1 Liver Research Center, Beijing Friendship Hospital, Beijing, 100050, China and 2 Section of Liver Disease and Nutrition, Alcohol Research and Treatment Center, Veterans Affairs Medical Center & Mt Sinai School of Medicine, 130 West Kingsbridge Road, Bronx, NY10468, USA
* Author to whom correspondence should be addressed: Tel.: +1 718 741 4244; Fax: +1 718 733 6257; E-mail: liebercs{at}aol.com
(Received 10 January 2005; accepted 3 February 2005; Advance Access publication 7 March 2005)
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ABSTRACT |
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INTRODUCTION |
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MATERIALS AND METHODS |
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Cell culture and treatment
Two human hepatoma HepG2 cell lines, described in Xu et al. (2003), were used: the 2E1 cells, which are HepG2 cells transfected with human CYP2E1 cDNA (provided by Dr F. L. Gonzalez, NCI, Bethesda, MD), and the Neo cells, which are transfected with the empty pCI vector. All cell lines were grown in complete minimum essential medium containing 10% fetal bovine serum, a 1% penicillin/streptomycin mixture and 0.5 mg/ml of G418 (Life Technologies, Grand Island, NY) in a humidified atmosphere of 95% air with 5% CO2 at 37°C.
Neo and 2E1 cells were incubated with 100 mM ethanol for 5 days at 37°C; 100 mM ethanol for 5 days was selected to mimic some chronic effects of ethanol. The cells were treated with, or without, DLPC suspended in bovine serum albumin (BSA) 2 h before the addition of ethanol (100 mM). Dishes or plates were kept air tight with parafilm to minimize the evaporation of ethanol. The ethanol concentration was stable for 5 days as detected by gas chromatography (GC-8500).
As previously described (Aleynik et al., 1999),
2 x 104 cells were plated onto 24-well plates and after the respective treatments, the medium was removed, and cell viability was evaluated by the MTT assay (Scudiero et al., 1988
). After a 2 h incubation at 37°C with MTT, the medium was removed and 0.5 ml of 100% dimethyl sulfoxide was added to each well to solubilize the blue formazan. The absorbance of the converted dye was measured at a wavelength of 570 nm with a background subtraction at 630 nm. Viability was expressed as percentage of control.
ELISA apoptosis detection
Apoptosis assay was performed using an ELISA apoptosis detection kit AK-120 (Biomol Research Laboratories, Inc., Plymouth Meeting, PA). A total of 5000 cells per well was seeded in a 96-well microplate. The cells were treated with DLPC and ethanol, then fixed and stained for apoptosis cells, according to the manufacturer's protocol.
Determination of hydrogen peroxide
Production of reactive oxygen species, mainly hydrogen peroxide and other organic peroxides, was monitored spectrofluorometrically by the DCF-DA method (Colell et al., 1998). DCF-DA was added to the culture plates at a final concentration of 20 µl/l, and incubated for 30 min at 37°C in darkness. The cells were washed and harvested, and read immediately in a fluorescence spectrophotometer at 488 nm for excitation and at 525 nm for emission. Results were expressed as percentage of control.
Preparation of cell homogenate
Cells (1 x 106) were cultured in 100-mm culture dishes. After treatment with DLPC and ethanol, they were scraped with a rubber policeman, collected and homogenized in 0.5 ml cold 2-(N-morpholino)ethanesulphonic acid (MES) buffer, centrifuged at 10 000 g for 15 min at 4°C, and the supernatant was saved for the GSH assay.
Preparation of mitochondria
Cells (1 x 106) were cultured in 100-mm culture dishes. After treatment with DLPC and ethanol, they were washed twice with phosphate-buffered saline (PBS), scraped from the dishes, and suspended in 1 ml cold isolation buffer (0.25 M sucrose, 10 mM Tris and 1 mM EDTA, pH 7.4). Cells were sonicated at 4°C. The homogenate was centrifuged at 3000 r.p.m. for 5 min at 4°C. The supernatants were centrifuged at 8500 r.p.m. for 10 min at 4°C. The mitochondrial pellet was washed in 1 ml isolation buffer with 0.5% BSA and then resuspended in 0.5 ml cold MES buffer for GSH assay.
GSH assay
Cell homogenate GSH and mitochondrial GSH were determined with the glutathione assay kit (Cayman Chemical, Ann Arbor, MI), according to the manufacturer's specifications.
Measurement of mitochondrial energization
Mitochondrial energization was determined according to Pastorino and Hoek (2000) as the retention of the dye 3,3'-dihexyloxacarbocyanine (DiOC6) (Molecular Probes Inc., Eugene, OR). Cells were loaded with 100 nmol/l of DiOC6 during the last 30 min of treatment. The cells were then washed twice in PBS. The level of retained DiOC6 was measured on a fluorescence plate reader at 488 nm for excitation and at 500 nm for emission.
Measurement of CYP2E1
CYP2E1 was determined by western blot in the 2E1 cells as described previously (Lieber et al., 2004).
Statistical analysis
Data (n = 6 for each parameter) were expressed as mean ± SEM and analyzed by one-way ANOVA, followed by post-hoc StudentNewmanKeuls tests for multiple comparisons between treatment groups; P < 0.05 was considered to be significant.
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RESULTS |
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DISCUSSION |
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The combination of innocuity and high efficacy against several toxic manifestations of ethanol makes DLPC an attractive candidate for therapeutic trials in humans. Furthermore, the preparation used in the present study is now available for human consumption. For these reasons, it would be of interest to assess the beneficial effects of DLPC in patients with alcoholic liver disease.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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