1. Volume 124, Issue 3, Pages 708-724 (March 2003)
Acetaldehyde impairs mitochondrial glutathione transport in HepG2 cells through endoplasmic reticulum stress 
Josep M. Lluis, Anna Colell, Carmen García–Ruiz, Neil Kaplowitz, José C. Fernández–Checa 
Gastroenterology 
Volume 124, Issue 3, Pages (March 2003)
DOI: /gast
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2. Fig. 1
Scanning electron micrographs of HepG2 cells. HepG2 cells were cultured in the (A) absence or (B and C) presence of acetaldehyde (100 μmol/L) for 3 days and then processed for electron microscopy as described in Materials and Methods. m, mitochondria; L, lipid droplets. (C) Acetaldehyde-treated HepG2 cells show altered mitochondria appearance (arrows). Bars = 0.5 μm.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Electron microscopic analyses of mitochondria from HepG2 cells. Representative electron micrographs of final mitochondrial fraction prepared from control or acetaldehyde-treated HepG2 cells are shown. Enlarged mitochondria are observed in acetaldehyde-treated cells. Bar = 0.5 μm.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Regulation of cytosolic GSH and mGSH by acetaldehyde. HepG2 cells cultured (A and B) in the absence or presence of acetaldehyde (30–100 μmol/L) or (C and D) for various periods of time with 100 μmol/L acetaldehyde were fractionated into (A and C) cytosol or (B and D) mitochondria for measurement of GSH in either fraction. To determine whether acetaldehyde stimulates peroxide levels, cells were incubated with 2'-7'-dichlorofluorescein diacetate for 1 hour after the end of the incubation period with acetaldehyde and dichlorofluorescin fluorescence was determined in a fluorometer (E). Alternatively, cells were treated with antimycin A (AA, 20 μmol/L) for 2 hours and then incubated with 2'-7'-dichlorofluorescein diacetate for an additional hour for dichlorofluorescin fluorescence determination (E). Results are the mean ± SD of n = 5 individual experiments. *P < 0.05 vs. control in the absence of acetaldehyde.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Kinetics of GSH transport in isolated mitochondria from HepG2 cells. Mitochondria from control (○) or 100 μmol/L acetaldehyde (■) for 3 days were prepared and used for GSH transport in the presence of ATP (3 mmol/L). The rate of transport of GSH was linear up to 1 minute, and the rate of transport at 30 seconds was plotted as a function of the substrate. Kinetic parameters were determined by nonlinear least-squares fit to the data by sum of 2 Michaelis–Menten functions. The Vmax and Michaelis constant parameters of both the high- and low-affinity components are summarized in the table. The Vmax is expressed as nmol GSH · mg protein−1 · 30 seconds−1 and Michaelis constant as mmol/L. Data were generated from n = 5 individual mitochondrial preparations per condition. The inset shows the initial rate of GSH transport expressed as nmol · mg protein−1 · 30 seconds−1 into mitochondria from control cells in the presence or absence of ATP. *P < 0.05 vs. control.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Steady-state fluorescence anisotropy and lipid composition of mitochondria from acetaldehyde-treated HepG2 cells. (A) Mitochondrial preparation from control (□) or acetaldehyde-exposed (■) (100 μmol/L for 3 days) HepG2 cells were labeled with different fluorescent probes, and fluorescence anisotropy was determined as described in Materials and Methods. (B) Mitochondrial lipid extracts were prepared as described in Materials and Methods to determine the levels of triglycerides, phospholipids, and total cholesterol. Results are the mean ± SD of n = 5 individual experiments. *P < 0.05 vs. control.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Fatty acid composition of mitochondrial phospholipids. Mitochondrial lipids from control (■) or acetaldehyde-treated cells (□) were extracted and fractionated into phosphatidylcholine (PC), phosphatidylethanolamine (PE), cardiolipin (Card), and phosphatidylinositol (PI) by thin-layer chromatography. Individual phospholipid classes were scraped from plates, and fatty acid methyl esters were resolved by gas chromatography. Results are the mean ± SD of 3 individual experiments.
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Order parameter and initial transport rate of GSH in isolated mitochondria from HepG2 cells. HepG2 cells were treated with acetaldehyde (100 μmol/L for 3 days) and mitochondria fraction was used for order parameter probed by (A) DPH or initial rate of GSH transport at (B) 1 and (C) 10 mmol/L. Results are the mean ± SD of n = 5 individual experiments. *P < 0.05 vs. control; &P < 0.05 vs. acetaldehyde.
Gastroenterology  , DOI: ( /gast )
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9. Fig. 8
Cholesterol enrichment of mitochondria from rat liver. Rat liver mitochondria were incubated with 100 μL of cholesterol/bovine serum albumin complex (CH) (300–320 mg/mL fatty acid free bovine serum albumin plus 4.4 mg cholesterol/mL) for 1 minute at 4°C or bovine serum albumin alone (control). After washing and centrifugation, mitochondria were used for (A) cholesterol determination or (B) order parameter. Results are the mean ± SD of n = 4 individual experiments. *P < 0.05 vs. control.
Gastroenterology  , DOI: ( /gast )
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10. Fig. 9
Initial rate of GSH or ADP transport in cholesterol-enriched mitochondria. Cholesterol-enriched mitochondria were prepared as indicated in the legend to Figure 8 and used for the initial rate of (A) GSH transport or (B) ADP as described in Materials and Methods. Results are the mean ± SD of n = 4 individual experiments. *P < 0.05 vs. control.
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11. Fig. 10
Cholesterol content in intact mitochondria and mitoplasts. ■, Distribution of cholesterol in mitochondria and mitoplasts from (A) cholesterol-enriched rat liver mitochondria or (B) acetaldehyde-treated HepG2 cells. □, Data from (A) control rat liver mitochondria or (B) mitochondria from HepG2 cells. Results are the mean ± SD of 3 individual experiments. *P < 0.05 vs. corresponding controls.
Gastroenterology  , DOI: ( /gast )
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12. Fig. 11
Effect of disulfiram on mGSH and cholesterol content in acetaldehyde-treated HepG2 cells. Mitochondrial fraction from HepG2 cells (control) or HepG2 cells treated with acetaldehyde (100 μmol/L for 3 days) with or without the continued presence of disulfiram (100 μmol/L, 6-hour preincubation before addition of acetaldehyde) was prepared as indicated in Materials and Methods and used for determination of (A) GSH or (B) total cholesterol. Results are the mean ± SD of n = 4 individual experiments. *P < 0.05 vs. control.
Gastroenterology  , DOI: ( /gast )
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13. Fig. 12
Expression of SREBP-1 and GADD153 by acetaldehyde in HepG2 cells. Cellular extracts from HepG2 cells after treatment with acetaldehyde (100 μmol/L for 3 days) were used for immunoblot analyses of (A) SREBP-1 and (B) GADD153. Additional incubations included the presence of 5 mmol/L homocysteine for 5 hours and 10 μg/mL tunicamycin for 8 hours. (C) HepG2 cells after acetaldehyde (100 μmol/L) incubation for 1–3 days were used for lipid extraction to determine the total cholesterol and the data expressed as percentage of control (no acetaldehyde). (D) HepG2 cells were incubated with acetaldehyde (100 μmol/L for 5 days) with or without the presence of lovastatin (1 μmol/L) and the level of cholesterol in mitochondrial fraction determined. Results are the mean ± SD of 4–5 individual experiments. *P < 0.05 vs. control; &P < 0.05 vs. acetaldehyde.
Gastroenterology  , DOI: ( /gast )
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14. Fig. 13
Survival of HepG2 cells to TNF exposure after pretreatment with acetaldehyde. HepG2 cells were pretreated with acetaldehyde (100 μmol/L) for various periods and then left untreated or treated with TNF (280 ng/mL) for an additional 48 hours. Cell survival was determined by MTT assay. Alternatively, cells were treated with CsA (2 μmol/L for 1 hour) or GSH-EE (2 mmol/L for 30 minutes) after the acetaldehyde preincubation period before addition of TNF. Furthermore, lovastatin (1 μmol/L) was added at day 2 after treatment with acetaldehyde and left for the next 3 days before addition of TNF. Results are the mean ± SD of n = 4 individual experiments. *P < 0.05 vs. control in the absence of TNF.
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions