Department of Liver and Pancreatic Diseases and
1 Department Laboratory Medicine, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgium
(Received 30 December 2002; first review notified 21 March 2003; in revised form 2 April 2003; accepted 16 April 2003)
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ABSTRACT |
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INTRODUCTION |
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It has been suggested that SDH might also be beneficial in alcohol intoxication (Varga et al., 1991). Ethanol is predominantly metabolized in the liver by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (Castaneda and Kinne, 2000
). At low doses of ethanol, the cytochrome P450 2E1 isoenzyme, previously referred to as microsomal ethanol-oxidizing system or MEOS, is in general not important (Lieber, 1994
), but is induced by ethanol at high concentrations (Roberts et al., 1995
). Via the cytochrome P450 2E1-pathway, ethanol is involved in the generation of reactive oxygen species. In addition, ethanol accounts for increased acetaldehyde production. Both factors are thought to be responsible for most of the increased hepatotoxicity of ethanol at higher doses (Lieber, 1993
; Kurose et al., 1997
). Beneficial effects of SDH towards alcohol toxicity have been investigated, in vivo, in a rat model (Valenzuela et al., 1989
; Comoglio et al., 1995
) and in vitro by using isolated rat hepatocytes (Corrazi et al., 1982
; Carini et al., 1992
). However, data from in vitro studies using human hepatocytes are sparse. In the present study we examined, for the first time, cell injury induced by repeated exposure to ethanol (over a period of 14 days) of primary human hepatocytes. We monitored cell viability and the release into the medium of cellular enzymes, and we investigated whether SDH could protect liver cells against the cytotoxic effects of ethanol.
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MATERIALS AND METHODS |
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Experimental procedure
Legalon®Sil was obtained from MADAUS AG (Cologne, Germany). One vial Legalon®Sil contained 528.5 mg of silibinin-C-2',3'-dihydrogensuccinate disodium salt (SDH) (corresponding to 350 mg silibinin). SDH was dissolved in sterile phosphate-buffered saline (PBS) at a concentration of 2 mM.
On day -2, cells were isolated and seeded in 75-cm2 culture flasks. After 1824 h the cells were washed with PBS to remove dead and non-attached cells and the medium was refreshed. On day 0, the medium was collected, used for control analysis and replaced with fresh medium containing ethanol (8.5, 17 or 34 mM) or SDH (5, 10 or 20 µM) or a combination of 34 mM ethanol with SDH. In parallel, cells were incubated in WEM-C as controls. On days 3, 5, 7, 10, 12 and 14, the medium was collected and replaced by fresh medium with the same concentration of ethanol and/or SDH. All incubations were carried out in triplicate.
In order to investigate whether pre-incubation of human hepatocytes had any additional effect on their behaviour towards exposure to ethanol, 5, 10 or 20 µM SDH was added to the cells 6 h before ethanol (34 mM) was introduced in the culture. Medium was collected and refreshed as indicated above.
Sample collection and analysis
At the given time-points the medium was collected, centrifuged for 10 min at 3000 r.p.m. (1100 g) at 4°C. Aliquots of the medium were stored at -20°C until further analysis. The release of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and -glutamyl transferase (
-GT) into the culture medium was determined on a Roche/Hitachi 747 analyser using Roche reagents. LDH is expressed as U/l released into the medium in 24 h in a 75-cm2 culture flask (equal to 13 x 106 human hepatocytes). Human albumin and fibrinogen, secreted into the culture medium, were determined by ELISA using specific monoclonal antibodies, as described previously (Fourneau et al., 1997
).
Statistical analysis of results
This was performed using Students t-test.
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RESULTS |
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DISCUSSION |
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Little is known of how primary (human) hepatocytes respond in a more chronic situation of repeated exposure to ethanol. We observed a significant release of LDH into the medium from day 7 onwards (Fig. 1A), when primary human hepatocytes were cultured in a medium containing 34 mM (0.2% vol./vol.) ethanol but not at 8.5 or 17 mM. The LDH in the medium remained elevated up to day 14, although the medium was refreshed regularly. Under these conditions, a slight decrease of albumin and fibrinogen secretion was observed (Fig. 3
). With the diminished availability of human liver tissue for experimental studies, we also tested the human hepatoma variant cell line HepG2-BD5 (van Pelt et al., 2003
). We found that these cells were tolerant to at least 12.75-fold higher doses of ethanol and showed decreased LDH-release upon incubation with ethanol. This makes these cells unsuitable as an alternative to primary cells for this type of research.
In the present study we observed that incubation of primary human hepatocytes with 20 µM SDH completely blocked the release of LDH induced by 34 mM ethanol (Fig. 1B). This could involve the protection by SDH of cell membranes from radical-induced damage, because the LDH release was not related to mortality of the hepatocytes. One argument against ethanol-induced cell death in this experimental setting is that, during the entire experiment, no increase was observed of AST, ALT or
-GT in the medium of cultures with or without ethanol (data not shown). The second argument is that protein synthesis by the hepatocytes, at the end of the experiment (as a measure for the number of cells), was not significantly different for the various medium combinations with ethanol and SDH when compared with cells in the control medium (Fig. 3
). Therefore, the LDH measured in the medium must have leaked out or been transported from hepatocytes that were still viable. Interestingly, when primary hepatocytes were incubated with 20 µM SDH alone, total protein production was increased by around 30%. This increase was significant for fibrinogen but not for albumin (Fig. 3
). Preincubation of the cells for 6 h with SDH prior to ethanol addition, moved forward the protective effect on the LDH release from day 7 to day 3 for a dose of either 5 or 10 µM SDH. At a dose of 20 µM SDH, preincubation of the cells did not have an additional protective effect on LDH release (Figs 1B
and 2
).
We can conclude from this study that SDH may have a beneficial effect on human hepatocytes when they are exposed to ethanol. In addition, the effect of SDH on protein synthesis in the absence of ethanol suggests a stimulating effect of this compound on cells. Whether these in vitro observations also have clinical significance remains to be clarified. The mechanism by which SDH might have a protective effect on the liver could involve multiple biochemical events (Valenzuela and Garrido, 1994; Wellington and Jarvis, 2001
). This may explain the conflicting results obtained from in vitro and in vivo (patient) studies. Carefully designed in vitro studies using primary (human) hepatocytes are required to evaluate further the role of SDH at different levels, including ethanol transport, metabolism, toxicity and oxidative stress.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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