Pharmacological Research Communications, Vol. 18, No. 8, 1986 699

LIPID PEROXIDATION INDUCED BY N-ACETYLCYSTEINE

IN ISOLATED RAT HEPATOCYTES

Maria Dore, Luigi Atzori and Luigi Congiu

Institute of General Pathology, University of

Cagliari, 09100 Cagliari, Italy

Received in final form 20 June 1986

SUMMARY. In isolated rat hepatocytes N-acetylcysteine induces

an increase of lipid peroxidation, as evaluated by the malon-

dialdehyde production and diene conjugation. Lipid peroxida-

tion did not result in increased cell mortality. Antioxidants

and free radicals scavengers completely protect toward lipid

peroxidation induced by N-acetylcysteine.

INTRODUCTION

Cysteine and its derivative, as N-acetylcysteine, are

extensively used for therapeutical purposes as mucolytics

(Grassi et al .,1973), in order to reduce the urotoxic side

effects of cyclophosphamide (Tolley, 1977) and as a treatment

of paracetamol overdose (Piperno and Bersenbruegge, 1976).

N-acetylcysteine and other sulfur-containing compounds are

also used experimentally to obtain protective effects against

several intoxications (Mitchell at al., 1976).The effective-

ness of N-acetylcysteine is due to its transformation into

cysteine that in turn can act as a precursor in the glutathio -

ne synthesiscReed, 1985).It has been widely demonstrated that

this thiol may bind many reactive intermediates that could

react with cellular macromolecules when glutathione is depleted.

Indeed, hepatotoxicity of several compounds may be enhanced

0031-6989/86/080699-07/$03.00/O 01986Theltalian Pharmacological Society

700 Pharmacological Research Communications, Vol. 18. No. 8, 1986

by depletion of glutathione (Reed and Beatty, 198O).It has

been demonstrated (Saez et. al., 1982; Searle and Tomasi, 1982;

Vin'a et al., 1983) that cysteine, in the presence of metal

ions, can rapidly undergo autoxidation with the formation of

H202 or free radicals (thiyl and hydroxyl radicals). Free ra -

dicals are considered to be very effective in the promotion

of lipid peroxidation of cellular membranes (Wilson, 1978).

Through this mechanism several compounds can produce structur -

al tissue injury. Antioxidants or free radical scavengers, by

inhibiting lipid peroxidation, may protect against pathologi-

cal alterations (Slater, 1978).

The aim of the present study was to investigate whether N-ace - tylcysteine has any effect on lipid peroxidation of isolated

rat hepatocytes.

MATERIALS AND METHODS

Male Wistar rats, 250-3009, allowed free access to

food and water were used throughout all the experiments.

Hepatocytes were isolated according to Gravela et al. (1977).

N-acetylcysteine(NAC), promethazine hydrochloride(PMH), butyl

ated hydroxyanisole(BHA) and butylated hydroxytoluene(BHT)

were purchased from Sigma.Collagenase was purchased from

Boehringer-Mannheim. Propyl gallate and Vitamin E(VitE)

were purchased from BDH. Cell viability was determined by Try

pan blue exclusion. Lipid peroxidation was evaluated by deter -

mining the chromogen formation derived from the reaction of

malondialdehyde(MDA) with thiobarbituric acid(TBA) at 535 nm

(Poli et al., 1979). Diene conjugation of lipid extracted from

hepatocytes was evaluated by the method of Corongiu et a1.(1983).

Results are expressed as mean values + standard error (S.E.). -

The data were analyzed by Student's t-test.

Pharmacological Research Communications, Vol. 18, No. 8, 1986 701

RESULTS

MDA content of isolated hepatocytes started to increase

after 60 min of incubation with NAC(0.5 to 2 mM). After 60

min the increase in MDA content was significantly higher than

in controls (Fig.11 for all NAC concentrations.

1 4 0 30 60 120(mW

TIME OF INCUBATION

Fig.l-Effect of NAC on MDA content in isolated rat hepatocytes.

3 ml of cell suspension (lo6 cell/ml) were incubated at 37°C.

for different times in the presence of NAC 0.5 mM (@I; 1 mM

(A) and 2 mM (ml; (-- -I control.Each point was the mean

value + S.E. of 3 experiments in duplicates. Significantly dif - -

ferent from controls : * pcO.05; ** p<O.O05; *** p<O.OOl.

MDA content decreased 120 min after addition of 1 and 2 mM

NAC, but the values are still higher than in controls. At

the lowest concentration of NAC there was a further increase

of MDA content at 120 min. Under these conditions cellular

viability did not differ from control cells until seven hours

(data not shown). Upon addition of BHT, BHA, PMH, PG and VitE

to cell suspensions incubated in the presence of NAC(1 mM),

702 Pharmacological Research Communications, Vol. 18. No. 8. 1986

the MDA content was significantly lower than in cells treated

with NAC alone (Fig.2).

Fig.2-Effect of PMH ( 50 PM); BHT ( 25 PM 1. BHA ( 25 IIM 1 and VitE

( 125 PM 1 on lipid peroxidation induced by NAC ( 1 mM 1 after

60 min of incubation in isolated hepatocytes.

X=Statistical difference from control ( p <O.OOl 1; XX=Statistical

difference from NAC alone ( p <O.OOl 1.

When the data were compared to the control, MDA content of

the hepatocytes incubated with VitE and NAC was significantly

higher (p=O.O05). Lipid peroxidation, as evaluated by the

TBA test, was confirmed by measuring conjugated dienes. As

shown in Fig.3, the diene conjugated signal is absent in

the lipid extracts of control hepatocytes, whereas after

addition of NAC (1 mM) there is a typical peak at 233 nm,

indicative of a conjugated diene shift. Considering an ab -

sence of signal at 233 nm, addition of PMH to hepatocytes

incubated with NAC results in total inhibition of diene con -

jugation.

Pharmacological Research Communications, Vol. 18, No. 8, 1986

Fig.3-Normal (a) and second derivative (b) UV spectra of lipids extracted

from isolated hepatocytes. A, control; B, after addition of 1 mM

NAC; C, after addition of 1 mM NAC plus 50 VM oromethazine. The

arrows shoaw a peak at 233 nm that indicates the conjugated-diene

shift in the second derivateve spectrum. In C there is no peak at

233 nm.

DISCUSSION

Our results indicate that NAC addition to isolated

hepatocytes increases the lipid peroxidation as measured

by the TBA test for MDA and diene conjugation. Moreover, this

prooxidant effect of NAC is inhibited by antioxidants or free

radicals scavengers. NAC concentrations used in our in vitro

experiments are higher than mean plasma concentrations reached

in clinical therapy. In fact, as showed by Renzi et a1.,(1985)

45 min after administration of 140 mg/kg NAC the plasma level

of NAC ranged from 11.5 to 20.1 vg/ml .,while in our experiments

the concentration corresponds to 120 pg/ml. The prooxidant ef-

fect of NAC can be explained considering that NAC after deace-

tylation may be transformed into cysteine. This later compound

704 Pharmacological Research Communications, Vol. 18, No. 8, 1986

is known to readily autoxidize with production of thiyl and

hydroxyl free radicals, (Saez et al., 1982; Searle and Tomasi,

1982; ViEa et al., 1983) shown to promote lipid peroxidation

of membrane lipids.NAC's failure to cause cell damage is not

surprising since several investigators (Reed and Fariss, 1984;

Younes and Siegers, 1984; Smith et al., 1983; Mitchell et al.,

1976; Stacey and Klaassen, 1979) have demonstrated how lipid

peroxidation is not always responsible for cell damage. Stacey

and Klaassen (1981) showed that vanadium, while producing lipid

peroxidation in isolated hepatocytes, had a minimal effect on

the structural integrity of cell membrane.

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