l i m i n a r y c y t o c h r o m e P-450 i n d u c t i o n , t h u s t h e y r e p r e s e n t a simpler m o d e l f o r s t u d y i n g t h e p e r o x i d a t i v e e f f e c t s o f such a drug. T h e t i m e course o f p a r a c e t a m o l - i n d u c e d G S H d e p l e t i o n and MDA s t i m u l a t i o n in isolated m o u s e liver cells s h o w e d significant level o f lipid p e r o x i d a t i o n associated t o a severe loss in the cellular G S H already a f t e r 60 min f r o m t h e a d d i t i o n o f 0.5 and 1 mM o f p a r a c e t a m o l (Figs. 5a and 5b). T h e d i e n e c o n j u g a t i o n b a n d was also c o n s i s t e n t l y observed in t h e phospholipids e x t r a c t e d f r o m t h e same cell p r e p a r a t i o n (Fig. 4). As r e p o r t e d b y Mold6us [ 1 4 ] , t h e G S H c o n t e n t o f m o u s e h e p a t o c y t e s did n o t decrease b y m o r e t h a n 3 0 - - 4 0 % o f the initial values even a f t e r 2 h o f i n t o x i c a t i o n with p a r a c e t a m o l . Nevertheless, these cells s h o w e d an o n s e t o f lipid p e r o x i d a t i o n earlier t h a n rat h e p a t o c y t e s , p r o b a b l y because o f species-related differences in the m i n i m u m G S H level r e q u i r e d t o p r e v e n t t h e spreading o f u n s a t u r a t e d f a t t y acids o x i d a t i o n . Plasma m e m b r a n e leakage o f L D H as a result o f p a r a c e t a m o l t r e a t m e n t o f m o u s e and 3 - m e t h y l c h o l a n t h r e n e - i n d u c e d rat h e p a t o c y t e s d e m o n s t r a t e d a close t e m p o r a l association with t h e p e r o x i d a t i v e events. Such an e f f e c t was m o r e evident in m o u s e liver cells, w h e r e a significant e n h a n c e m e n t o f L D H release was p r e c e d e d b y t h e increase in MDA p r o d u c t i o n . On t h e o t h e r hand, n o significant variations in t h e L D H leakage was p r e s e n t in the cells receiving 5 m M p a r a c e t a m o l , w h e r e MDA f o r m a t i o n was also practically absent (Figs. 1 and 5). Covalent binding o f activated p a r a c e t a m o l m e t a b o l i t e s t o p r o t e i n s o f either m o u s e and 3 - m e t h y l c h o l a n t h r e n e - i n d u c e d rat h e p a t o c y t e s is r e p o r t e d in Fig. 6. In b o t h species t h e dosing with 5 m M p a r a c e t a m o l s h o w e d t h e

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Fig. 5. Time course o f (a) p a r a e e t a m o l - i n d u c e d GSH d e p l e t i o n , (b) M D A f o r m a t i o n and (c) LDH release in isolated m o u s e h e p a t o c y t e s i n c u b a t e d u p t o 120 min at 37°C. P a r a c e t a m o l c o n c e n t r a t i o n s w e r e as follows: o, n o n e ; A, 0.5 raM; e, 1 mM; *, 5 mM. LDH release is expressed as a p e r c e n t a g e o f t h e t o t a l e n z y m e c o n t e n t as previously d e s c r i b e d [ 15]. Each p o i n t r e p r e s e n t s t h e m e a n o f 3- 4 e x p e r i m e n t s _+ S.D.

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T I M E (rain} Fig. 6. C o v a l e n t b i n d i n g o f reactive p a r a c e t a m o l m e t a b o l i t e s t o t h e p r o t e i n s o f (a) 3m e t h y l c h o l a n t h r e n e - i n d u c e d a n d (b) u n t r e a t e d m o u s e h e p a t o c y t e s , i n c u b a t e d r e s p e c t i v e l y for 180 a n d 120 rain at 37°C. [ 14C ] P a r a c e t a m o l c o n c e n t r a t i o n s were ; *; 0.5 m M ; o, 1 raM; =, 2.5 r a M ; . , 5 raM. E a c h p o i n t r e p r e s e n t s t h e m e a n s o f 3 e x p e r i m e n t s -+ S.D. T A B L E II E F F E C T O F a - T O C O P H E R O L A N D DPPD ON GSH D E P L E T I O N , M D A P R O D U C T I O N , L D H R E L E A S E A N D C O V A L E N T B I N D I N G O F A C T I V A T E D M E T A B O L I T E S IND U C E D BY P A R A C E T A M O L T R E A T M E N T IN I S O L A T E D R A T H E P A T O C Y T E S I N D U C E D WITH 3 - M E T H Y L C H O L A N T H R E N E Isolated h e p a t o c y t e s u s p e n s i o n was i n c u b a t e d for 180 m i n at 37°C. T h e values are t h e m e n a s of 4--5 d e t e r m i n a t i o n s +_ S.D. Treatment

GSH nmol/106 cells

Noi~.e 40.1 P a r a c e t a m o l (1 m M ) 13.0 + ~ - t o c o p h e r o l (100 t~M) 17.3 + DPPD (1 ~M) 19.6

_+ 4.2 +_ 3.9 +_ 2.3 +_ 2.7

MDA nmol/106 cells

LDH % of total

1.5 -+__0.18 4.6+__0.70 0.3+__0.04 0.4+__0.08

18 +__2.4 38+__2.8 16+__1.4 20+__2.1

Covalent binding nmol/mg _proteins 0.51 0.46 0.35

-+__ 0.07 +__ 0.05 +__ 0.06

260 TABLE III EFFECT OF ~-TOCOPHEROL AND DPPD ON GSH DEPLETION, MDA PRODUCTION, LDH RELEASE AND COVALENT BINDING OF ACTIVATED METABOLITES INDUCED BY PARACETAMOL TREATMENT IN ISOLATED MOUSE HEPATOCYTES Liver cell suspension was incubated for 120 rain at 37°C. The values are the means of 3 different cell preparations +_S.D. Treatment

GSH nmol/106 cells

MDA nmol/106 cells

LDH % of total

Covalent binding nmol/mg proteins

None Paracetamol (1 raM) + a-tocopherol (100 uM) + DPPD (1 uM)

55.2 +_3.8 17.3 +_4.5 19.1 +_3.2 18.4 +_2.5

1.2 +_0.30 4.2 _+0.36 0.4 +_0.12 0.6 +_0.10

11 +_1.5 39 +_3.4 13 +_4.1 19 +_3.7

0.70 +_0.090 0.67 +_0.030 0.65 +_0.023

highest protein binding o f labelled metabolites, which did n o t correlate with the slight cellular lesions found in the same cells. This discrepancy between drug activation and its toxic effects was further investigated in order t o assess th e role played by the peroxidative reactions. Table II shows that the addition o f a - t ocopherol and DPPD to 3-methylcholanthrene-induced rat h e p a t o c y t e s completely prevent MDA form at i on due to 1 mM paracetamol, w i t h o u t affecting its metabolic activation as indicated by GSH depletion and covalent binding of radioactive intermediates. C o n c o m i t a n t with the inhibition of MDA formation, these antioxidants greatly reduced LDH leakage f r om h e p a t o c y t e plasmamembranes. Comparable results were also obtained following a - t o c o p h e r o l and DPPD addition to paracetamol poisoned mouse liver cells (Table III) to indicate t hat lipid p er o x id atio n is a general event, responsible for some of the hepatic lesions during the early phases o f paracetamol intoxication. DISCUSSION Several authors have reported that paracetamol stimulates lipid peroxidation, as measured in terms o f ethane expiration in mice [10,11] and o f MDA p r o d u c t i o n in rat and mouse liver homogenates [10,12] or postmitochondrial supernatants [13]. Comparable results have been here obtained with both mouse and 3-methylcholanthrene-induced rat hepatocytes where th e occurrence o f lipid per oxi d at i on follows a marked reduct i on o f intracellular GSH c o n t e n t , due to the reaction of paracetamol activated metabolites with the tripeptide. Concerning this aspect the drug activation rate plays a critical role in the onset of lipid p e r o x i d a t i o n by influencing the decrease o f GSH levels; only rat hepat ocyt es isolated from 3-methylcholanthrene-induced animals show a significant MDA p r o d u c t i o n , which is further increased b y the non,specific stimulation o f paracetamol metabolism due to acetone addition [24]. Conversely isolated mouse liver cells, which readily activate paracetamol [14], do n o t require preliminary c y t o c h r o m e P-450

261

induction to promote lipid peroxidation, even at drug concentrations lower than those effective in rat hepatocytes. In the cells from both animal species a lag period of 60--90 min is evident before MDA detection; this phase differentiates the peroxide formation induced by several GSH
262 have been found effective in protecting from N-OH-acetaminophen-induced injury, suggesting that peroxidative reactions are involved [ 26]. At present, the role played by the two mechanism of lesion, i.e. covalent binding of reactive intermediates and peroxidative reactions, in the pathogenesis of hepatocyte death induced by paracetamol is not completely clear. Recent data pointed out the toxicological significance of some aldehydic end products of lipid peroxidation [27,28] the intracellular concentration of which might justify their involvement in causing cell lesions in course of CC14 and ADP-Fe 3÷ treatment (Poll et al., unpublished results). In the case of paracetamol, however, the paradoxical protection exerted by large doses of the drug does not allow the consideration of lipid peroxidation as the only pathogenetic event [29]. Thus it is reasonable to admit that both mechanisms are able to induce derangement of different cellular functions, leading to hepatocyte necrosis. Nevertheless we suggest that the occurrence of peroxidative reactions could be a critical event, in the sense that it would damage the cells rapidly enough to precede the destructive effects consequence of macromolecules arylation. The presence of such a mechanism might therefore explain the reported cases of hepatic injuries in patients with enhanced susceptibility to paracetamol, as well as in alcoholics, in which the drug activation is abnormally increased [30]. ACKNOWLEDGEMENTS

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