1. Volume 115, Issue 6, Pages 1541-1551 (December 1998)
Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor 
Anna Colell, Carmen García-Ruiz, Merce Miranda, Esther Ardite, Montse Marí, Albert Morales, Fernando Corrales, Neil Kaplowitz, José C. Fernández-Checa 
Gastroenterology 
Volume 115, Issue 6, Pages (December 1998)
DOI: /S (98)
Copyright © 1998 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Compartmentation of cellular GSH in hepatocytes from pair-fed and ethanol-fed rats. Freshly isolated hepatocytes from pair- and ethanol-fed rats with or without SAM supplementation in the liquid diet were fractionated as indicated in Materials and Methods to obtain the cytosol and mitochondria. Total GSH in these fractions was determined as described in Materials and Methods. Results are means ± SD of pair-fed (n = 5) and ethanol-fed (n = 6) rats without SAM and for each group with SAM supplementation. *P < 0.05 vs. pair-fed without SAM; **P < 0.05 vs. ethanol-fed without SAM.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Survival and hydrogen peroxide generation in hepatocytes exposed to TNF-α and effect of SAM supplementation. Hepatocytes isolated from pair-fed (○) or ethanol-fed (□) rats with or without SAM supplementation were cultured overnight. ●, Pair-fed + SAM; ■, ethanol-fed + SAM. Cells were then incubated with increasing concentrations of TNF-α (250–10,000 U/mL; 9.2–370 ng/mL) for 12 hours. (A) Cell viability was determined by trypan blue exclusion and GST activity released in extracellular medium. (B) Parallel culture dishes were incubated with DCFDA to determine DCF fluorescence to monitor the levels of hydrogen peroxide generation as described in Materials and Methods. Results are means ± SD of pair-fed (n = 5) and ethanol-fed (n = 6) rats without SAM and for each group with SAM supplementation. Data from the ethanol-fed group were significantly different from pair-fed control and ethanol-fed SAM-supplemented values.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Relationship of survival and hydrogen peroxide generation in hepatocytes exposed to TNF-α. Survival and DCF fluorescence from the indicated groups of rats, determined as described in Figure 2, were plotted, and linear regression was performed by computer analyses. Survival and DCF data for the pair-fed group with and without SAM administration were similar and hence were pooled. Slopes were , , and for pair-fed (○), ethanol and SAM supplementation (■), and ethanol-fed (□), respectively. Results are means ± SD for pair-fed (n = 5) and ethanol-fed (n = 6) rats without SAM and for each group with SAM supplementation.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Effect of antioxidants on TNF-induced oxidative stress. Freshly isolated hepatocytes from pair-fed and ethanol-fed rats were incubated with 50 μmol/L BHT and 50 μmol/L vitamin E or vehicle, dimethyl sulfoxide, or vegetable oil for 30 minutes and then exposed to 10,000 U/mL TNF-α for the indicated time in Fisher's medium in the continued presence of antioxidants. Aliquots of cells were taken to determine DCF fluorescence, and cell survival was assessed by trypan blue exclusion. DCF and viability of pair-fed cells were unchanged with TNF-α exposure in the presence of BHT and vitamin E. Results are means ± SD of 3 cell preparations for each group. *P < 0.05 vs. pair-fed control and ethanol-fed plus BHT and vitamin E.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Morphological appearance and DNA integrity of hepatocytes incubated with TNF-α. (A) Cells from pair-fed and ethanol-fed rats were cultured and incubated with 10,000 U/mL TNF-α (a and b, respectively) for 12 hours and then exposed to Hoechst and observed under a fluorescence microscope to determine chromatin appearance. (B) Parallel aliquots were taken to estimate DNA fragmentation (Mrk., DNA size markers). The positive control for apoptosis (c) shows the appearance of cultured hepatocytes whose total GSH levels were depleted by preincubation with diethyl maleate (0.8 mmol/L for 15 minutes followed by washing to remove excess diethyl maleate) before treatment of cells with 10,000 U/mL TNF-α and stained with the same fluorochrome. cn, condensed nuclei; fn, fragmented nuclei. (Original magnification 200×.)
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Restoration of mitochondrial pool of GSH by GSH-EE. Freshly isolated rat hepatocytes from pair-fed and ethanol-fed rats were fractionated into cytosol and mitochondria for GSH determination as described for Figure 1. Parallel cell suspensions were incubated with GSH-EE at 2 mmol/L for 20–30 minutes and then washed to remove excess ester. Cells were subsequently fractionated to isolate cytosol and mitochondrial fraction by Percoll centrifugation as described in Materials and Methods, and GSH in each compartment was measured. Results are means ± SD of 3 and 4 cell preparations from pair-fed and ethanol-fed rats, respectively. *P < 0.05 vs. pair-fed in the absence of GSH-EE; **P < 0.05 vs. ethanol-fed without GSH-EE; #P < 0.05 vs. pair-fed.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

8. Fig. 7
(A) Survival and (B) hydrogen peroxide generation induced by TNF-α and effect of GSH-EE. Hepatocytes from pair-fed and ethanol-fed rats were incubated in the presence of 10,000 U/mL TNF-α or its vehicle for 4 hours in Fisher's medium. Cell suspensions for each group were incubated with GSH-EE (2 mmol/L) to increase the cellular GSH levels or ethanol (2 mmol/L). Compartmentation of cytosol and mitochondrial GSH after incubation with GSH-EE was determined as in Figure 6. At the end of the incubation period, viability and DCF fluorescence were determined as described in Materials and Methods. Results are means ± SD of 3 and 4 cell preparations from pair-fed and ethanol-fed rats, respectively. *P < 0.05 vs. pair-fed and ethanol-fed groups in the absence of TNF-α; **P < 0.05 vs. ethanol-fed group in the presence of TNF-α.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

9. Fig. 8
TNF-induced NF-κB activation and expression of CINC in hepatocytes. Hepatocytes from each group were cultured and exposed to the indicated dose of TNF-α overnight. (A) Nuclear extracts were isolated and analyzed by electrophoretic mobility shift assay to detect NF-κB activation. (B) Parallel nuclear extracts were incubated with the indicated antibodies against p65 and p50 of NF-κB/rel members and analyzed by electrophoretic mobility shift assay for detection of supershifted bands denoted as SS. (C) Total RNA was isolated and hybridized with CINC cDNA, and its levels were expressed in relationship to 18 S used as reference control. Expression of CINC was quantitated by densitometry. Results for CINC expression are means ± SD of 3 and 4 cell preparations from pair-fed and ethanol-fed rats, respectively. *P < 0.05 vs. pair-fed cells without TNF; **P < 0.05 vs. corresponding pair-fed hepatocytes incubated with TNF-α.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

10. Fig. 9
Effect of SAM supplementation on NF-κB activation and CINC expression induced by TNF-α. Hepatocytes from pair-fed and ethanol-fed rats supplemented with SAM during feeding were cultured and treated with TNF-α as in Figure 8. (A) Nuclear extracts were then examined for activation of p50/p65 heterodimer and p50 homodimers of NF-κB. (B) Total RNA was isolated and hybridized with CINC cDNA, (C) and its levels were expressed in relationship to 18 S used as reference control. Expression of CINC was quantitated by densitometry. Results for CINC expression are means ± SD of 3 and 4 cell preparations from pair-fed and ethanol-fed rats, respectively.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

11. Fig. 10
Sensitization of normal hepatocytes by HP to TNF-α. Freshly isolated normal hepatocytes were incubated with HP (1 mmol/L for 5 minutes) and washed thoroughly. Cells were then fractionated into (A) cytosol and (B) mitochondria to determine GSH content in these fractions. Subsequently, cells were exposed to 10,000 U/mL TNF-α for the indicated time. Aliquots were taken for determination of (C) DCF fluorescence and (D) viability. Cytosol and mitochondrial GSH in the absence of TNF-α did not change over the course of the incubation. Results are means ± SD of 3 cell preparations. *P < 0.05 vs. control cells.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions