1. Volume 117, Issue 1, Pages 181-190 (July 1999)
An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice 
Abdellah Mansouri*, Isabelle Gaou*, Caroline de Kerguenec*, Sabine Amsellem*, Delphine Haouzi*, Alain Berson*, Alain Moreau‡, Gérard Feldmann‡, Philippe Lettéron*, Dominique Pessayre*, Bernard Fromenty* 
Gastroenterology 
Volume 117, Issue 1, Pages (July 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Assessment of the time course of hepatic mtDNA and nDNA by slot blot hybridization. Mice were killed 1, 2, 4, 24, or 48 hours after intragastric administration of water (control mice) or 5 g of ethanol/kg body wt. Hepatic DNA (200 ng) was blotted on a nylon membrane, hybridized with a 10.9-kb mtDNA probe, stripped, and rehybridized with a mouse C0t-1 nDNA probe. (A) Slot blot analysis of DNA of a control mouse (lane 0) and 5 mice killed 1, 2, 4, 24, or 48 hours after ethanol administration. nDNA is unchanged, whereas mtDNA is markedly decreased at 2 hours. (B) mtDNA/nDNA ratio. For each mouse, blot intensities were determined by densitometry analysis and the mtDNA/nDNA hybridization ratio was calculated. Results (mean ± SEM for 5–8 treated mice) are expressed as percentages of control values (36 control mice). *Significantly different from control mice, P < 0.05.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Assessment of the proportions of the three main mtDNA forms by Southern blotting. (A) Mice were killed 2 hours after intragastric administration of water or ethanol (5 g/kg), and Southern blots of total hepatic DNA (1.5 μg in control mice, 7 μg in intoxicated mice) were hybridized with a 10.9-kb mtDNA probe. In the first lane, control DNA was cut with SacI, yielding a linear 16.3-kb mtDNA standard. In lanes C1–C3, DNA of 3 control mice mainly contains supercoiled and circular mtDNA molecules. In lanes A1–A3, DNA of 3 intoxicated mice shows disappearance of supercoiled mtDNA and increased linear mtDNA. (B) Relative proportions of the three main mtDNA forms at various times after ethanol administration. Mice were killed 1, 2, 4, or 24 hours after intragastric administration of water or ethanol. The percentages of supercoiled (•), full-length linear (▵), and circular (○) forms of mtDNA were determined by densitometry analysis of Southern blot autoradiographs. Results are means ± SEM for 27 control mice (time 0) and 7–9 intoxicated mice at each time point (times 1–24). *Significantly different from control mice, P < 0.05.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Assessment of mtDNA damage by long PCR amplification. Mice were killed 2 hours after intragastric administration of water or ethanol (5 g/kg). (A) Hepatic DNA from 2 control mice (C1 and C2) and 2 alcoholized mice (A1 and A2) was subjected to long PCR to coamplify a long (8636-bp) PCR product and a short (316-bp) product. Next, 20 μL of the PCR medium was electrophoresed on 1.6% agarose gels with ethidium bromide, and gels were photographed under UV transillumination. In the intoxicated mice, amplification of the 8636-bp PCR product is decreased, whereas the 316-bp fragment is normally amplified. M1 and M2 are 1-kb DNA ladder and HindIII-digested phage λ standards, respectively (GIBCO BRL). (B) Hepatic DNA of 9 control mice and 9 intoxicated mice was subjected to long PCR, and photographs of agarose gels were scanned to assess the long fragment/short fragment intensity ratio. *Significantly different from control mice, P < 0.05.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Acute ethanol intoxication decreases the amounts of mitochondrial transcripts in the liver. (A) Control mice (C1–C3) or alcoholized mice (A1–A3) were killed 2 hours after intragastric administration of water or ethanol (5 g/kg), and total hepatic RNA was subjected to Northern blot hybridization using, consecutively, a mitochondrial 12S rRNA probe, a mitochondrial ND5 mRNA probe, and a β-actin mRNA probe. (B) Mitochondrial transcripts/β-actin mRNA hybridization ratios at various times after ethanol administration. Autoradiographs were scanned to assess the relative amounts of the different RNA species. The 12S rRNA/β-actin mRNA ratio (○) and ND5 mRNAs/β-actin mRNA ratio (•) (mean ± SEM for 4–6 intoxicated mice) are expressed in percentage of control values (8 control mice). *Significantly different from control mice, P < 0.01.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 5
[3H]Thymidine incorporation into nDNA and mtDNA. Groups of 15 mice were killed 0, 2, 3, 4, 10, 12, or 24 hours after ethanol administration and 2 hours after [3H]thymidine administration. Results are means ± SEM for 5 DNA pools (each from 3 pooled livers). *Significantly different from control mice, P < 0.05.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Prevention of ethanol-induced mtDNA depletion by 4-methylpyrazole and melatonin. 4-Methylpyrazole (1 mmol/kg), melatonin (10 mg/kg), or saline was injected intraperitoneally 30 minutes before intragastric administration of ethanol (5 g/kg) or water. Two hours after ethanol or water administration, hepatic mtDNA/nDNA ratio was assessed by slot blot hybridization. Results (mean ± SEM for 5–11 treated mice) are expressed as percentages of control values (15 control mice). *Significantly different from control mice, P < †Significantly different from ethanol-intoxicated mice, P < MP, 4-methylpyrazole; Melat, melatonin; E, ethanol.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions