jnh Journal of Nutrition and Health J Nutr Health 2288-3886 2288-3959 The Korean Nutrition Society 10.4163/jnh.2015.48.5.390 jnh-48-390 Research Article Effects of chronic alcohol and excessive iron intake on mitochondrial DNA damage in the rat liver∗ ParkJung-Eun LeeJeong-Ran ChungJayong † Department of Food and Nutrition, Kyung Hee University, Seoul 02447, Korea †To whom correspondence should be addressed. tel: +82-2-961-0977, e-mail: jchung@khu.ac.kr

This work was supported by grants from the Basic Science Research Program of the National Research Foundation (NRF-2010-0011226).

 012015 19012015 485390 397 18August2015 24September2015 8October2015 Copyright © 2015 The Korean Nutrition Society 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose:

In this study, we investigated the effects of chronic alcohol and excessive iron intake on mitochondrial DNA (mtDNA) damage and the progression of alcoholic liver injury in rats.

 Methods:

Twenty-four Sprague-Dawley male rats were divided into four groups (Control, EtOH, Fe, and EtOH + Fe), and fed either control or ethanol (36% of total calories) liquid diet with or without 0.6% carbonyl iron for eight weeks. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, liver malondialdehyde concentrations were measured by colorimetric assays. Liver histopathology was examined by Hematoxylin-eosin staining of the fixed liver tissues. The integrity of the hepatic mtDNA and nuclear DNA was measured by long-range PCR. The gene expression levels of cytochrome c oxidase subunit 1 (Cox1) and NADH dehydrogenase subunit 4 (Nd4) were examined by real-time PCR.

 Results:

Serum ALT and AST activities were significantly higher in the EtOH+Fe group, as compared to the Control group. Similarly, among four groups, liver histology showed the most severe lipid accumulation, inflammation, and necrosis in the EtOH + Fe group. PCR amplification of near-full-length (15.9 kb) mtDNA showed more than 50% loss of full-length product in the liver of the EtOH + Fe group, whereas amounts of PCR products of a nuclear DNA were unaffected. In addition, the changes in the mtDNA integrity showed correlation with reductions in the mRNA levels of mitochondrial gene Cox1 and Nd4.

 Conclusion:

Our data suggested that the liver injury associated with excessive iron and alcohol intake involved mtDNA damage and corresponding mitochondrial dysfunction.

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Effects of ethanol and iron on serum ALT and AST activities and liver MDA concentrations. (A) Serum ALT activity, (B) Serum AST activity, (C) Liver MDA concentration. Values are expressed as mean ± SE (n = 6 for each group). Different superscripts show statistical significance (p < 0.05).

Liver histology in ethanol and/or iron fed rats (x400). (A) Hematoxylin and eosin (H&E) staining, (B) Perls’ Prussian blue staining (iron staining). Arrows show inflammatory cell infiltrations.

Effects of ethanol and iron on the integrity of mitochondria DNA (mtDNA) and nuclear DNA. (A) Full-length (15.9 kb) mtDNA PCR products, (B) 16.1 kb nuclear transferrin receptor DNA PCR products. Values are expressed as mean ± S.E (n = 6 for each group). Different superscripts show statistical significance (p < 0.05).

Effects of ethanol and iron on the mRNA levels of Cox1 and Nd4 in the rat liver. (A) Gapdh, (B) Cox1, (C) Nd4. Values are expressed as mean ± S.E (n = 6 for each group). Different superscripts show statistical significance (p < 0.05).

Nucleotide sequence for PCR primers

Gene   Sequence Size
15.9 kb fragment of mtDNA forward CCT CCC ATT CAT TAT CGC CGC CCT TGC 15.9 kb
  reverse GAT GGG GCC GGT AGG TCG ATA AAG GAG  
200 bp fragment of mtDNA forward CCT CCC ATT CAT TAT CGC CGC CCT TGC 200 bp
  reverse GTC TGG GTC TCC TAG TAG GTC TGG GAA  
Transferrin receptor forward GCA TAT TGG AAC ACT TGT GAG GGT GG 16.1 kb
  reverse AGA AGA CAT GCG ATT AGA TGC CAG AA  
β-globin forward CCA ATC TGC TCA CAC AGG 265 bp
  reverse CAC CTT TCC CCA CAG G  

Body and liver weights, liver iron concentrations, and blood iron parameters after 8-week feeding of ethanol and iron in rats

  Control EtOH Fe EtOH + Fe
Initial body weight (g) 191.7 ± 2.1 195.6 ± 5.8 194.2 ± 4.0 194.4 ± 6.1
Final body weight (g) 378.7 ± 8.4 373.6 ± 12.5 374.7 ± 10.4 354.0 ± 13.7
Liver weight (g) 8.9 ± 0.3a 12.9 ± 0.5b 9.6 ± 0.3a 11.8 ± 0.5b
Liver index (g/100 g bw) 2.4 ± 0.0a 3.4 ± 0.1c 2.6 ± 0.1b 3.3 ± 0.0c
Hematocrit (%) 44.9 ± 1.3 46.5 ± 0.3 50.0 ± 0.8 45.4 ± 2.4
Hemoglobin (g/dL) 14.7 ± 0.2 15.0 ± 0.3 14.7 ± 0.3 15.5 ± 0.3
Liver iron (µg/g dry wt) 310.0 ± 43.7a 213.2 ± 40.6a 3685.4 ± 526.4c 1884.0 ± 231.8b

Values are expressed as mean ± SE (n = 6 for each group). Means with different superscripts are significantly different at p < 0.05.