Animals and ethanol treatment
. SV/129-background CYP2E1 knockout (Cyp2e1
) (Lee et al., 1996
) and humanized CYP2E1 transgenic knockin mice (Cyp2e1
) (Cheung et al., 2005
) were kindly provided by Dr Frank J. Gonzalez (Laboratory of Metabolism, National Cancer Institute, Bethesda, MD). Colonies of these mice were established at Mount Sinai, and the offspring of these mating pairs were used in this study. The SV129 wild-type (WT) mice were purchased from Charles River Laboratory. The C57BL/J6 background CYP2A5 knockout (Cyp2a5
) mouse colony was established at Mount Sinai by rederivation from male Cyp2a5
mice (Zhou et al., 2010
) (kindly provided by Dr Xinxin Ding, Wadsworth Center, New York State Department of Health, Albany, NY) and female C57BL/J6 WT mice (purchased from Charles River Laboratory). All mice were housed in temperature-controlled animal facilities with 12-h light/12-h dark cycles and were permitted consumption of tap water and Purina standard chow ad libitum
until being fed the liquid diets. The mice received humane care, and experiments were carried out according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals and with approval of the Mount Sinai Animal Care and Use Committee. Liver tissues from WT and Nrf2 knockout (Nrf2
) mice fed dextrose or ethanol were kindly provided by Drs J. Lamlé and A. Vogel (Lamlé et al., 2008
All mice were initially fed the control liquid dextrose diet (Bio-Serv, Frenchtown, NJ) for 3 days to acclimate them to the liquid diet. Then, the mice were fed either the liquid ethanol diet (Bio-Serv) or the control liquid dextrose diet as described by Lieber and DeCarli (1972)
for 3 weeks. The content of ethanol was gradually increased every 3–4 days from 10% (1.77% [vol/vol]) of total calories to 20% (3.54% [vol/vol]), 25% (4.42% [vol/vol]), 30% (5.31% [vol/vol]), and finally 35% of total calories (6.2% [vol/vol]). For experiments involving less than 2 weeks of feeding, the mice were directly subjected to the diet containing ethanol as 35% of total calories. The control mice were pair-fed the control dextrose diet on an isoenergetic basis. For 3-week antioxidant intervention experiments, during the 3 weeks of feeding with ethanol or dextrose liquid diets, NAC (75 mg/kg body weight, ip) and Vc (125 mg/kg body weight, ip) were injected daily into the ethanol-fed SV129 WT mice. For CYP2E1 inhibitor chlormethiazole (CMZ) intervention experiments, during 2 weeks of ethanol feeding, CMZ (50 mg/kg body weight, ip) was injected every other day into the ethanol-fed SV129WT mice (Lu et al., 2008
). For 2-day antioxidant intervention experiments, NAC and Vc were first injected the day before ethanol diet feeding, and then NAC was injected at 150 mg/kg body weight, ip, once a day or Vc was injected at 125 mg/kg body weight, ip, every 12 h. The ethanol-fed mice had access to their rations ad libitum
, and the conditions of WT, knockout, and humanized transgenic mice were comparable. The amount of food consumed by the Cyp2e1
, and Cyp2e1
mice and the SV129 and C57BL/J6 background WT mice was approximately the same.
The mice were sacrificed by cervical dislocation under anesthesia, and the livers were rapidly excised into fragments and washed with cold saline. The liver tissue aliquots were stored at –80°C for further assays. Liver homogenates were prepared in ice-cold 0.15M KCl and stored at –80°C in aliquots. Liver triglycerides (TG) were assayed using commercial available kits (POINTE Scientific Inc., Canton, MI). Liver samples were fixed in 10% formalin solution and embedded in paraffin. Liver sections (5-µm thick) were stained with hematoxylin and eosin for pathological evaluation.
For the binge ethanol model, the mice were gavaged with ethanol once at 6 g/kg body weight (0.2 ml of 38% ethanol (v/v) per 10 g body weight), and the mice were sacrificed 3, 6, 9, and 14 h after the gavage.
Preparation of hepatic microsomes. Hepatic microsomes were prepared by placing liver aliquots in 0.15M KCl and homogenization in a Polytron homogenizer for 10 strokes. The homogenate was centrifuged at 9000 g for 20 min, and then the resulting supernatant fraction was centrifuged further at 105,000 g for 60 min. The resulting pellets (microsomes) were resuspended in 50mM sodium phosphate buffer (pH 7.4). All procedures were carried out on ice.
Isolation of primary hepatocytes
. The hepatocytes were isolated as previously described (Lu et al., 2004
). The cells were seeded in collagen-coated plates. After overnight preculture, the cells were treated with 100mM ethanol for 36 h. The cells were observed under an inverted microscope, photos of the cells were taken, and then the cells were subjected to Oil Red O staining. Some of the cells were collected for Western blotting analysis or TG assay.
. The production of acetaldehyde from ethanol by microsomes was assayed as previously described (Cederbaum and Cohen, 1984
). The ethanol oxidation buffer containing 100mM potassium phosphate buffer, pH 7.4, 25mM ethanol, and 1mM NADPH was supplemented with 1mM sodium azide to inhibit catalase activity and with 0.1mM deferoxamine to inhibit microsomal hydroxyl radical formation because both catalase and hydroxyl radical contribute to ethanol oxidation (Cederbaum, 1991
). Microsomal protein (50 µg) was added to 0.1 ml of the reaction buffer and was incubated for 15 min at 37°C. Reactions were carried out in center-well flasks containing 0.15 ml of 15mM semicarbazide HCl in 180mM potassium phosphate, pH 7.4, in the center well. Reactions were terminated by the addition of trichloroacetic acid (TCA) to a final concentration of 4.5% (w/v). The sealed flasks were incubated overnight at room temperature to allow diffusion of acetaldehyde into the center well. The absorbance of the aldehyde-semicarbazone complex was determined at 224 nm.
Cytochrome P450 2E1 and 2A5 activity
. CYP2E1 activity was measured by the rate of oxidation of 1mM p
-nitrophenol to p
-nitrocatechol by 100 µg of microsomal protein for 15 min at 37°C (Lu and Cederbaum, 2006a
). CYP2A5 activity was measured by assessing COH activity with 100µM coumarin as substrate plus 100 µg of microsomal protein and incubation for 15 min at 37°C (Lu and Cederbaum, 2006a
Measurement of reduced glutathione levels
. Liver homogenate was mixed with TCA to a final concentration of 5%, and the mixture was incubated at 4°C for 30 min to extract glutathione (GSH). The TCA extracts (10 µl) were added to 200 µl of methanol containing 1 mg/ml o
-phthaldehyde and then were incubated for 15 min at 37°C in the dark. Fluorescence was measured at 350/420 nm (excitation/emission). The concentration of GSH was determined from a GSH standard curve (Lu and Cederbaum, 2006b
Determination of thiobarbituric acid reactive substances
. In brief, hepatic homogenates supplemented with butylated hydroxytoluene to prevent further oxidation during assay were incubated with 0.2 ml of TCA [15% (wt/vol)]-TBA [0.375% (wt/vol)]-HCl (0.25 N) solution in a boiling water bath for 10 min. After centrifugation at 524 × g for 5 min, the resulting supernatant was used to determine the formation of thiobarbituric acid reactive substances (TBARS) by evaluating absorbance at 535 nm. TEP (1,1,3,3-tetraethoxypropane) treated as above served as a standard (Lu et al., 2005
Western blotting. Hepatic proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. After 1 h of blocking with 2% fat-free milk, membranes were incubated overnight with anti-CYP2E1 IgG (a gift from Dr Jerome Lasker, Hackensack Biomedical Research Institute, Hackensack, NJ), anti-CYP2A5 IgG (a gift from Dr Risto Juvonen, Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland), and antibodies against Nrf2, lamin, Calnexin, heme oxygenase 1 (HO-1), or β-actin (Santa Cruz) followed by 1-h incubation with peroxidase secondary anti-rabbit, anti-chick, and anti-goat antibodies (Millipore), respectively. Chemiluminescence was detected by Image Reader LAS-4000 (Fujifilm) after adding PierceECL Western Blotting Substrate (Thermo Scientific, Rockford, IL). All specific bands were quantified with the Automated Digitizing System (ImageJ gel programs, version 1.34S; National Institutes of Health, Bethesda, MD).
Transcriptional activity of Nrf2. Nuclear proteins were isolated from frozen liver tissues using the NE-PER nuclear protein extraction kit (Thermo Scientific) according to the manufacturer’s protocol. Nrf2 DNA binding activity was assessed using a TransAM Transcription Factor ELISA kit (Active Motif, catalog no. 50296). This kit contains 96-well plates coated with oligonucleotides containing the antioxidant sensitive response element (ARE) consensus binding site (5′-GTCACAGTGACTCAGCAGAATCTG-3). Nuclear protein (2.5 µg) was added to each well. Nrf2 bound to the imbedded ARE oligonucleotides on the 96-well plates was detected colorimetrically by incubating the plates with a primary antibody against Nrf2 followed by incubating with a secondary antibody conjugated to horseradish peroxidase. Nuclear extracts from COS-7 cells transfected with Nrf2 were used as a positive control for Nrf2 binding activity.
Statistics. Results are expressed as means ± SEMs. Statistical evaluation was carried out by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. p < 0.05 was considered as statistically significant.