DUAL EFFECT OF ETHANOL ON CELL DEATH IN PRIMARY CULTURE OF HUMAN AND RAT HEPATOCYTES

RAFAEL CASTILLA, RAÚL GONZÁLEZ, DALIA FOUAD, ENRIQUE FRAGA and JORDI MUNTANÉ*

Unidad Clínica Aparato Digestivo, Hospital Universitario Reina Sofía, Córdoba, Spain

* Author to whom correspondence should be addressed at: Unidad de Investigación, Unidad Clínica Aparato Digestivo, Hospital Universitario Reina Sofía, Av. Menendez Pidal s/n, E-14004 Córdoba, Spain. Tel.: +34 957 011070; Fax: +34 957 010452; E-mail: jordi.muntane.exts{at}juntadeandalucia.es

(Received 16 September 2003; in revised form 28 February 2004; accepted 9 March 2004)


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aims: In-vivo and in-vitro studies have shown that ethanol induces hepatocyte damage. The aim of the present study was to evaluate the effect of a broad range of ethanol concentrations on apoptosis and necrosis in primary culture of human and rat hepatocytes. Methods: Human and rat hepatocytes were isolated from human hepatectomies and male Wistar rats (200–250 g) using the classical collagenase perfusion method. After stabilization of cell culture, ethanol (0–10 mmol/l) was administered and the parameters were measured 24 h after ethanol addition. Apoptosis was studied by DNA fragmentation, iodide propidium–DNA staining, caspase-3 activity and annexin V binding in hepatocytes. Necrosis was evaluated by lactate dehydrogenase (LDH) release. Malondialdehyde (MDA) and GSH/GSSG were used as parameters of oxidative stress. Results: Ethanol enhanced dose-dependently all the parameters associated with apoptosis in human and rat hepatocytes. Low or high ethanol concentrations induced an opposite action against cell necrosis in cultured hepatocytes. Low concentrations of ethanol (1–2 mmol/l) reduced LDH release from human and rat hepatocytes. However, the highest ethanol concentration (10 mmol/l) induced a sharp increase in cell necrosis. The effect of ethanol on cell necrosis was related to lipid peroxidation in hepatocytes. Conclusions: Ethanol differentially regulates apoptosis or necrosis in cultured hepatocytes. Although ethanol exerted a dose-dependent induction of apoptosis, low ethanol concentrations were able to reduce basal lipid peroxidation and necrosis in hepatocytes. The highest ethanol concentration (10 mmol/l) induced apoptosis and necrosis in human and rat cultured hepatocytes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alcohol-dependent liver disease (ALD) is the most common form of liver dysfunction in the western world. ALD is also the major cause of chronic illness and death associated with alcohol misuse (Frank and Raicht, 1985Go; French, 1996Go). In addition, excessive alcohol intake is also associated with gastric mucosa damage (Kvietys et al., 1990Go), inhibition of platelet aggregation (Mikhailidis et al., 1983Go), alteration of red blood cell membranes (Rottenberg, 1986Go), deregulation of immune response (Deviére et al., 1988Go; Nelson et al., 1989Go) and inhibition of cell growth (Clarren and Smith, 1978Go). Other experimental studies have also demonstrated a beneficial effect of ethanol against coronary heart disease (Amarasuriya et al., 1992Go; Miyamae et al., 1998Go; Xia et al., 1998Go), drug-related neuronal dysfunction (Boobis et al., 1975Go; Padilla et al., 1992Go) and drug-related hepatotoxicity (Tredger et al., 1985Go).

The aim of the present study was to evaluate the effect of a broad range of ethanol concentrations on cell death in cultured human and rat hepatocytes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials
All reagents were from Sigma Chemical (St Louis, MO) unless otherwise stated. William's medium E was from AppliChem (Darmstadt, Germany). Antibiotic–antimycotic solution and fetal bovine serum were from Life Technologies (Paisley, UK). All experimental animals received humane care and the study protocols and human hepatectomies were obtained following the institution's guidelines and supervised by the Ethic Committee.

Preparation of primary hepatocytes and cell culture
Human hepatocytes were isolated from hepatectomies obtained from patients (two men and two women; 52 ± 15 years old) submitted to surgical intervention for the resection of primary or secondary liver tumours. Rat hepatocytes were isolated from livers of male Wistar rats (seven animals, 200–250 g) anaesthetized by intraperitoneal administration of sodium thiopental. Human and rat hepatocytes were isolated by in-situ collagenase perfusion of liver (Seglen, 1976Go). Livers were perfused first with an oxygenated solution I (10 mmol/l HEPES, 6.7 mmol/l KCl, 145 mmol/l NaCl and 2.4 mmol/l EGTA), pH 7.4 at 37°C, and then with solution II (100 mmol/l HEPES, 6.7 mmol/l KCl, 67 mmol/l NaCl, 10 g/l albumin, 4.8 mmol/l CaCl2 and 0.05% collagenase A), pH 7.4 at 37°C. In order to reduce ischaemia-reperfusion syndrome during human hepatocyte isolation antioxidants were added (100 µmol/l sorbitol, 100 µmol/l manitol and 100 µmol/l GSH) to solution I and II. Thereafter, tissue was gently minced and cell suspension filtered through a nylon mesh. Hepatocytes were centrifuged and washed three times at 50 g for 5 min in William's medium E, pH 7.4, supplemented with 1 µmol/l insulin, 0.6 µmol/l hydrocortisone, 15 mmol/l HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin, 2 mmol/l glutamine and 26 mmol/l NaHCO3. Cell viability determined by trypan blue exclusion was consistently higher than 85%. Hepatocytes (150 000 cells/cm2) were plated in a Petri dish coated with collagen type I (Iwaki, Gyouda, Japan) and cultured in supplemented William's medium E, pH 7.4, containing 5% fetal bovine serum. After 2 h, culture medium was removed and replaced by fresh supplemented medium without fetal bovine serum and the culture was maintained for 24 h without treatment. After stabilization of cell culture, ethanol (0–10 mmol/l) was administered and the parameters were measured 24 h after ethanol addition.

DNA fragmentation in hepatocytes
The fragmentation of DNA in hepatocytes was evaluated in agarose gel and by the presence of oligonucleosomes in cell extract detected by commercial ELISA kit. For the first procedure, the whole hepatocyte population, including the floating cells obtained from collected culture medium, was treated with 1 ml lysis buffer (100 mmol/l Tris-HCl, 5 mmol/l EDTA, 150 mmol/l NaCl and 0.5% sarkosyl), pH 8.0 at 4°C for 10 min. Samples were incubated with RNAse (50 µg/ml) at 37°C for 2 h and proteinase K (100 µg/ml) at 48°C for 45 min. DNA was obtained by phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma Chemical) extraction and precipitated with cold isopropanol (1:1) at –20°C for 12 h. DNA was recovered by centrifugation of the sample at 20 800 g at 4°C for 10 min. Thereafter, the precipitate was washed with 70% ethanol, dried and re-suspended in Tris-EDTA buffer (10 mmol/l Tris, 1 mmol/l EDTA) at pH 8.0. Samples (100 µg DNA) were analysed on 1.5% agarose gel with ethidium bromide (0.5 µg/ml).

For the second procedure a cell death detection ELISA kit was used (Roche Diagnostics, Mannheim, Germany) that evaluated the presence of mono- and oligonucleosomes in cell lysate. The whole hepatocyte population, including the floating cells obtained from collected culture medium, was also used in this assay, and it followed the working procedure described by the manufacturer.

Quantification of DNA hypoploidy by flow cytometry analysis
Apoptosis was also evaluated by the measurement of the percentage of hypodiploid cells, with lower DNA content, in relation to the percentage of hepatocytes in cell cycle (Muntane et al., 1998Go). Hepatocytes were recovered using a nonenzymatic cell dissociation solution (Sigma Chemical). The whole hepatocyte population, including the floating cells obtained from collected culture medium, was washed twice with phosphate buffer solution (PBS) (137 mmol/l NaCl, 2.7 mmol/l KCl, 4.3 mmol/l Na2HPO4) pH 7.4 at 284 g at 4°C for 5 min. The cell fraction was permeabilized and fixed in ethanol (70%) for 4 h at 4°C. Afterwards, hepatocytes were washed in PBS and incubated with RNAse A (5 U/ml) for 10 min at room temperature and with propidium iodide (20 µg/ml) for 10 min at room temperature. After incubation, the DNA content in hepatocytes was evaluated using a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, USA). DNA fluorescence pulse processing was used to discriminate between single cells and aggregates of cells (doublet discrimination) by evaluating the FL2-width versus FL2-area scatter plot. Light scatter gating was used to eliminate smaller debris from analysis. DNA content was displayed on a four-decade logarithmic scale. An analysis gate was set to limit the measurement of hypoploidy to an area of 2-fold loss of DNA content.

Caspase-3-associated activity
The whole hepatocyte population, including the floating cells obtained from collected culture medium, was treated with 800 µl of lysis buffer (10 mmol/l HEPES, pH 7.9, 10 mmol/l KCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 0.6% Nonidet NP-40, 5 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l DTT) for 10 min on ice. After, samples were vortexed and centrifuged at 15 000 g for 1 min at 4°C. The supernatant, as cytoplasmatic fraction, was recovered and frozen at –80°C for the further measurement of caspase-3-associated activity. Caspase-3 associated activity was measured in samples (25 µg protein) by a fluorometric assay using the peptide-based substrate ac-N-acetyl-Asp-Glu-Val-Asp-AFC (Ac-DEVD-AFC) (Bachem, Bubendorf, Switzerland). The linear increase in fluorescence (Ex 400 nm, Em 505 nm) of enzymatically released AFC was recorded over a period of 2 h at 37°C using a GENios Reader (TECAN, Salzburg, Austria).

Measurement of annexin V binding
The binding of annexin V to phophatidylserine sites in human and rat hepatocytes was followed using a commercial kit (Sigma Chemical). This assay is based on the binding of annexin V-FITC to hepatocytes and further quantitative determination by flow cytometry. The inclusion in the kit of propidium iodide to label the cellular DNA in necrotic cells allowed us to evaluate the percentage of apoptotic hepatocytes that binds annexin V.

Measurement of lactate dehydrogenase release
Lactate dehydrogenase (LDH) in the culture medium and hepatocytes was measured by modification of a colorimetric routine laboratory method (Taffs and Sitkovsky, 1991Go). Briefly, a volume of culture medium or cell lysate was incubated with 0.2 mmol/l ß-NADH and 0.4 mmol/l pyruvic acid diluted in PBS pH 7.4. It was also confirmed that addition of ethanol to the incubation mixture did not alter the oxidation of ß-NADH. LDH concentration in the sample was proportional to the linear decrease in the absorbance at 334 nm. LDH concentration was calculated using a commercial standard. The percentage of LDH released from hepatocytes was calculated as LDH present in culture medium in relation to total LDH obtained in culture medium and hepatocytes.

Evaluation of lipid peroxidation
The presence of malondialdehyde (MDA) in culture medium was used as an index of lipid peroxidation in hepatocytes following a procedure described previously (Wasowicz et al., 1993Go). Briefly, the samples (100 µl) were treated with trichloroacetic acid (10%) and centrifuged at 20 800 g at 4°C for 5 min. EDTA (1.34 mmol/l) and GSH (0.65 mmol/l) was added to the supernatant to prevent further lipid peroxidation caused during the assay. The samples were treated with 1 ml HCl (25%) and 1 ml of thiobarbituric acid (1% diluted in 50 mmol/l NaOH), and the mixture was heated at 100°C for 1 h. MDA was evaluated measuring the absorbance of the samples at 532 nm in a DU® 64 Spectrophotometer (Beckman Coulter, California, USA). Standard curve was prepared daily using 1,1,3,3-tetraethoxypropane (Sigma Chemical) diluted in ethanol as source of MDA.

Quantification of GSH/GSSG ratio
GSH and GSSG were quantified in hepatocytes following the procedure described by Asensi et al. (1994)Go. The whole hepatocyte population, including the floating cells obtained from collected culture medium, was treated with precipitating solution (12% perchloric acid, 40 mmol/l N-ethylmaleimide and 2 mmol/l bathophenanthroline disulfonic acid) at 4°C for 5 min. The samples were centrifuged at 20 800 g at 4°C for 5 min. Afterwards, 50 µl of glutamyl glutamate (1 mmol/l) (Sigma Chemical) as internal standard and 10 µl of m-cresol purple (1 mmol/l) (Sigma Chemical Co.) as pH indicator were added to the samples (500 µl). The pH of the solution was adjusted to 8.0–8.5 with KOH (2 mol/l) containing MOPS (0.3 mol/l) to prevent excessive alkalization. After centrifugation of the samples at 20 800 g at 4°C for 5 min, a volume (25 µl) was derivatized with 50 µl 1-fluoro-2,4-dinitrobenzene (1%) (Sigma Chemical) in a small glass tube. After 45-min incubation in the dark at room temperature, samples were dessicated under vacuum and stored at –20°C until injection. Afterwards, the samples were dissolved in 50 µl of methanol (80%) and injected (25 µl) into the HPLC system (Beckman Instruments) equipped with a Sperisorb NH2 column (20 x 04 cm, 5 µm particles) (Teknokroma, Barcelona, Spain). The flow rate was set at 1 ml/min. Two mobile phases were used: solvent A (80% methanol) and solvent B (0.5 mol/l sodium acetate in 64% methanol). After injection of the sample, the mobile phase was held at 80% solvent A and 20% solvent B for 5 min followed by a 10-min linear gradient up to 1% solvent A and 99% solvent B. Later, the mobile phase was held at 99% solvent B until GSSG had eluted. The concentration of GSH and GSSG was quantified using the areas below the corresponding HPLC peaks of the sample. Standard curve was drawn using commercial GSH and GSSG (Sigma Chemical).

Statistical analysis
Results are expressed as means with their corresponding standard errors of four to seven separate experiments. Differences between groups were assessed by one-way analysis of variance (ANOVA) using the least significant differences (LSD) test as multiple comparison analysis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of apoptosis by ethanol exposure in cultured rat hepatocytes
The effect of ethanol (0–10 mmol/l) in the degree of apoptosis in rat hepatocytes was evaluated. Ethanol gradually increased DNA fragmentation (Fig. 1) and caspase-3-associated activity (Fig. 2) in cultured hepatocytes. The measurement of histone-associated DNA fragments in cell extract also showed that ethanol gradually enhanced the presence of mono- and oligonucleosomes in cultured rat hepatocytes. In this sense, ethanol (1 mmol/l) significantly raised DNA fragments (2.34-fold ± 0.164) in comparison with control hepatocytes (P ≤ 0.05). The measurement of DNA content in hepatocytes by flow cytometry showed that the percentage of hypoploidy cells was significantly enhanced at the highest ethanol concentration (10 mmol/l) (Table 1) (P ≤ 0.05). The same method showed that hepatocytes in apoptosis came from hepatocytes in the G2+M cell cycle stage (Table 1). An early parameter of apoptosis such as annexin V binding to hepatocyte was also evaluated (Table 1). In this assay, intermediate ethanol concentrations (2 mmol/l) significantly enhanced the percentage of apoptotic hepatocytes that binds annexin V (P ≤ 0.05). Interestingly, hepatocytes treated with the highest ethanol concentration (10 mmol/l) reduced their ability to bind annexin V (P ≤ 0.05).



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Fig. 1. DNA fragmentation in rat hepatocytes treated with ethanol. DNA fragmentation was evaluated following the procedure described in Materials and methods. Ethanol gradually increases DNA fragmentation in rat hepatocytes. The image is representative of seven different experiments. Bp, base pairs (DNA molecular weight marker).

 


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Fig. 2. Caspase-3 associated activity in rat hepatocytes treated with ethanol. Caspase-3-associated activity was evaluated following the procedure described in Materials and methods. Ethanol gradually increases caspase-3-associated activity in rat hepatocytes. Data are the mean ± SD of seven experiments. *P ≤ 0.01 versus hepatocytes treated with lower ethanol concentration; #P ≤ 0.02 versus hepatocytes treated with lower ethanol concentration; {diamondsuit}P ≤ 0.05 versus hepatocytes treated with lower ethanol concentration.

 

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Table 1. Effect of ethanol on the percentage of hepatocytes in hypodiploid and G2+M stage, as well as the binding of annexin V to apoptotic cells in primary culture of rat hepatocytes

 
Induction of necrosis by ethanol exposure in cultured rat hepatocytes
The measurement of LDH released from hepatocytes is an index of cell necrosis (Fig. 3). Interestingly, LDH release was significantly reduced at intermediate ethanol concentration (0.2–5 mmol/l) (P ≤ 0.05). At 10 mmol/l ethanol a sharp increase in LDH release was observed in cultured rat hepatocytes (Fig. 3).



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Fig. 3. Lactate dehydrogenase (LDH) released from rat hepatocytes treated with ethanol. Data correspond to the percentage (%) of total LDH released from hepatocytes to the culture medium. The administration of low doses of ethanol (0.2–5 mmol/l) reduced LDH release, but 10 mmol/l ethanol induced a sharp increase in LDH release from rat hepatocytes. Data are the mean ± SD of seven experiments. *P ≤ 0.01 versus hepatocytes treated with lower ethanol concentration; #P ≤ 0.05 versus hepatocytes treated with lower ethanol concentration.

 
Evaluation of lipid peroxidation after ethanol exposure in cultured rat hepatocytes
The presence of MDA in the culture medium was used as an index of lipid peroxidation in hepatocytes (Fig. 4). MDA concentration was significantly reduced at an intermediate concentration of ethanol (0.02–5.00 mmol/l) (P ≤ 0.05). The highest ethanol concentration (10 mmol/l) induced a rise in MDA concentration over control hepatocytes (Fig. 4) (P ≤ 0.05).



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Fig. 4. Malondialdehyde (MDA) in culture medium from rat hepatocytes treated with ethanol. MDA was evaluated following the procedure described in Materials and methods. The administration of low doses of ethanol (0.2–5 mmol/l) reduced MDA concentration in culture medium, but 10 mmol/l ethanol induced a sharp increase in LDH release from rat hepatocytes. Data are the mean ± SD of seven experiments. *P ≤ 0.02 versus hepatocytes treated with lower ethanol concentration; #P ≤ 0.05 versus hepatocytes treated with lower ethanol concentration.

 
Effect of ethanol on GSH/GSSG ratio in cultured rat hepatocytes
The antioxidant status was evaluated by the ratio of GSH and GSSG in hepatocytes. Ethanol gradually decreased GSH/GSSG in hepatocytes, reaching statistical significance at the highest ethanol concentration (10 mmol/l) (93 ± 3.8) in comparison with the value obtained in control hepatocytes (144 ± 15.6) (P ≤ 0.05).

Effect of ethanol on cell death in cultured human hepatocytes
We present data corresponding to the effect of ethanol (0–10 mmol/l) in the degree of apoptosis and necrosis in cultured human hepatocytes. Ethanol (10 mmol/l) induced a significant increase in the presence of histone-associated DNA fragments (3.01-fold ± 0.300) and the percentage of hypodiploid cells (Table 2) in comparison with the values obtained in control hepatocytes (P ≤ 0.05). The presence of an early marker of apoptosis, such as annexin V binding, was also assessed in cultured human hepatocytes (Table 2). In this assay, intermediate ethanol concentrations (2 mmol/l) significantly enhanced the percentage of apoptotic hepatocytes that binds annexin V (P ≤ 0.05). Interestingly, hepatocytes treated with the highest ethanol concentration (10 mmol/l) reduced their ability to bind annexin V (P ≤ 0.05).


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Table 2. Effect of ethanol on the percentage of hepatocytes in hypodiploid and G2+M stage, as well as the binding of annexin V to apoptotic cells in primary culture of human hepatocytes

 
The effect of ethanol on necrosis in human hepatocytes was similar to that observed in rat hepatocytes. The administration of intermediate (1–2 mmol/l) or high (10 mmol/l) ethanol concentration exerted cytoprotection or exacerbation respectively against cell necrosis in cultured human hepatocytes (Fig. 5) (P ≤ 0.05).



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Fig. 5. Lactate dehydrogenase (LDH) release in human hepatocytes treated with ethanol (0–10 mmol/l). Data correspond to the percentage (%) of total LDH released by hepatocytes to the culture medium. The administration of low doses of ethanol (1–2 mmol/l) reduced LDH release, but 10 mmol/l ethanol induced a sharp increase in LDH release from human hepatocytes. Data are the mean ± SD of four experiments. *P ≤ 0.02 versus hepatocytes treated with lower ethanol concentration; #P ≤ 0.05 versus hepatocytes treated with lower ethanol concentration.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study showed that ethanol administered at concentrations below 10 mmol/l exerts a differential regulation on apoptosis and necrosis in cultured human and rat hepatocytes. In this sense, ethanol enhances apoptosis but reduces necrosis, probably through a protection against basal lipid peroxidation.

Alcohol-dependent liver disease (ALD) is the most frequent cause of cell death associated with alcohol misuse (Frank and Raicht, 1985Go). Several reports have shown that ethanol enhances in-vivo (García-Ruiz et al., 1994Go; Reinke et al., 1997Go; Rouach et al., 1997Go) and in-vitro (Lamb et al., 1994Go; Kurose et al., 1997Go; Baileym et al., 1998Go) hepatocyte cell death through stimulation of oxidative stress. Although free radicals from stimulated inflammatory cells may play an important role in alcohol-dependent liver toxicity (Koop et al., 1997Go), it seems that enhanced intracellular oxidative stress in hepatocytes during ethanol exposure limits cell viability. In ethanol-induced cytotoxicity, free radicals may be derived from the induction of cytochrome P4502E1 activity (Morimoto et al., 1993Go) or by mitochondrial dysfunction after reduction of GSH content and ATP generation (Cunningham et al., 1990Go; Collell et al., 1998Go). Necrosis and apoptosis have been demonstrated in rat hepatocytes treated with ethanol (50 mmol/l or higher) (Lamb et al., 1994Go; Higuchi et al., 1996Go). In our conditions, ethanol was able to induce apoptosis and necrosis in human and rat hepatocytes. Nevertheless, ethanol showed differential regulatory properties against apoptosis and necrosis. In this sense, ethanol increased cell apoptosis in a concentration-dependent fashion. In contrast, the administration of intermediate ethanol concentrations, 1–2 mmol/l and 0.2–5 mmol/l in human and rat hepatocytes respectively, reduced cell necrosis. The highest ethanol concentration (10 mmol/l) induced a sharp increase in hepatocyte necrosis. Although we have not measured intracellular free radical production, our data suggest that oxidative stress in terms of lipid peroxidation and antioxidant status induced by ethanol was more related to necrosis than to apoptosis in rat hepatocytes.

The administration of an acute dose of ethanol has been shown to exert cytoprotection in different human and experimental liver dysfunction (Sato et al., 1981Go; Banda and Quart, 1982Go). In this sense, although chronic ethanol consumption increases the risk of acetaminophen-associated liver injury, low doses of ethanol consumption prior to the ingestion of acetaminophen leads to a reduction of its related reactive metabolite in humans (Banda and Quart, 1982Go). The incubation of microsomes with ethanol has not been shown to significantly affect paracetamol activation (Tredger et al., 1985Go). It has been proposed that acute ethanol administration decreases the availability of NADPH required as cofactor for monooxygenation (Reinke et al., 1980Go). In our experiments, the addition of 0.2–5 mmol/l ethanol reduced MDA in culture medium, suggesting that ethanol was reducing the basal level of intracellular oxidative stress in hepatocytes. Cytochrome P4502E1, an isoform mostly involved in intracellular free radical production (Albano et al., 1991Go), activates acetaminophen to reactive metabolites which cause cell toxicity (Dai and Cederbaum, 1995Go). In our studies, the expression of cytochrome P452E1 was maintained for 48 h without decline after hepatocyte isolation (data not shown). These results suggest that the protection by ethanol against hepatocyte necrosis is probably due to a reduction of intracellular free radical production in a similar way by which ethanol is able to protect against acetaminophen cytotoxicity (Reinke et al., 1980Go; Banda and Quart, 1982Go; Tredger et al., 1985Go). Other factors, such as nitric oxide, which have been shown to inhibit cytochrome P4502E1 catalytic activity and its reactive oxygen radical formation (Gergel et al., 1997Go), do not seem to play a role in our system. We have not observed any changes in nitrite-plus-nitrate concentration in culture medium during ethanol treatment (data not shown).

In conclusion, the present study shows that although high ethanol concentration induces apoptosis and necrosis in human and rat hepatocytes, the exposure to low ethanol concentrations reduces cell necrosis, probably through a reduction of intracellular oxidative stress. More studies should be done to fully elucidate the intracellular mechanism by which ethanol exerts this cytoprotective effect.


    ACKNOWLEDGEMENTS
 
This study was supported by the Programa de Promoción de la Investigación en Salud (FIS 01/3069) and Red Temática Investigación Cooperativa (G03/015) del Ministerio de Sanidad y Consumo.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Albano, E., Tomasi, A., Persson, J. O., Terelius, Y., Goria-Gatti, L., Ingelman-Sundberg, M. and Dianzani, M. V. (1991) Role of ethanol-inducible cytochrome P450 (P450 IIE1) in catalysing the free radical activation of aliphatic alcohols. Biochemical Pharmacology 41, 1895–1902.[CrossRef][ISI][Medline]

Amarasuriya, R. N., Gupta, A. K., Civen, M., Horng, Y. C., Maeda, T. and Kashyap, M. L. (1992) Ethanol stimulates apolipoprotein A-I secretion by human hepatocytes: implications for a mechanism for atherosclerosis protection. Metabolism 41, 827–832.[ISI][Medline]

Asensi, M., Sastre, J., Pallardo, F. V., García, J., Estrela, J. M. and Viña, J. (1994) A high-performance liquid chromatography method for measurement of oxidized glutathione in biological samples. Analytical Biochemistry 217, 323–328.[CrossRef][ISI][Medline]

Baileym, S. M. and Cunningham, C. C. (1998) Acute and chronic ethanol increases reactive oxygen species generation and decreases viability in fresh, isolated rat hepatocytes. Hepatology 28, 1318–1326.[ISI][Medline]

Banda, P. W. and Quart, B. D. (1982) The effect of mild alcohol consumption on the metabolism of acetaminophen in man. Research Communication Chemical Pathology and Pharmacology 38, 57–70.[ISI]

Boobis, A. R., Gibson, A. and Stevenson, R. W. (1975) Ethanol protection against hemicholinium toxicity in mice. Biochemical Pharmacology 24, 485–488.[CrossRef][ISI][Medline]

Clarren, S. K. and Smith, D. W. (1978) The fetal alcohol syndrome. New England Journal of Medicine 298, 1063–1067.[ISI][Medline]

Collell, A., García-Ruiz, C., Miranda, A., Ardite, E., Mari, M., Morales, A., Corrales, F., Kaplowitz, N. and Fernandez-Checa, J. C. (1998) Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor. Gastroenterology 115, 1541–1551.[ISI][Medline]

Cunningham, C. C., Coleman, W. B. and Spach, P. I. (1990) The effects of chronic ethanol consumption on hepatic mitochondrial energy metabolism. Alcohol and Alcoholism 25, 127–136.[ISI][Medline]

Dai, Y. and Cederbaum, A. I. (1995) Cytotoxicity of acetaminophen in human cytochrome P4502E1-transfected HepG2 cells. Journal of Pharmacology Experimental Therapy 273, 1497–1505.

Deviére, J., Denys, C., Schandene, L., Romasco, F., Adler, M., Wybran, J. and Dupont, E. (1988) Decreased proliferative activity associated with activation markers in patients with alcoholic liver cirrhosis. Clinical Experimental Immunology 72, 377–382.[ISI][Medline]

Frank, D. and Raicht, R. F. (1985) Alcohol-induced liver diseases. Alcohol Clinical Experimental Research 9, 66–82.[ISI][Medline]

French, S. W. (1996) Ethanol and hepatocellular injury. Clinical Laboratory Medicine 16, 289–306.

García-Ruiz, C., Morales, A., Ballesta, A., Rodés, J., Kaplowitz, N. and Fernández-Checa, J. C. (1994) Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria in periportal and perivenous rat hepatocytes. Journal of Clinical Investigation 94, 193–201.[ISI][Medline]

Gergel, D., Misík, V., Riesz, P. and Cederbaum, A. I. (1997) Inhibition of rat and human cytochrome P4502E1 catalytic activity and reactive oxygen radical formation by nitric oxide. Archives of Biochemistry and Biophysics 337, 239–250.[CrossRef][ISI][Medline]

Higuchi, H., Kurose, I., Kato, S., Miura, S. and Ishii, H. (1996) Ethanol-induced apoptosis and oxidative stress in hepatocytes. Alcoholism: Clinical and Experimental Research 20, 340A–346A.[Medline]

Koop, D. R., Klopfenstein, B., Iimuro, Y. and Thurman, R. G. (1997) Gadolinium chloride blocks alcohol-dependent liver toxicity in rats treated chronically with intragastric alcohol despite the induction of CYP2E1. Molecular Pharmacology 51, 944–950.[Abstract/Free Full Text]

Kurose, I., Higuchi, H., Kato, S., Miura, S., Watanabe, N., Kamegaya, Y., Tomita, K., Takaishi, M., Horie, Y., Fukuda, M., Mizukami, K. and Ishii, H. (1997) Oxidative stress on mitochondria and cell membrane of cultured rat hepatocytes and perfused liver exposed to ethanol. Gastroenterology 112, 1331–1343.[ISI][Medline]

Kvietys, P. R., Twohig, B., Danzell, J. and Specian, R. D. (1990) Ethanol-induced injury to the rat gastric mucosa. Role of neutrophils and xanthine oxidase-derived radicals. Gastroenterology 98, 909–920.[ISI][Medline]

Lamb, R. G., Koch, J. C., Snyder, J. W., Huband, S. M. and Bush, S. R. (1994) An in vitro model of ethanol–dependent liver cell injury. Hepatology 19, 174–182.[CrossRef][ISI][Medline]

Mikhailidis, D. P., Jeremy, J. Y., Barradas, M. A., Green, N. and Dandona, P. (1983) Effect of ethanol on vascular prostacyclin (prostaglandin I2) synthesis, platelet aggregation, and platelet thromboxane release. British Medicine Journal (Clinical Research Edition) 287, 1495–1498.

Miyamae, M., Rodríguez, M. M., Camacho, S. A., Diamond, I., Mochly-Rosen, D. and Figueredo, V. M. (1998) Activation of epsilon protein kinase C correlates with a cardioprotective effect of regular ethanol consumption. Proceedings of the National Academy of Sciences of the United States of America 95, 8262–8267.[Abstract/Free Full Text]

Morimoto, M., Hagbjörk, A. L., Nanji, A. A., Ingelman–Sundberg, M., Lindros, K. O., Fu, P. C., Albano, E. and French, S. W. (1993) Role of cytochrome P4502E1 in alcoholic liver disease pathogenesis. Alcohol 10, 459–464.[CrossRef][ISI][Medline]

Muntané, J., Montero, J.L., Marchal, T., Perez-Seoane, C., Lozano, J.M., Fraga, E., Pintado, C.O., De la Mata, M. and Miño, G. (1998) Effect of PGE1 on TNF-{alpha} status and hepatic D-galactosamine-induced apoptosis in rats. Journal of Gastroenterology and Hepatology 13, 197–207.[ISI][Medline]

Nelson, S., Bagby, G. J., Bainton, B. G. and Summer, W. R. (1989) The effect of acute and chronic alcoholism on tumor necrosis factor and the inflammatory response. Journal of Infectious Diseases 160, 422–429.[ISI][Medline]

Padilla, S., Lyerly, D. L. and Pope, C. N. (1992) Subacute ethanol consumption reverses p-xylene-induced decreases in axonal transport. Toxicology 75, 159–167.[CrossRef][ISI][Medline]

Rottenberg, H. (1986) Membrane solubility of ethanol in chronic alcoholism. The effect of ethanol feeding and its withdrawal on the protection by alcohol of rat red blood cells from hypotonic hemolysis. Biochimica Biophysica Acta 855, 211–222.[ISI][Medline]

Reinke, L. A., Kaufman, F. C., Belinsky, S. A. and Thurman, R. G. (1980) Interactions between ethanol metabolism and mixed-function oxidation in perfused rat liver: inhibition of p-nitrosanisole O-demethylation. Journal of Pharmacology Experimental Therapy 213, 70–78.

Reinke, L. A., Moore, D. R. and McCay, P. B. (1997) Free radical formation in livers of rats treated acutely and chronically with alcohol. Alcoholism: Clinical and Experimental Research 21, 642–646.[ISI][Medline]

Rouach, H., Fataccioli, V., Gentil, M., French, S. W., Morimoto, M. and Nordmann, R. (1997) Effect of chronic ethanol feeding on lipid peroxidation and protein oxidation in relation to liver pathology. Hepatology 25, 351–355.[ISI][Medline]

Seglen, P. O. (1976). Preparation of isolated rat liver cells. Methods in Cell Biology 13, 29–83.[Medline]

Sato, C., Matsuda, Y. and Lieber, C. S. (1981) Increased hepatotoxicity of acetaminophen after chronic ethanol consumption in the rat. Gastroenterology 80, 140–148.[ISI][Medline]

Taffs, R. and Sitkovsky, M. (1991) In vitro assays for mouse B and T lymphocyte function. In Current Protocols in Immunology, Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M. and Strober, W., eds, pp. 3.16.1–8. Greene Publishing, Wiley Interscience, New York.

Tredger, J. M., Smith, H. M., Read, R. B., Portmann, B. and Williams, R. (1985) Effects of ethanol ingestion on the hepatotoxicity and metabolism of paracetamol in mice. Toxicology 36, 341–352.[CrossRef][ISI][Medline]

Wasowicz, W., Nève, J. and Peretz, A. (1993) Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum. Importance of extraction pH and influence of sample preservation and storage. Clinical Chemistry 39, 2522–2526.[Abstract/Free Full Text]

Xia, J., Allenbrand, B. and Sun, G. Y. (1998) Dietary supplementation of grape polyphenols and chronic ethanol administration on LDL oxidation and platelet function in rats. Life Sciences 63, 383–390.[CrossRef][ISI][Medline]





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