Toxicology Letters, 66 (1993) 7-12 0 1993 Elsevier

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TOXLET 02824

Role of lipid peroxidation in acetaminophen-induced hepatotoxicity: comparison with carbon tetrachloride

Toshinori Kamiyama, Chifumi Sato, Jian Liu, Kazuo Tajiri, Happei Miyakawa and Fumiaki Marumo
Second Department of Internal Medicine, School of Medicine and Division of Health Science, School of Allied Medical Science, Tokyo Medical and Dental University, Tokyo (Japan)

(Received 29 June 1992) (Accepted 26 August 1992)

Key words: Acetaminophen;

Carbon tetrachloride; Lipid peroxidation; Ethanol

SUMMARY The effect of acetaminophen on lipid peroxidation in vivo and in vitro was studied in rat liver and the data were compared with those with carbon tetrachloride. Carbon tetrachloride increased diene conjugates in vivo and thiobarbituric acid reactive substance production in vitro in the liver microsomal incubation. These changes were further enhanced by ethanol that has previously been shown to increase carbon tetrachloride-induced hepatotoxicity. On the other hand, acetaminophen did not increase diene conjugates in vivo and inhibited thiobarbituric acid reactive substance production in vitro. These effects were minimally affected by ethanol which has previously been shown to inhibit acetaminophen-induced hepatotoxicity. Thus, lipid peroxidation may play a minimal role in acetaminophen-induced hepatotoxicity in contrast with carbon tetrachloride-induced hepatotoxicity.

INTRODUCTION Several mechanisms have been postulated in drug-induced hepatotoxicity. Among them, covalent binding hypothesis and lipid peroxidation theory are two major postulated mechanisms of drug-induced hepatotoxicity. The role of covalent binding in acetaminophen-induced hepatotoxicity has been widely accepted [I]. Several reports, however, have suggested the role of oxygen radicals and lipid peroxidation in

Correspondence to: Chifumi Sate, M.D., 2nd Department of Internal Medicine, Faculty of Medicine, Tokyo Medical and Dental University, S-45, Yushima I-chome, Bunkyo-ku, Tokyo 113, Japan.

acetaminophen-induced hepatotoxicity [2,3], which is a matter of controversy. In carbon tetrachloride-induced hepatotoxicity, the role of lipid peroxidation has been extensively studied [4]. We have previously observed in the rat that acute ethanol administration inhibits acetaminophen-induced hepatotoxicity, whereas the same maneuver enhances carbon tetrachloride-induced hepatotoxicity [5]. Using this model, the role of lipid peroxidation in acetaminophen-induced hepatotoxicity was assessed in vivo and in vitro, and the data were compared with those in carbon tetrachloride-induced hepatotoxicity.
MATERIALS AND METHODS

Male Sprague-Dawley rats (body wt. 180-200 g) were purchased from Japan Laboratory Animals Inc. (Tokyo, Japan). The animals were housed in iron-mesh animal cages and given free access to water and laboratory chow. They were fasted for 18 h before use. Acetaminophen, carbon tetrachloride, ethanol, L-ascorbic acid, thiobarbituric acid (TBA), and cyclohexane were from Wako Pure Chemical Industries Inc. (Japan). NADPH was purchased from Sigma Chemical Co. (St. Louis, MO)
In vivo experiments

The rats were given ethanol (6 g/kg, i.p.) or saline. Six hours later, they were given an intraperitoneal dose of acetaminophen (1 g/kg; 0.25 M supersaturated solution at 40C) or carbon tetrachloride (1 ml/kg; dissolved in olive oil at a 1:l dilution). Two hours thereafter, the animals were sacrificed by decapitation and the livers were immediately perfused with an ice-cold 1.15% KC1 containing 1 mM EDTA. Then the livers were excised and homogenized in 1.15% KC1 containing 1 mM EDTA. The mitochondrial fraction and microsomal fraction were separated by centrifugation [6] and lipids were extracted from each fraction according to Folch et al. [7]. Diene conjugates were measured as a marker for lipid peroxidation [6].
In vitro experiments

Washed liver microsomes were prepared as previously reported [S] either with 1.15% KC1 or 1.15% KC1 containing 1 mM EDTA. NADPH-dependent (enzymatic) and Fe*-ascorbic acid-dependent (non-enzymatic) lipid peroxidation was measured. The incubation mixture for NADPH-dependent lipid peroxidation contained 0.1 M potassium phosphate buffer (pH 7.4), 1 mg/ml microsomal proteins, 0.4 mM NADPH and compounds as indicated in a total volume of 1 ml. For Fe*-ascorbic acidinduced lipid peroxidation, 0.02 mM Fe,SO,, 0.01 mM EDTA, and 0.4 mM ascorbic acid were added instead of NADPH. Incubation was started by adding either NADPH or ascorbic acid, and was carried out for 15 min in a shaking water bath at 37 C, and the reaction was terminated by adding 1 ml of ice-cold 30% trichloroacetic acid. Lipid peroxidation was assessed by measuring TBA reactive substances [9]. Protein concentrations were measured according to Lowry et al. [lo].

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Fig. 1. Effects of acetaminophen and carbon tetrachloride on diene conjugates production in rat liver mitochondria in vivo. The rats were given ethanol (6 g/kg, i.p.) or saline. Six hours later they were injected with acetaminophen (1 g/kg, i.p.) or carbon tetrachloride (1 ml/kg, i.p.) and were sacrificed 2 h thereafter. Values are mean + SE (n = 4-S). * Indicates P < 0.05. ** Indicates P c 0.01.

Statistics Statistical significance was assessed by Students t-test and P values less than 0.05 are considered statistically significant.
RESULTS

In vivo experiments Diene conjugates were measured as a marker of lipid peroxidation in vivo. Acetaminophen administration did not affect diene conjugates in either mitochondria (Fig. 1) or microsomes (Fig. 2), and the effect of ethanol was minimal. Carbon tetrachloride treatment, however, tended to increase diene conjugates in both fractions and the increase was significant in combination with a prior administration of ethanol (Figs. 1 and 2). In vitro experiments NADPH-dependent and Fe2-ascorbic acid-induced lipid peroxidation were measured in liver microsomes as TBA reactive substances. Acetaminophen apparently inhibited this reaction in the enzymatic system (Table I) and showed no apparent effect in the non-enzymatic system (Table II). These effects of acetaminophen were

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Fig. 2. Effects of acetaminophen and carbon tetrachloride on diene conjugates production in rat liver microsomes in vivo. The rats were given ethanol (6 g/kg, i.p.) or saline. Six hours later they were injected with acetaminophen (I g/kg, i.p.) or carbon tetrachloride (1 ml/kg, i.p.) and were sacrificed 2 h thereafter. Values are mean f SE (n = 4-5). * Indicates P < 0.05. ** Indicates P < 0.01.

not significantly affected by the addition of ethanol (Tables I and II). On the other hand, carbon tetrachloride increased lipid peroxidation and ethanol further enhanced this reaction in both systems (Tables I and II).
DISCUSSION

The present study shows that acetaminophen does not enhance lipid peroxidation in vivo and rather inhibited lipid peroxidation in vitro. On the other hand, carbon tetrachloride enhances lipid peroxidation in vivo and in vitro, and this effect of carbon tetrachloride is more apparent in the presence of ethanol. We have previously reported that acute ethanol administration enhances carbon tetrachloride-induced hepatotoxicity [5]. Therefore, enhanced lipid peroxidation in the present study correlates well with the in vivo toxicity study. In that study acetaminophen-induced hepatotoxicity was inhibited by acute ethanol administration [5] and covalent binding of acetaminophen to proteins was decreased [ 111. In the present study lipid peroxidation was not increased by acetaminophen, and ethanol administration did not decrease lipid peroxidation, suggesting that lipid peroxidation may not play a significant role in the hepatotoxicity of acetaminophen. In the previous

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TABLE I EFFECTS OF ACETAMINOPHEN AND CARBON TETRACHLORIDE ENT LIPID PEROXIDATION IN RAT LIVER MICROSOMES I Control Acetaminophen ccl, Ethanol CCI, + acetaminophen Ccl, + ethanol Acetaminophen + ethanol 24.3 ? 2.3 10.9 f os* 33.2 f 2.1* 28.1 IT0.7 15.8 + 2.3* 36.9 _+l.l* 11.6 f 1.8* II 3.5 + 2.4 f 6.5 + 4.4 + 4.0 + 7.6 f 3.3 f 0.5 0.6* 0.6* 0.4 0.4 OS** 0.2 ON NADPH-DEPEND-

Rat liver microsomes were incubated as described in Materials and Methods. Values are expressed as nmol TBA reactive substance/mg protein/l5 min and mean f SE (n = 4). I, prepared in 1.15% KCl; II, prepared in 1.15% KC1 containing 1 mM EDTA. * Indicates P < 0.05 compared with controls. ** Indicates P < 0.05 compared with CC&.

study, covalent binding of acetaminophen to proteins was decreased by ethanol treatment and correlated well with the hepatotoxicity [l 11. The present study is in accordance with previous data [12] which demonstrated a minimal increase of ethane after acetaminophen administration. Other studies were mostly carried out in the mouse and under different experimental conditions such as vitamin E deficiency or fasting [3]. Therefore, although lipid peroxidation may occur under some experimental conditions, it does not necessarily correlate with hepatotoxicity.

TABLE II EFFECTS OF ACETAMINOPHEN AND CARBON TETRACHLORIDE ON Fe2/L-ASCORBIC ACID-DEPENDENT LIPID PEROXIDATION IN RAT LIVER MICROSOMES Control Acetaminophen ccl, Ethanol CC& + acetaminophen CC& + ethanol Acetaminophen + ethanol 3.7 + 4.1 f 5.1 + 4.1 f 4.0 f 6.8 f 3.9 f 0.3 0.2 0.2* 0.2* 0.4 0.4* 0.4

Rat liver microsomes were incubated as described in Materials and Methods. Values are expressed as nmol TBA reactive substance/mg protein/l5 min and mean f SE (n = 8). Microsomes were prepared in 1.15% KCl. * Indicates P < 0.05 compared with control.

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In conclusion, the role of lipid peroxidation in acetaminophen-induced hepatotoxicity appears to be minimal, although it plays an important role in carbon tetrachloride-induced hepatotoxicity.
ACKNOWLEDGEMENT

Part of this study was supported by Grant-in-aid 02304040 and 03670355 from the Japanese Ministry of Education, Science, and Culture.
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