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Acetaminophen-induced liver injury and oxidative stress: protective effect of propofol

Kostopanagiotou, Georgia Ga; Grypioti, Agni Da,b,c; Matsota, Paraskevia; Mykoniatis, Michael Gb; Demopoulos, Constantinos Ac; Papadopoulou-Daifoti, Zoeb; Pandazi, Agelikia

European Journal of Anaesthesiology (EJA): July 2009 - Volume 26 - Issue 7 - p 548–553
doi: 10.1097/EJA.0b013e32831c8a01
Original Articles – General
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Background and objective We evaluated the effects of propofol on oxidative stress and acute liver injury and regeneration produced by acetaminophen administration in rats.

Methods Acetaminophen (3.5 g kg−1) was administered by gastric tube to 50 adult male Wistar rats. One minute before acetaminophen, propofol was administered intraperitoneally (60 mg kg−1) to 25 rats and diethyl ether to the other 25 animals. All rats were sacrificed. Markers of oxidative stress (malondialdehyde levels, cholesterol/high-density lipoprotein cholesterol fraction and glutathione-S-transferase-π activity), liver injury (aspartate aminotransferase alanine aminotransferase and alkaline phosphatase and histological signs of inflammation and in-situ apoptosis) and liver regeneration (rate of [3H]thymidine incorporation into hepatic DNA, activity of liver thymidine kinase and mitotic index in hepatocytes) were determined. Unpaired Student's t-test and one-way analysis of variance were used for statistical analysis and a P value of 0.05 or less was considered significant.

Results All markers of oxidative stress were significantly decreased in propofol-treated animals. Biochemical and histological markers of liver injury and regeneration in propofol-treated animals did not show any significant decrease compared with those observed in the control group.

Conclusion The antioxidant capacity of propofol, verified in our study, did not manage to prevent liver injury and accelerate regeneration after acetaminophen administration in rats.

aSecond Department of Anesthesiology, Attikon Hospital, Greece

bDepartment of Pharmacology, Medical School, Greece

cDepartment of Chemistry, National and Kapodistrian University of Athens, Athens, Greece

Accepted 16 September, 2008

Correspondence to Georgia G. Kostopanagiotou, MD, PhD, 1 Rimini Str., Chaidari, 12462 Athens, Greece Tel: +30 210 583 23 71; fax: +30 210 532 64 13; e-mail: banesthclin@attikonhospital.gr

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Introduction

The liver is an important target for the toxicity of drugs in terms of oxidative stress. Although considered well tolerated at therapeutic doses, an overdose of acetaminophen (N-acetyl-p-amino-phenol or paracetamol) produces a centrilobular hepatic necrosis that can lead to fatal fulminant hepatic failure. The proportion of acetaminophen-induced liver failure cases has increased between 1998 (21%) and 2003 (51%) in the United States and remains a significant concern in other countries also [1]. Liver injury by acetaminophen overdose is caused by increased production of reactive oxygen species, antioxidant (glutathione) depletion and cytokine upregulation [2–4].

Previous studies have shown that antioxidants may prevent acetaminophen-induced liver injury [5,6]. Propofol, a highly lipid-soluble general anaesthetic, has been found to possess antioxidant activity in vitro and in vivo[7–11]. Also, propofol has been found to improve survival in patients with severe intracranial hypertension in the setting of fulminant hepatic failure, when used for sedation of these patients in the critical care setting [12].

Therefore, this study was designed to determine the possible protective effects of propofol on acetaminophen-induced acute liver injury and regeneration, evaluating its ability to reduce oxidative stress after acetaminophen administration in rats.

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Methods

Experimental animal model

Written approval from the Aretaion Hospital review board was obtained for the performance of the experiments.

  1. Animals: All animals received proper care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals, prepared by the Academy of Sciences, and published by the National Institutes of Health (Institute of Laboratory Animal Resources Commission on Life Sciences, 1996). Fifty adult male Wistar rats (Hellenic Pasteur Institute, Athens, Greece), weighing 160–200 g each, were used in our study. They had free access to food and water, were kept in an air-conditioned room at 21°C, with a 12/12 h light/dark cycle and were fasted for 12 h before acetaminophen treatment.
  2. Treatment: A single dose of acetaminophen (3.5 g kg−1 body weight) suspended in saline solution was administered by gastric tube to all animals. The dose of acetaminophen administered in our animal model of hepatotoxicity in rats is based on other studies using this acetaminophen model [13]. One minute before acetaminophen administration 25 rats were treated with propofol (propofol-lipuro 1%, B. Braun Melsungen AG, Germany) (60 mg kg−1 body weight) intraperitoneally and the other 25 animals were anaesthetized with diethyl ether. All rats were sacrificed in groups of five animals at different time points (8, 16, 24, 32 and 40 h after acetaminophen treatment). One hour before sacrifice, all animals received intraperitoneally 25 μCi of [3H]thymidine/100 g body weight (Amersham Corp., Buckinghamshire, UK). Immediately after exsanguinations, the livers were removed, cleaned and weighed. Small portions of the livers were kept frozen at −80°C in order to be analysed for their DNA content and their thymidine kinase activity, whereas another portion was separated for histological examination.
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Estimation of oxidative stress

  1. Malondialdehyde levels: Malondialdehyde levels were determined in liver homogenate with a commercial test kit for lipid peroxidation (Malondialdehyde-586 assay; Oxis International Inc., Portland, Oregon, USA).
  2. Cholesterol/high-density lipoprotein (HDL) cholesterol fraction: All serum samples were processed for the determination of the levels of cholesterol and HDL cholesterol using a random-access chemistry analyser (RA-1000; Technicon Instruments Corp., Tarrytown, New York, USA).
  3. Glutathione-S-transferase-π activity: Glutathione-S-transferase-π activity was determined in serum samples with a commercial ELISA test kit for oxidative stress (glutathione-S-transferase-π ELISA kit Art. No. K7960; Immundiagnostik AG, Bensheim, Germany).
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Estimation of liver injury

Blood samples were collected from all animals via cardiac puncture. The samples were allowed to clot and the serum was removed by centrifugation at 1000 × g for 10 min at room temperature. All serum samples were sterile, haemolysis-free and were processed for the determination of the enzymatic activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), using a random-access chemistry analyser (RA-1000; Technicon Instruments Corp.).

For liver histopathology analysis, midsections of the left lobes of the liver were processed for light microscopy. This processing consisted of specimen fixation in 4% buffered neutral formalin solution for 24 h, embedding in paraffin wax, slicing sections at 5 μm of thickness and staining them with haematoxylin–eosin. The liver tissue sections were independently examined and scored; the examiner was unaware of the group to which the specimen belonged. The degree of inflammation was not calculated but was estimated by the histopathology specialist; the degree was expressed as the mean of 10 high-power fields, chosen at random, and has been classified on a scale of 0–3 (normal, 0; mild, 1; moderate, 2; severe, 3) [14].

The extent of in-situ apoptosis was determined on formalin-fixed, paraffin-embedded liver tissue sections by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL). DNA strand breaks were assessed by TdT-dependent incorporation of dUTP using the Horseradish Peroxidase-DAB In situ Detection System (Enzo Life Science, New York, USA) according to the manufacturer's instructions. Five randomly selected fields from each tissue section were examined at an original magnification of 200×, and the percentage of positively stained hepatocytes was calculated.

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Estimation of liver regenerative capacity

Liver regeneration was evaluated by assessing the rate of [3H]thymidine incorporation into hepatic DNA, the enzymatic activity of liver thymidine kinase and the mitotic index in hepatocytes.

  1. Liver DNA synthesis: Liver portions were homogenized in ice-cold deionized water and the DNA was extracted and quantified in accordance with the method of Munro and Fleck [15], as modified by Kyprianidis et al.[16]. The specific activity of DNA was calculated from the radioactivity measured with a liquid scintillation counter (1211 Rackbeta; LKB-Wallac, Turku, Finland) and the amount of DNA that was determined colorimetrically [16,17]. Results were expressed as counts/minute incorporated (cpm) μg−1 of hepatic DNA.
  2. Liver thymidine kinase activity: The enzymatic activity of thymidine kinase was determined by the method of Kahn et al.[18] in the supernantant obtained after homogenization of liver portions following by ultracentrifugation at 105 000 × g for 1 h at 4°C with an ultracentrifuge (model L5-75; Beckman Instruments, Fullerton, California, USA). Duplicate aliquots of each sample were spotted onto diethylaminoethyl cellulose discs (DEAE Cellulose, Whatman Grade DE 81). The discs were counted for their radioactivity content in a liquid scintillation counter (1211 Rackbeta; LKB-Wallac). The protein was determined by the method of Lowry et al.[19]. The activity of the enzyme thymidine kinase was expressed as cpm min−1 mg−1 of protein.
  3. Hepatocyte mitotic activity: Hepatocyte mitotic activity was estimated on liver tissue sections stained with haematoxylin–eosin and assessed as the number of nuclei in metaphase observed/10 high-power fields (one high-power field is an area in the cell appropriate for histological estimation), chosen at random.
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Statistical analysis

The results are expressed as means ± SEM. All values were obtained from at least four animals. The statistical analysis was performed using unpaired Student's t-test and one-way analysis of variance. The results were considered significant if the P value was less than or equal to 0.05.

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Results

Liver oxidative stress

Malondialdehyde (indicator of lipid peroxidation and biochemical marker of oxidative stress) levels in propofol-treated animals were decreased when compared with those observed in the control group of rats during all time points examined (Fig. 1). Furthermore, the cholesterol/HDL cholesterol fraction (indicator of oxidative stress) was lessened at all time intervals examined compared with control values (Fig. 2). The activity of glutathione-S-transferase-π, one of the most important defence systems against peroxidases and electrophilic agents, showed a significant decrease during all time points examined in propofol-treated rats in comparison with the values obtained in the control group of rats. The enzyme (glutathione-S-transferase-π) activity showed a constant value in propofol-treated rats (Fig. 3).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Fig. 3

Fig. 3

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Liver injury

All biochemical parameters examined (AST, ALT and ALP) in propofol-treated animals did not show any significant decrease compared with those observed in the control group of rats. The data demonstrated that AST and ALT activities in propofol-treated animals were not altered significantly at any time interval examined compared with control group values (Figs 4 and 5). ALP activity in propofol-treated rats did not show a significant decrease at 24 and 40 h in comparison with the control group values (Fig. 6).

Fig. 4

Fig. 4

Fig. 5

Fig. 5

Fig. 6

Fig. 6

No considerable changes in liver histology were noted during the time course of our observation. The degree of inflammation was stable in propofol-treated animals at all time intervals examined compared with control animals (Fig. 7). The apoptotic process followed exactly the same time course as that of inflammation. The presence and number of apoptotic figures in propofol-treated animals were the same as those observed in the control group between 24 and 40 h, being prominent in hepatocytes in the vicinity of necrotic areas (Fig. 8).

Fig. 7

Fig. 7

Fig. 8

Fig. 8

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Liver regenerative activity

The rate of [3H]thymidine incorporation into hepatic DNA in rats receiving acetaminophen and propofol in comparison with control rats receiving only acetaminophen is presented in Fig. 9. The rate of regenerating activity after propofol treatment was unaltered compared with the rate without treatment (control animals) at all time intervals.

Fig. 9

Fig. 9

Constant values were also observed in thymidine kinase activities in propofol-treated animals compared with those observed in control animals at all time intervals examined after treatment (Fig. 10).

Fig. 10

Fig. 10

Hepatocyte mitotic activity did not show any change after propofol treatment in comparison with control rats. During all time points examined, mitoses were the same in all animals (both control and propofol-treated animals) (Fig. 11).

Fig. 11

Fig. 11

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Discussion

We chose the rat model because liver damage caused in rats by acetaminophen administration resembles human liver damage in both its biological and its morphological aspects [20]. The acetaminophen dose was chosen according to previous studies, as a dose inducing nonlethal toxicity in rats [13,21]. We should clarify that the dose we used is much greater than the dose considered lethal in humans, but is nonlethal for rats. Nonlethal toxicity involves both necrosis and apoptosis of hepatocytes [22,23]. Both procedures were observed in our study after acetaminophen administration, because we found increased activity of serum hepatic enzymes (AST, ALT and ALP) indicative of hepatic necrosis, as well as histological indications of apoptosis. The inability of propofol to decrease serum hepatic AST, ALT (indicative of necrosis) and ALP (indicative of cholestasis) activities is in good agreement with the standing liver injury observed between 24 and 40 h, prominent by the unchanging degree of apoptosis and inflammation observed at those time points. Therefore, propofol administration had no beneficial effect on indicators either of necrosis or of apoptosis, although it reduced all markers of hepatic oxidative stress.

The inability of propofol to prevent liver injury and accelerate liver regeneration after acetaminophen overdose in our experiment might be explained by the various mechanisms involved in liver injury caused by acetaminophen intoxication. Metabolic conversion of acetaminophen to N-acetyl-p-benzoquinonimine generated by cytochrome P-450-mediated oxidases is believed to be the initial step in acetaminophen toxicity. This toxic metabolite depletes the intracellular pool of glutathione, a nonprotein thiol with oxidant scavenger and redox-regulating capacities [2,24]. We observed decreased levels of glutathione-S-transferase-π activity after propofol administration, but we did not measure levels of glutathione. The scavenging activity of propofol might not be adequate to overcome the depletion of glutathione.

The increased production of reactive oxygen species that leads to mitochondrial damage is said to be another significant source of liver injury [3]. In our experiment, acetaminophen-induced oxidative stress was evidenced by high levels of hepatic malondialdehyde, increased values of the cholesterol/HDL cholesterol fraction and increased activity of glutathione-S-transferase-π observed in the control animals. Propofol administration reduced the above indicators of hepatic oxidative stress, confirming previous observations about its antioxidant activity [7–11].

Cytokines, chemokines and vascular endothelial growth factor also seem to be involved in hepatocyte injury and regeneration following acetaminophen toxicity [4,24]. We did not measure cytokine levels in our study; we did however, evaluate histological markers of inflammation and regeneration, processes that are associated with cytokines. The fact that propofol administration did not seem to affect inflammation and regeneration in our study may partly explain its inability to prevent injury. On the contrary, other studies have shown that propofol can inhibit cytokine responses to endotoxaemia in vitro and in vivo and after gut ischaemia/reperfusion-induced liver injury [25–28].

In accordance with our study, Shimono et al.[29] observed no protective effect of propofol in hepatic injury induced by hypoxia/reoxygenation in rat liver slices. Other studies, however, showed hepatoprotective effects of propofol against halothane-induced liver injury and against gut ischaemia/reperfusion-induced liver injury in rats [28,30].

The protective effects of propofol are dose and tissue-dependent, probably because of differences in the inherent susceptibility of each tissue [11,29]. Liver is a tissue sensitive to propofol protective effects and Runzer et al.[11] have shown that the relative response of tissues to the protective effects of propofol is liver > kidney > heart > lung. However, they state that the effects of propofol on organ functional preservation, after an ischaemia/reperfusion challenge, have not been clearly elucidated [11]. As far as dose is concerned, we used the dose of propofol that was effective in halothane-induced injury, but it proved inadequate for our acetaminophen intoxication model. Because we did not measure plasma concentrations, we are not able to conclude whether our dose reached effective plasma levels. Therefore, further studies examining the effect of different propofol doses on acetaminophen-induced liver injury are needed in order to investigate whether propofol can protect liver in this situation.

In conclusion, the antioxidant capacity of propofol, verified in our study, did not manage to prevent liver injury and accelerate regeneration after acetaminophen administration in rats.

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Keywords:

acetaminophen; antioxidants; liver injury; propofol

© 2009 European Society of Anaesthesiology