PRIMARY HUMAN HEPATOCYTES ARE PROTECTED AGAINST PROLONGED AND REPEATED EXPOSURE TO ETHANOL BY SILIBININ-DIHEMISUCCINATE

Alcohol & Alcoholism Vol. 38, No. 5, pp. 411–414, 2003 doi:10.1093/alcalc/agg099, available online at www.alcalc.oupjournals.org PRIMARY HUMAN HEPATOCYTES ARE PROTECTED AGAINST PROLONGED AND REPEATED EXPOSURE TO ETHANOL BY SILIBININ-DIHEMISUCCINATE JOS F. VAN PELT*, CHRIS VERSLYPE, TINA CRABBÉ, ZAHUR ZAMAN1 and JOHAN FEVERY Department of Liver and Pancreatic Diseases and 1Department 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) Abstract — Aims and methods: We investigated the effect of silibinin-C-2′,3′-dihydrogensuccinate (SDH) on primary human hepatocytes when exposed to ethanol for 14 days. At regular intervals, the medium was refreshed and liver enzymes and secreted protein in the medium were determined. Results: The ethanol-induced release of lactate dehydrogenase (at 34 mM ethanol) was completely blocked by 20 µM SDH. SDH itself stimulated fibrinogen release and had no toxic effect. Conclusions: We can conclude that SDH has a beneficial effect on human hepatocytes when exposed to ethanol in vitro. 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. INTRODUCTION Silymarin is a flavonoid extracted from the milk thistle Silibum marianum. The biologically active isomer has been identified as silibinin-C-2′,3′-dihydrogensuccinate (SDH) and is commercially available under the name Legalon®. It has been used in the treatment of acute poisoning by the mushroom Amanita phalloides (Trost and Halbach, 1978; Hruby et al., 1983), and a variety of liver diseases, mainly in Central and Southern Europe (Ferenci et al., 1989). Although the hepatoprotective properties of SDH and its derivatives have been reported both from in vitro and in vivo studies (Salmi and Sarna, 1982; Varga et al., 1991; Carini et al., 1992; Pares et al., 1998), its mechanism of action still has not been established. Different mechanisms have been proposed. Silymarin (or its derivatives) might function as an antioxidative-scavenger of free radicals (mainly active towards HO and HOCl and less so for H2O2 or O2–); it might function as a regulator of membrane permeability and stability or of transport across the membrane; or it might function as a stimulator of polymerase I and rRNA transcription, leading to increased rRNA synthesis (Valenzuela and Garrido, 1994; Wellington and Jarvis, 2001). 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 ethanoloxidizing 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 MATERIALS AND METHODS Isolation and culture of primary human hepatocytes Human hepatocytes were isolated from liver obtained from post-mortem organ donors when the tissue could not be used for transplantation. For these experiments, permission from the ‘Medical Ethical Committee for experimentation using human tissues with informed consent of the family’ had been obtained. A two-step collagenase (Roche; Mannheim, Germany) perfusion method was used as described previously (Rijntjes et al., 1986). Donor 1 was an 18-year-old male, cells after isolation had a viability of 80% as determined by the trypan blue exclusion test. Donor 2 was a 30-year-old male, the cells after isolation had a viability of 79%. Both donors died as a result of trauma and had no history of ethanol misuse. The cells were seeded at a density of 175 × 103 viable cells per cm2 in plastic culture flasks coated with human liver bio-matrix (Rijntjes et al., 1986). Cells were cultured in Williams’ E medium supplemented with: 10% fetal calf serum, 2 mM L-glutamine, 50 nM dexamethasone, 0.02 U/ml insulin, 2.5 mg/ml fungizone, 100 mg/ml vancomycin, 50 mg/ml gentamycin, 100 mg/ml streptomycin, and 100 U/ml penicillin (WEM-C). Two days after cell seeding, cells were incubated as described in ‘Experimental procedure’. 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. *Author to whom correspondence should be addressed at: Laboratory Hepatology, Department of Liver and Pancreatic Diseases, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium, tel.: +32-16–345835, fax: +32-16–345846, E-mail address: 411 Alcohol & Alcoholism 38 © Medical Council on Alcohol 2003; all rights reserved 412 J. F. VAN PELT et al. On day –2, cells were isolated and seeded in 75-cm2 culture flasks. After 18–24 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. A 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 relea (...truncated)