1. Volume 123, Issue 4, Pages 1278-1290 (October 2002)
Impaired adaptive resynthesis and prolonged depletion of hepatic mitochondrial DNA after repeated alcohol binges in mice 
Christine Demeilliers, Caroline Maisonneuve, Alain Grodet, Abdellah Mansouri, Richard Nguyen, Marina Tinel, Philippe Lettéron, Claude Degott, GéRard Feldmann, Dominique Pessayre, Bernard Fromenty 
Gastroenterology 
Volume 123, Issue 4, Pages (October 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Hepatic mtDNA and nDNA levels assessed by slot blot hybridization. (A) Representative blots. Mice received one intragastric daily dose of water or ethanol (5 g/kg diluted in water) each day for 4 consecutive days and were killed 2, 24, 48, or 96 hours after the fourth dose. Hepatic DNA (200 ng) was blotted on a nylon membrane, hybridized with an 8.6-kilobase mtDNA probe, stripped, and rehybridized with a mouse C0t-1 nDNA probe. Whereas nDNA is unchanged in intoxicated mice, mtDNA is decreased at 2, 24, or 48 hours but normal at 96 hours. (B) Time course of the mtDNA/nDNA ratio. Mice were killed 0, 2, 12, 24, 36, 48, or 96 hours after the fourth intragastric dose of ethanol or water. For each mouse, blot intensities were determined by densitometry analysis and the mtDNA/nDNA hybridization ratio was calculated. Results (mean ± SEM for 6–12 intoxicated mice) are expressed as a percentage of the respective control values (5 control mice for each time point). Note that the 0-hour value corresponds to the persistent effects of the third ethanol dose. *Significantly different from control mice (P < 0.05).
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Assessment of hepatic mtSSB transcripts by ribonuclease protection assay. (A) Representative gels. Mice were killed 24 hours after the fourth intragastric administration of water (C1–C3) or ethanol (5 g/kg) (A1–A3). Total hepatic RNA was extracted and ribonuclease protection assays were performed to assess the 330–base pair protected fragment of mtSSB mRNA and the major 245–base pair protected fragment of β-actin mRNAs. (B) Time course of the mtSSB/β-actin mRNAs ratio. For each RNA sample, this ratio was calculated by using the intensity of the 330–base pair mtSSB protected fragment and that of the major 245–base pair β-actin protected fragment. Results in mice killed 2, 12, 24, or 48 hours after the fourth ethanol dose (mean ± SEM for 5–7 intoxicated mice) are expressed as a percentage of control values (9 control mice). *Significantly different from control mice (P < 0.05).
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
In organello mtDNA synthesis. (A) Validation of the assay in mitochondria isolated from control mice. [3H]dTTP (20 μCi/mL) incorporation into mtDNA was measured in mitochondria incubated at 37°C for 5 hours with the diverse substrates indicated in Materials and Methods and with or without 0.3 mmol/L aphidicolin (Aph.), 10 mmol/L N-ethylmaleimide (NEM), and 10, 50, or 100 μmol/L dideoxycytidine triphosphate (ddCTP). These compounds are expected to inhibit (NEM and ddCTP) or not inhibit (Aph.) mitochondrial polymerase γ. At the end of the reaction, mitochondria were centrifuged and washed 3 times before counting the incorporated radioactivity. Results (mean ± SEM for 6 determinations) are expressed as the percentage of control values without polymerase inhibitors (10 determinations). (B) Effects of alcohol. Mice were killed 2 or 24 hours after the fourth intragastric administration of water or ethanol (5 g/kg), and mitochondria were isolated to measure in vitro mtDNA synthesis. Results (mean ± SEM for 9–12 intoxicated mice) are expressed as a percentage of the respective control values (9–11 control mice for each time point). *Significantly different from control values (P < 0.05).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Assessment of mtDNA damage by long PCR amplification. (A) Representative agarose gels. The hepatic DNA from 3 control mice (C1–C3), 2 mice killed 2 hours after the fourth ethanol dose (A1 and A2), and 2 mice killed 24 hours after the fourth ethanol dose (A3 and A4) was subject to long PCR to coamplify a long (8636 base pairs) mtDNA fragment and a short (316 base pairs) mtDNA fragment with commercial polymerases. Twenty microliters of the PCR reaction was electrophoresed on ethidium bromide–containing 1.3% agarose gels, which were photographed under UV transillumination. Amplification of the 8636–base pair product is decreased in intoxicated mice, whereas the 316–base pair fragment is normally amplified. M, HindIII-digested phage λ DNA. (B) Quantification. The hepatic DNA of control mice or mice killed 2 or 24 hours after the fourth ethanol intoxication was subject to long PCR, and photographs of agarose gels were scanned to assess the long fragment/short fragment intensity ratio. Results (mean ± SEM for 7–11 intoxicated mice) are expressed as a percentage of the respective control values (7–10 control mice). *Significantly different from control mice (P < 0.05).
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Effect of exonuclease III supplementation on long PCR amplification. The hepatic DNA of one control mouse (C1) and 2 mice killed 2 hours after the fourth ethanol dose (A1 and A2; different from those used in Figure 4) was subject to long PCR to amplify a long (8636 base pairs) mtDNA fragment without (exo 0) or with 25 U of exonuclease III (exo +). The 0.8% agarose gel was loaded with 20 μL of the PCR products. As a positive control, a DNA sample (D1) that had been depurinated in vitro was also studied. Increased amplification of the 8636–base pair mtDNA fragment in the presence of exonuclease III reflects the presence of apurinic/apyrimidinic sites and/or DNA strand breaks. M, HindIII-digested phage λ DNA.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
CYP2E1 protein. (A) Representative immunoblots. Liver microsomes of 3 control mice (C1–C3) and 3 mice killed 4 hours after the fourth ethanol dose (A1–A3) were immunoblotted with rabbit polyclonal CYP2E1 antibodies. (B) Quantification. Mice were killed 2, 4, 24, and 48 hours after the fourth intragastric administration of water or ethanol (5 g/kg), and the CYP2E1 protein band was quantified on immunoblots. Results (mean ± SEM for 3–4 intoxicated mice) are expressed as the percentage of control values (10 control mice). *Significantly different from control mice (P < 0.05).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Liver ultrastructure. Mice were killed (A and B) 2 hours or (C and D) 24 hours after the fourth ethanol intoxication. Small lipid droplets (L) and abnormal mitochondria (arrows) are visible in some hepatocytes. Mitochondrial abnormalities consist of elongated or hypertrophied organelles with dilatation of cristae. N, nucleus. Bars = 1 μm. (Original magnification: A, 6000×; B–D, 20,000×.)
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions