DLPC ATTENUATES ALCOHOL-INDUCED CYTOTOXICITY IN HEPG2 CELLS EXPRESSING CYP2E1

YOUQING XU1, MARIA A. LEO2 and CHARLES S. LIEBER2,*

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)


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aims: Alcoholic liver injury was shown to result largely from oxidative stress generated by ethanol metabolism via cytochrome P4502E1 (CYP2E1). Our aim was to determine whether this could be overcome by using dilinoleoylphosphatidylcholine (DLPC), an innocuous antioxidant extracted from soybeans. Methods: To address this question, we determined whether DLPC protects against alcohol-induced cytotoxicity in HepG2 cells expressing CYP2E1. A HepG2 subclone (2E1) expressing CYP2E1 and a control subclone (Neo) were exposed for 2 h to DLPC (10 µM), and then 100 mM ethanol was added for 5 days. Results: Ethanol significantly decreased cell viability in the 2E1 cells and increased apoptosis. These alterations were attenuated by DLPC with the most significant effects in the 2E1 cells. This was accompanied by a reduction of the ethanol-induced oxidative stress, including diminished hydrogen peroxide production in the 2E1, but not in the Neo cells. The mitochondrial membrane potential was significantly diminished by ethanol in both cells. It was also improved after adding DLPC, but only in the 2E1 cells. In these cells, mitochondrial glutathione (GSH) was also partially restored by DLPC, which significantly inhibited the CYP2E1 induction by ethanol. Conclusion: DLPC opposes the cytotoxicity induced by alcohol in HepG2 cells expressing CYP2E1, a protective action due, at least in part, to an attenuation of the alcohol-induced oxidative stress and the alteration in the mitochondrial membrane potential. On account of these beneficial effects of DLPC and its innocuity, it is now germane to assess its therapeutic action in alcoholics.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alcoholic liver injury results largely from oxidative stress generated by ethanol metabolism via cytochrome P4502E1 (CYP2E1) (Lieber, 1997Go, 1999Go). This oxidative stress is caused by the generation of reactive oxygen species during alcohol metabolism. The consequences of the oxidative stress, such as lipid peroxidation (Lieber, 1997Go, 1999Go) and decreased glutathione (GSH), particularly mitochondrial GSH (Hirano, 1992Go), play an important role in alcohol-induced liver damage. CYP2E1 inhibitors were found to oppose this effect of ethanol but the compounds tested so far have insufficient inhibitory activity or intrinsic toxicity, which precludes their chronic use in humans (Lieber, 1997Go, 1999Go). In contrast, dilinoleoylphosphatidylcholine (DLPC), the main and active component of polyenylphosphatidylcholine extracted from soybeans, attenuated the apoptosis of hepatocytes induced by ethanol feeding in rats (Mak et al., 2003Go). It also exhibited a remarkable specificity in its antioxidant and cytoprotective properties (Aleynik et al., 1999Go). In the present study, our aim was to determine to what extent DLPC has a protective effect on the alcohol-induced CYP2E1-mediated cytotoxicity by assessing its action in the HepG2 cells expressing CYP2E1.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Ethanol (95%) was purchased from Pharmco Products Inc. (Brookfield, CT) and 2',7'-dichlorofluorescein diacetate (DCF-DA) from Molecular Probes, Inc. (Eugene, OR). DLPC was obtained from Avanti Polar Lipid Inc. (Alabaster, AL). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was purchased from Sigma Chemical Co. (St Louis, MO) and DCF-DA was purchased from Molecular Probes, Inc. (Eugene, OR).

Cell culture and treatment
Two human hepatoma HepG2 cell lines, described in Xu et al. (2003)Go, 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., 1999Go), ~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., 1988Go). 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., 1998Go). 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)Go 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., 2004Go).

Statistical analysis
Data (n = 6 for each parameter) were expressed as mean ± SEM and analyzed by one-way ANOVA, followed by post-hoc Student–Newman–Keuls tests for multiple comparisons between treatment groups; P < 0.05 was considered to be significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In preliminary experiments, the Neo and 2E1 cells were cultured for 5 days in a medium containing 10 µM DLPC or an equal volume of vehicle (control). At the end of the treatment, cell viability was determined by the MTT assay. As expected, there was no significant difference among the various groups, and DLPC was not toxic in either the Neo or 2E1 cells. However, ethanol significantly decreased cell viability in both cells, but more in the 2E1 ones, and DLPC corrected the ethanol-induced effect (Fig. 1).



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Fig. 1. Effect of ethanol and DLPC (dilinoleoylphosphatidylcholine) on cell viability. Ethanol decreased cell viability, particularly in 2E1 cells (P < 0.01 vs Neo cells); DLPC significantly opposed the effect of ethanol in 2E1 cells (P < 0.01), ethanol vs ethanol + DLPC. *P < 0.05, **P < 0.01 ethanol vs control in Neo and 2E1 cells, respectively.

 
The decreased viability was paralleled by increased apoptosis, again with a greater effect in 2E1 cells, and DLPC attenuated this toxic effect of ethanol (Fig. 2). Since one of the mechanisms of apoptosis is an alteration of the mitochondria, with a specific involvement of mitochondrial membranes, this parameter was measured in the two cell lines and documented by the retention of the DiOC6 dye. Ethanol decreased the membrane potential, particularly in the 2E1 cells, and DLPC opposed this effect (Fig. 3).



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Fig. 2. Effect of DLPC on ethanol-induced apoptosis. Ethanol significantly increased apoptosis in both Neo (**P < 0.01) and 2E1 cells (***P < 0.001), but more in the 2E1 cells (P < 0.05); DLPC attenuated this ethanol-induced effect in the 2E1 cells (*P < 0.05).

 


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Fig. 3. Effect of DLPC on mitochondrial membrane potential. This parameter was significantly affected by ethanol in Neo and, particularly, in the 2E1 cells (***P < 0.001), with a significant difference between the two cell types (P < 0.05); DLPC corrected the ethanol-induced effect in the 2E1 cells (**P < 0.01).

 
One of the mechanisms of ethanol toxicity is also its increase in the production of hydrogen peroxide. This action was greatest in the 2E1 cells and DLPC reduced this ethanol effect (Fig. 4). When GSH was measured in the homogenate of the two cell lines, no difference was found, but in the mitochondrial fraction, GSH was significantly reduced by alcohol in both cell types, with a more prominent effect in those transfected with CYP2E1. DLPC opposed this alcohol effect but only in the 2E1 cells (Fig. 5).



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Fig. 4. Effect of DLPC on hydrogen peroxide (measured by DCF-DA). Compared with controls, ethanol favored the production of hydrogen peroxide in both Neo (**P < 0.01) and 2E1 cells (***P < 0.001), with a significantly greater effect in the 2E1 cells (P < 0.05); DLPC corrected, in part, the production due to ethanol in the 2E1 cells (**P < 0.01).

 


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Fig. 5. Effect of DLPC on GSH (glutathione). No significant effect was observed in the hGSH (homogenate GSH), whereas in the mGSH (mitochondrial GSH) fraction, ethanol significantly reduced GSH in both cells (***P < 0.001); this decrease was greater in the 2E1 cells (P < 0.01), and DLPC corrected the ethanol-induced reduction in these cells (**P < 0.01).

 
Finally, as expected, ethanol increased CYP2E1 protein (determined by western blot), whereas DLPC abolished this induction (Fig. 6).



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Fig. 6. Effect of DLPC on CYP2E1 in 2E1 Cells. Ethanol significantly increased CYP2E1, as shown by western blot (*P < 0.05), and DLPC prevented this induction (P < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study revealed that DLPC is protective against the toxicity of the oxidative stress generated by ethanol in HepG2 cells transfected with CYP2E1. The mechanism of the DLPC effect involves specifically the CYP2E1 induction by ethanol, which is suggested by the fact that DLPC was mostly active in the 2E1 cells. The beneficial effects of DLPC reported in the present study extend other favorable effects of DLPC against the toxicity of ethanol, revealed in several prior studies both in vivo (Navder and Lieber, 2002Go) and also in vitro in other hepatic cells or cell lines (Cao et al., 2002aGo,bGo,cGo,dGo), as well as in human lipoproteins (Navder et al., 2000Go). These favorable effects of DLPC were secondary, at least in part, to its antioxidant properties and its attenuation of the ethanol-induced oxidative stress resulting from CYP2E1 induction, a major mechanism of liver injury (Lieber, 2004Go). However, the mechanism of prevention by DLPC of the ethanol-associated CYP2E1 induction has not yet been elucidated. In any event, DLPC is also a physiological phospholipid and it is a major constituent of membranes and other tissue components (Lieber et al., 1994Go). Its depletion was observed in the livers of baboons fed with alcohol chronically and its repletion was beneficial (Lieber et al., 1994Go).

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.


    ACKNOWLEDGEMENTS
 
These studies were supported, in part, by NIH grant AA1115, the Department of Veterans Affairs, the Kingsbridge Research Foundation and the Christopher D. Smithers Foundation. The Investigators are grateful to Mr A. Ponomarenko for his graphic contributions and Ms Y. Rodriguez for her secretarial assistance.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aleynik, S. I., Leo, M. A., Takeshige, U. et al. (1999) Dilinoleoylphosphatidylcholine is the active antioxidant of polyenylphosphatidylcholine. Journal of Investigative Medicine 47, 507–512.[ISI][Medline]

Cao, Q., Mak, K. M. and Lieber, C. S. (2002a) Dilinoleoylphosphatidylcholine prevents transforming growth factor-ß1-mediated collagen accumulation in cultured rat hepatic stellate cells. Journal of Laboratory Clinical Medicine 139, 202–210.[CrossRef][ISI]

Cao, Q., Mak, K. M. and Lieber, C. S. (2002b) Dilinoleoylphosphatidylcholine decreases LPS-induced TNF-{alpha} generation in Kupffer cells of ethanol-fed rats: respective roles of MAPKs and NF-{kappa}B. Biochemical Biophysical Research Communication 294, 849–853.[CrossRef][ISI]

Cao, Q., Mak, K. M. and Lieber, C. S. (2002c) DLPC decreases TGF-ß1-induced collagen mRNA by inhibiting p38 MAPK in hepatic stellate cells. American Journal of Physiology Gastrointestinal Liver Physiology 283, G1051–G1061.[ISI]

Cao, Q., Mak, K. M. and Lieber, C. S. (2002d) Dilinoleoylphosphatidylcholine decreases acetaldehyde-induced TNF-{alpha} generation in Kupffer cells of ethanol-fed rats. Biochemical Biophysical Research Communication 299, 459–464.[CrossRef][ISI]

Colell, A., Garcia-Ruiz, C., Miranda, M. et al. (1998) Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor. Gastroenterology 115, 1541–1551.[ISI][Medline]

Hirano, T., Kaplowitz, N., Tsukamoto, H. et al. (1992) Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats. Hepatology 16, 1423–1427.[ISI][Medline]

Lieber, C. S. (1997) Cytochrome P4502E1: its physiological and pathological role. Physiology Review 77, 517–544.[Abstract/Free Full Text]

Lieber, C. S. (1999) Microsomal ethanol-oxidizing system (MEOS), the first 30 years (1968–1998)—a review. Alcohol: Clinical Experimental Research 23, 991–1007.[ISI][Medline]

Lieber, C. S. (2004) CYP2E1: From ASH to NASH. Hepatology Research 28, 1–11.[CrossRef][ISI][Medline]

Lieber, C. S., Robins, S. J., Li, J. et al. (1994) Phosphatidylcholine protects against fibrosis and cirrhosis in the baboon. Gastroenterology 106, 152–159.[ISI][Medline]

Lieber, C. S., Leo, M. A., Mak, K. M. et al. (2004) Model of nonalcoholic steatohepatitis. American Journal of Clinical Nutrition 79, 502–509.[Abstract/Free Full Text]

Mak, K. M., Wen, K., Ren, C. et al. (2003) Dilinoleoylphosphatidylcholine reproduces the antiapoptitic actions of polyenylphosphatidylcholine against ethanol-induced hepatocyte apoptosis. Alcohol: Clinical Experimental Research 27, 997–1005.[ISI][Medline]

Navder, K. P. and Lieber, C. S. (2002) Dilinoleoylphosphatidylcholine is responsible for the beneficial effects of polyenylphosphatidylcholine on ethanol-induced mitochondrial injury in rats. Biochemical Biophysical Research Communication 291, 1109–1112.[CrossRef][ISI]

Navder, K. P., Baraona, E. and Lieber, C. S. (2000) Dilinoleoylphosphatidylcholine protects human low density lipoproteins against oxidation. Atherosclerosis 152, 89–95.[CrossRef][ISI][Medline]

Pastorino, J. G. and Hoek, J. B. (2000) Ethanol potentiates tumor necrosis factor-{alpha} cytotoxicity in hepatoma cells and primary rat hepatocytes by promoting induction of the mitochondrial permeability transition. Hepatology 31, 1141–1152.[CrossRef][ISI][Medline]

Scudiero, D. A., Shoemaker, R. H., Paul, K. D. et al. (1988) Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research 48, 4827–4833.[Abstract]

Xu, Y., Leo, M. A. and Lieber, C. S. (2003) Lycopene attenuates arachidonic acid toxicity in HepG2 cells overexpressing CYP2E1. Biochemical Biophysical Research Communication 303, 745–750.[CrossRef][ISI]





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