만성 알코올과 철분의 과잉 섭취가 흰쥐의 간 세포 미토콘드리아 DNA 손상에 미치는 영향

국문 초록 (Abstract)

본 연구에서는 Sprague-Dawley 종 랫트 수컷을 대조군, EtOH군, Fe군, EtOH + Fe군으로 나누어, 알코올과 철분을 액상 사료로 8주간 공급한 후, 간 조직과 간 세포 mtDNA의 손상 정도를 알아보았다. EtOH + Fe군은 대조군, EtOH군, Fe군의 다른 세 군에 비해 혈청 ALT와 혈청 AST 수치가 가장 유의적으로 높았으며, 간 조직 검사의 결과에서도 다수의 지방구, 염증성 세포 침입 및 조직의 괴사가 관찰되는 등 가장 심한 간 손상이 확인되었다. DNA 손상 여부를 긴 영역 PCR을 사용하여 분석한 결과, 만성적인 알코올과 철분에 의한 노출은 간 세포의 mtDNA 손상을 유발하는 것으로 나타났으며, 핵 DNA에는 영향을 미치지 않았다. 또한 미토콘드리아의 호흡에 관여하는 Cox1과 Nd4 유전자 발현 정도를 real-time PCR으로 분석한 결과, 알코올 또는 철분은 간 세포의 Cox1 mRNA와 Nd4 mRNA 수준을 유의적으로 낮추는 것으로 나타났다. 이상의 결과는 만성 알코올 또는 과잉의 철분에 의한 간 손상에 mtDNA 손상 및 미토콘드리아 기능 저하가 관여함을 제시한다.

다국어 초록 (Multilingual Abstract)

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.

목차 (Table of Contents)

• ABSTRACT
• 서론
• 연구방법
• 결과
• 고찰
• 요약
• References

참고문헌 (Reference)

1 김형태, "성인 음주자의 영양소 섭취실태: 2011 국민건강영양조사 자료 분석" 대한영양사협회 19 (19): 343-355, 2013

2 Kwak CS, "The effects of smoking and alcohol drinking on nutritional status and eating habits in adult males" 5 (5): 161-171, 2000

3 Day CP, "Steatohepatitis: a tale of two “hits”?" 114 (114): 842-845, 1998

4 Takeyama Y, "Role of Kupffer cell-derived reactive oxygen intermediates in alcoholic liver disease in rats in vivo" 20 (20): 335A-339A, 1996

5 Brandon-Warner E, "Rodent models of alcoholic liver disease: of mice and men" 46 (46): 715-725, 2012

6 Tang Y, "Quercetin prevents ethanol-induced dyslipidemia and mitochondrial oxidative damage" 50 (50): 1194-1200, 2012

7 Larosche I, "Prolonged ethanol administration depletes mitochondrial DNA in MnSOD-overexpressing transgenic mice, but not in their wild type littermates" 234 (234): 326-338, 2009

8 Becker U, "Prediction of risk of liver disease by alcohol intake, sex, and age: a prospective population study" 23 (23): 1025-1029, 1996

9 Wu D, "Oxidative stress mediated toxicity exerted by ethanol-inducible CYP2E1" 207 (207): 70-76, 2005

10 Aliev G, "Oxidative stress mediated mitochondrial and vascular lesions as markers in the pathogenesis of Alzheimer disease" 21 (21): 2208-2217, 2014

11 Loguercio C, "Oxidative stress in viral and alcoholic hepatitis" 34 (34): 1-10, 2003

12 Cederbaum A, "Nrf2 and antioxidant defense against CYP2E1 toxicity" 5 (5): 1223-1244, 2009

13 Richter C, "Normal oxidative damage to mitochondrial and nuclear DNA is extensive" 85 (85): 6465-6467, 1988

14 Boland ML, "Mitochondrial dysfunction in cancer" 3 : 292-, 2013

15 Blake R, "Mitochondrial dysfunction and complications associated with diabetes" 1840 (1840): 1404-1412, 2014

16 Lakshmi Devi S, "Mitochondrial damage, cytotoxicity and apoptosis in iron-potentiated alcoholic liver fibrosis: amelioration by taurine" 38 (38): 869-879, 2010

17 Yakes FM, "Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress" 94 (94): 514-519, 1997

18 Gao X, "Mitochondrial DNA damage in iron overload" 284 (284): 4767-4775, 2009

19 Wallace DC, "Mitochondria and cancer" 12 (12): 685-698, 2012

20 Ganne-Carrié N, "Liver iron is predictive of death in alcoholic cirrhosis: a multivariate study of 229 consecutive patients with alcoholic and/or hepatitis C virus cirrhosis: a prospective follow up study" 46 (46): 277-282, 2000

21 Ministry of Health and Welfare, "Korea Health Statistics 2013: Korea National Health and Nutrition Examination Survey" Korea Centers for Disease Control and Prevention 2014

22 Xiong S, "Iron-dependent activation of NF-κB in Kupffer cells: a priming mechanism for alcoholic liver disease" 30 (30): 107-113, 2003

23 Silva M, "Iron dextran increases hepatic oxidative stress and alters expression of genes related to lipid metabolism contributing to hyperlipidaemia in murine model" 2015 : 272617-, 2015

24 Masola B, "Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase" 9 (9): 363-374, 1995

25 Tsukamoto H, "Experimental liver cirrhosis induced by alcohol and iron" 96 (96): 620-630, 1995

26 Choi JS, "Effects of excess dietary iron and fat on glucose and lipid metabolism" 24 (24): 1634-1644, 2013

27 Whitfield JB, "Effects of alcohol consumption on indices of iron stores and of iron stores on alcohol intake markers" 25 (25): 1037-1045, 2001

28 Ministry of Health and Welfare, "Community health survey 2014" Korea Centers for Disease Control and Prevention 2015

29 Demori I, "Combined effects of high-fat diet and ethanol induce oxidative stress in rat liver" 40 (40): 185-191, 2006

30 Statistice Korea, "Cause of death statistics 2013" Statistice Korea

31 Lu Y, "CYP2E1 potentiation of LPS and TNFα-induced hepatotoxicity by mechanisms involving enhanced oxidative and nitrosative stress, activation of MAP kinases, and mitochondrial dysfunction" 5 (5): 149-167, 2010

32 Ohkawa H, "Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction" 95 (95): 351-358, 1979

33 Mathews S, "Animals models of gastrointestinal and liver diseases. Animal models of alcoholinduced liver disease: pathophysiology, translational relevance, and challenges" 306 (306): G819-G823, 2014

34 Ayala-Torres S, "Analysis of gene-specific DNA damage and repair using quantitative polymerase chain reaction" 22 (22): 135-147, 2000

35 Stewart S, "Alcoholic liver disease: new insights into mechanisms and preventative strategies" 7 (7): 408-413, 2001

36 Valerio LG Jr, "Alcohol mediates increases in hepatic and serum nonheme iron stores in a rat model for alcoholinduced liver injury" 20 (20): 1352-1361, 1996

37 Olynyk J, "A long-term study of the interaction between iron and alcohol in an animal model of iron overload" 22 (22): 671-676, 1995