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1 J. Exp. Biomed. Sci. 14 (2008) Malondialdehyde Level by Ethanol Exposure in Mouse According to the ALDH2 Enzyme Activity Chung-Jong Lee, Yong-Dae Kim, Sung-Hoon Kim, Sang-Yong Eom, Yan Wei Zhang and Heon Kim Department of Preventive Medicine and Medical Research Institute, College of Medicine, Chungbuk National University, Cheongju , Korea Excessive alcohol consumption is associated with increased risks of many diseases including cancer. Individuals who regularly consume excessive quantities of alcohol have a greater risk of developing head and neck cancers such as esophageal, pharyngeal and oral cavity cancers if they are deficient in ALDH2 expression compared to normal populations. We evaluated lipid peroxidation in Aldh2 +/+ and Aldh2 -/- mice after they had been subjected to acute ethanol exposure. Malondialdehyde (MDA) level in liver tissue was evaluated as a biomarker of oxidative lipid peroxidation. Although the ethanol treatment did not increase the hepatic MDA level both in Aldh2 +/+ mice and in Aldh2 -/- mice, the MDA level was significant higher in the Aldh2 -/- mice than in the Aldh2 +/+ group. The MDA level was also significantly correlated with olive tail moment in blood and the level of 8-OHdG in liver tissue. This is a strong evidence to support our hypothesis that oxidative stress is more intense in Aldh2 -/- mice than in Aldh2 +/+ mice. Our results suggest that ALDH2-deficient individuals may be more susceptible than wild-type ALDH2 individuals to ethanol-mediated liver disease, including cancer. Key Words: Aldehyde dehydrogenase 2, Malondialdehyde, Oxidative stress, Knockout mice 서 과다한음주는암을비롯한다양한질환의위험을증가시킨다 (Blum, 2001; Dey and Cederbaum, 2006). 체내에유입된에탄올은알코올탈수소효소 (alcohol dehydrogenase; ADH) 에의해아세트알데히드로대사된후다시알데히드탈수소효소 2 (aldehyde dehydrogenase 2; ALDH2) 에의해아세트산으로대사되어배출된다 (Agarwal and Goedde, 1992). 에탄올의대사과정중에는다량의 free radical이생성되어체내에서산화적스트레스를유발하는것으로알려져있고 (Bondy, 1992; Nordmann et al., 1992; Ishii et al., 1997), 알코올의급성투여가활성산소의생성을증가시킨다는보고도있다 (Reinke et al., 1991; Bondy and Orozco, 1994). * 논문접수 : 2008년 2월 15일수정재접수 : 2008년 3월 15일 교신저자 : 김용대, ( 우 ) 충북청주시흥덕구개신동 12번지, 충북대학교의과대학예방의학교실 Tel: , Fax: ydkim@chungbuk.ac.kr 론 한국인을포함한아시아인종의약 50% 는 ALDH2 gene의점돌연변이로인해 ALDH2 활성이저하된유전자형을지니고있다 (Kee et al., 2003; Oyama et al., 2005). ALDH2 활성이저하된사람은과도한양의알코올을섭취할경우, ALDH2 활성이높은사람에비해식도암이나구강암등의발생율이유의하게높은것으로알려져있다 (Yokoyama et al., 2002). 아직까지그정확한이유는밝혀지지않았으나유력한가설중하나는 ALDH2 효소의활성에따라체내에서의에탄올대사과정이변화되고이로인해산화적스트레스의발생정도가서로다를가능성이다. 본연구진은선행연구를통하여에탄올투여에의한산화적유전자손상정도가 ALDH2 효소의활성이결핍된경우에더욱심각하다는연구결과를보고한바있다 (Kim et al., 2005; Kim et al., 2007). 그러나산화적스트레스로인한유전자손상지표로가장많이이용되는 8-OHdG는측정을위한전처리과정에서자연적인산화반응에의해서인위적으로도생성될가능성이있다는단점이있어서에탄올대사에의해발생되는산화적스트레스를보다정확하게평가하기위해서는산화적유전자손상이외에지질과산화에미치는영향도함께고

2 려되는것이타당하다. 따라서, 본연구에서는 ALDH2 knockout 마우스를이용하여에탄올을경구투여한후간조직내 malondialdehyde (MDA) 농도를측정하여산화적과산화지질생성과 ALDH2 효소활성과의관련성을규명하고자하였다. 재료및방법 1. 실험동물 Aldh2 -/- 마우스 (Kitagawa et al., 2000) 를일본의 Kawamoto 교수로부터분양받아실험에사용하였다. 12 주령의수컷 Aldh2 +/+ 및 Aldh2 -/- (C57BL/6J strains) 를 plastic mouse cages에서 12시간 (07:00~19:00) 은밝게, 나머지 12시간 (19:00~07:00) 은어두운상태로유지시켰으며배양실의온도는 23~25 를유지하였다. 동물실험과관련된모든조작은 'Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health' 의규정에따라시행하였다. 2. Aldh2 genotyping 및 ethanol 투여마우스꼬리부분에서 genomic DNA를추출하여 PCRamplification 방법으로 Aldh2 genotype을결정하였다 (Isse et al., 2002). 각군에 7마리씩의마우스를배정하여알코올투여군에는 40% ethanol (2 g/kg/day) 을 7일간매일 1회경구투여하였으며대조군에는동량의생리식염수를경구투여하였다. 3. 간조직에서의 malondialdehyde(mda) 농도측정 MDA 농도는 Ohkawa 등 (1979) 의방법을다소수정하여사용하였다. 마우스로부터분리한간조직 0.1 g 정도를 20 mm Tris-HCl buffer (ph 7.4) 1 ml에넣고초음파로분쇄하고 3000 g 4 에서 15분간원심분리한다음상층액 50 µl를취하여 0.05% BHT 50 µl, 0.1 N HNO µl, 42 mm TBA 150 µl를넣고 30초간잘섞어주었다. 100 에서 1시간동안방치한후 5분간 ice에방치하였다. n-butanol 300 µl를넣고 30초간잘섞어준후 12,000 g에서 5분간원심분리하고상층액 250 µl를고압액체크로마토그래피 (HPLC) 방법으로분석하였다. HPLC는 Model SP930D 펌프 (Young Lin, Anyang, Korea) 와 Model SIL-10ADvp 자동시료주입기 (Shimadzu, Kyoto, Japan), 그리고 Model SPD-10Avp 자외선검출기 (Shimadzu), Autochro-3000 Data system (Young Lin) 등으로구성된것을사용하였다. 컬럼은 Tosoh사 (Tokyo, Japan) 의 TSK gel ODS-80, mm 역상컬럼을이용하였다. 이동상용액으로는 50 mm potassium monobasic phosphate:acetonitrile : methanol (65:15: 20, ph 6.8) 용액을사용하였으며, 분당 1 ml의속도로흘려주었다. 자외선검출기파장은 532 nm를사용하였다. 4. 통계분석통계분석은윈도우용 SPSS (Version 12.0) 를사용하였다. 양군간의 MDA 농도의평균은 Mann Whitney U test 를이용하여분석하였다. MDA 농도와 olive tail moment 및 8-OHdG 농도와의관련성을평가하기위하여상관분석을시행하였다. P-값이 0.05 이하인경우를유의한것으로판정하였다. 결과및고찰 IARC 자료에따르면사람에서아세트알데히드의발암성은충분히가능성이있다 (IARC, 1999). 일본인에서음주에의한암발생은 ALDH2 유전자다형성과관련이있는것으로알려져있다 (Yokoyama et al., 1998; Yokoyama et al., 2001; Yokoyama et al., 2002). 즉, Aldh2*2 ( 비활성형 ) 유전자형을가진사람은 Aldh2*1 ( 활성형 ) 을가진사람에비해식도암의위험도가 7~12배가량높은것으로보고되었다. Yokoyama 등 (Yokoyama et al., 2002) 은적어도하나의 Aldh2*2 유전자를가진사람은혈중아세트알데히드농도가상대적으로높으며, 이것이알코올과관련된식도암에서중요한원인으로작용할가능성이매우크다고보고한바있다. 알코올에의한간독성은활성산소의생성이매우중요한원인으로알려져있다. Zhang 등 (2004) 은최근배양세포 (myocyte) 에아세트알데히드를노출시키면활성산소의생성이증가된다고보고하였으며 Isse 등 (2005) 의연구에서도 Aldh2 knockout 마우스는 wildtype 마우스에비해혈중알데히드농도가유의하게높다고보고하였다. 이러한연구결과들은 ALDH2 활성이에탄올에의한산화적스트레스발생에큰영향을줄가능성을시사해준다. 본연구진은이러한가설을입증하기위해서에탄올을투여한마우스에서산화적유전자손상지표인 8-OHdG의농도및 CYP2E1 효소의발현정도를측정하여 ALDH2 효소의활성에따른차이를평가한바있다 (Kim et al., 2005; Kim et al., 2007). 그결과, 요중 8-OHdG

3 Table 1. Analysis of variance (2-way ANOVA) of the effects of the Aldh2 genotype and ethanol exposure on the MDA level in liver tissue Variables Aldh2 genotype Ethanol exposure MDA level in liver tissue F= P<0.001 F=2.619 N.S Fig. 1. Malondialdehyde level in liver tissue of mice treated with saline or 2 g/kg/day of ethanol for 7 days. Seven different livers for each treatment group were individually analyzed by HPLC method. Statistical analysis was performed by Mann Whitney U test. 의농도및간조직의 CYP2E1 발현은 Aldh2 -/- 마우스가 Aldh2 +/+ 마우스에비해유의하게높은것을확인하였다. CYP2E1은체내에탄올대사에관여하는 MEOS (microsomal ethanol oxidizing system) 의대표적인효소로활성산소의발생과밀접한관련이있는것으로알려져있다 (Lieber, 1999). 따라서 ALDH2 효소의결핍은이에대한보상작용으로 MEOS의발현을상대적으로증가시키며, 이로인해활성산소의발생이더욱증가되었을가 능성이높다. 그러나한종류의산화적스트레스지표를평가하여이러한가능성을입증했다고보기는어렵다. 유전자손상지표인 8-OHdG가가장많이사용되는산화적스트레스지표이기는하지만, 측정을위한전처리과정에서인위적인생성가능성이있다는단점이있어서보다객관적이고정확한산화적스트레스를평가하기위해서는 8-OHdG 이외의다른지표를이용한평가가추가적으로필요할것으로생각된다. MDA는지질과산화에대한지표로서 8-OHdG와더불어대표적인산화적스트레스지표로이용된다 (Esterbauer et al., 1991). 포화지방산에서생성되는과산화지질은매우불안정하여복잡한형태의다른화합물로대사되는데, 여기에는 MDA 등의카보닐화합물이포함되며, 이와같은과산화지질의증가는다양한종류의만성질환발생과관련이있는것으로알려져있다 (De Maria et al., 1996; Browne and Beal, 2006). 본연구에서는 7일간의에탄올투여후간조직내의 MDA 농도를측정함으로써 ALDH2 효소의활성이에탄올에의한산화적스트레스발생에미치는영향을평가하고자하였다. 그결과, 예상과는다르게 Aldh2 +/+ 마우스의경우, 간조직에서의 MDA 농도는에탄올투여전 0.48±0.08에서투여후 0.40±0.08 µm/µg protein으로통계적인차이를보이지않았으며, Aldh2 -/-의경우에도투여전 0.61±0.09에서투여후 0.54±0.07 µm/µg protein으로에탄올투여에따른유의한차이는나타나지않았다 (Fig. 1). 이러한결과는에탄올의투여가 MDA의생성을증가시킨다는기존의연구결과들과는다른것이지만아세트알데히드에노출시킨마우스의경우, 요중 8-OHdG의농도는유의한증가를보였으나혈중 MDA의농도에는변화가없었다는 Ogawa 등 (2006) 의연구결과와일치하는것이었다. 또한 Tokunaga 등 (2003) 의연구에서도톨루엔에노출시킨마우스의폐, 간및신장조직에서 8-OHdG의농도는유의하게증가한반면, 이들기관에서의 MDA 농도는유의한변화가없다고보고한바있다. 대표적인산화적스트레스의두지표로알려진 8-OHdG와 MDA의결과가왜다른지에대해서는명확하지않다. 두지표가갖는민감도의차이때문일가능성이있으나이에대한추가연구가진행되어야할것으로본다. 한편, 본연구에서의흥미로운결과는 Aldh2 -/- 마우스의간조직내 MDA 농도는 Aldh2 +/+ 마우스에비해통계적으로유의하게높았다는것이다. 이는에탄올을투여하지않은생리식염수투여군 (0.48 vs 0.61, P=0.018) 의경우와에탄올투여군 (0.40 vs 0.54, P=0.013) 모두에서나타났다 (Fig. 1). 뿐만아니라, 2-way ANOVA 를이용하여에탄올과 ALDH2 효소의활성중각각의영향을배제한상태에서 MDA 농도에미치는영향을분석해본결과 ALDH2 효소의활성이유의하게 MDA 농도에영향을미치는것으로나타났다 (P<0.001, Table 1). 이결과는본연구진이기존에보고한몇편의논문결과 (Kim et al., 2005; Kim et al., 2007) 와일치하는결과이다. 기존의연구에의하면 Aldh2 -/- 마우스는 Aldh2 +/+ 마우스에비해간조직의 8-OHdG 및 CYP2E1 발현정도그리고유전자손상지표인 olive tail moment 수치등이유의하게높은것으로나타났다. 더구나이러한양상은

4 A B Fig. 2. The relationships between the olive tail moment in blood and malondialdehyde level in liver tissue of mice treated with saline or 2 g/kg/day of ethanol for 7 days (A) and the level of 8-OHdG in liver tissue and malondialdehyde level (B). R indicates correlation coefficient. Statistical analysis was performed by Pearson correlation test. Histograms show levels of olive tail moments in blood and 8-OHdG in liver tissue (Kim et al., 2007). 에탄올을투여하지않은군에서도관찰되어본연구결과와정확히일치하였다. 아직이에대한정확한원인은알수없으나가능성있는가설중하나는포유동물의체내에서는에탄올섭취가없더라도음식물등을통해서적지않은양의에탄올대사가자체적으로이루어진다는것이다 (McManus et al., 1960). 또한, CYP2E1 효소는에탄올이외의다른많은물질에의해서도그발현이유도될수있는것으로알려져있다 (Gonzalez, 2005). 결국, ALDH2 활성이결핍된마우스는체내의에탄올대사과정중발생되는아세트알데히드를대사시키기위해 CYP2E1 등의 MEOS 발현이증가되고, 이는활성산소의생성을촉진시켜산화적스트레스의증가가유도되는것으로판단된다. 한편, Matsumoto 등 (2007) 은 ALDH2가결핍된마우스에에탄올을일회경구투여한후간조직의 MDA 농도를측정한연구에서 Aldh2 -/- 마우스의경우는 Aldh2 +/+ 마우스에비해서에탄올투여후의 MDA 농도가상 대적으로낮은것으로보고하여본연구와는다소상반된결과를나타내었다. 그러나 Matsumoto 등 (2007) 의연구가본연구와는달리에탄올을 1회투여하고 12시간이후에단한번만 MDA를측정했다는점을감안한다면, 두연구의결과를직접비교하는것은무리가있다. 왜냐하면, 에탄올을일회투여한후에시간에따라나타나는체내의생화학적변화양상은반복투여에의한변화양상과는상당한차이가있을것이기때문이다. Matsumoto 등 (2007) 의연구에서에탄올투여전의 MDA 농도는 Aldh2 -/- 마우스가 Aldh2 +/+ 마우스보다다소높은경향을보여본연구의결과와유사하였으나통계적으로유의성을보이지는않았다. 이러한차이는연구자간의실험적인편차에의한것일수도있고, MDA의측정방법이본연구에서는 HPLC의방법을사용한반면, Matsumoto 등 (2007) 의연구에서는 ELISA의방법을이용하였기때문에이로인한차이일가능성도생각해볼수있다. 그러나이에대한보다정확한원인은추가

5 적인연구를통해서규명해야할것이다. 기존에보고한이들지표들과 (Kim et al., 2007) 간조직에서의 MDA 농도와의관련성을알아보기위하여상관분석을실시하였다 (Fig. 2). 그결과, 혈중 olive tail moment와간조직의 8-OHdG 농도는간조직의 MDA 농도와각각상관계수 0.484와 0.583의통계적으로유의한관련성을보였다. 이것은본연구에서평가한산화적스트레스지표들이측정과정에서의오류에의한것이아닌실제의생성정도를반영하고있음을의미한다. 본연구의결과 ALDH2 효소활성의결핍은간조직에서의 MDA 생성을유의하게증가시키는것으로확인되었으며, 이는 ALDH2 효소활성의저하가에탄올에의한산화적스트레스에더욱취약하다는가설을뒷받침해주는중요한증거가될수있을것으로생각된다. 감사의글이논문은 2006년도충북대학교학술연구지원사업의연구비지원에의하여연구되었음 (This work was supported by Chungbuk National University Grant in 2006). REFERENCES Agarwal DP, Goedde HW. Pharmacogenetics of alcohol metabolism and alcoholism. Pharmacogenetics : Blum HE. Hepatocellular carcinoma: susceptibility markers. IARC Sci Publ : Bondy SC, Orozco J. Effects of ethanol treatment upon sources of reactive oxygen species in brain and liver. Alcohol : Bondy SC. Ethanol toxicity and oxidative stress. Toxicol Lett : Browne SE, Beal MF. Oxidative damage in Huntington's disease pathogenesis. Antioxid Redox Signal : De Maria N, Colantoni A, Fagiuoli S, Liu GJ, Rogers BK, Farinati F, Van Thiel DH, Floyd RA. Association between reactive oxygen species and disease activity in chronic hepatitis C. Free Radic Biol Med : Dey A, Cederbaum AI. Alcohol and oxidative liver injury. Hepatology : 63S-74S. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med : Gonzalez FJ. Role of cytochromes P450 in chemical toxicity and oxidative stress: studies with CYP2E1. Mutat Res : International Agency for Research on Cancer (IARC). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans Vol. 71, Part Two. IARC, Lyon. Ishii H, Kurose I, Kato S. Pathogenesis of alcoholic liver disease with particular emphasis on oxidative stress. J Gastroenterol Hepatol : 272S-282S. Isse T, Matsuno K, Oyama T, Kitagawa K, Kawamoto T. Aldehyde dehydrogenase 2 gene targeting mouse lacking enzyme activity shows high acetaldehyde level in blood, brain, and liver after ethanol gavages. Alcohol Clin Exp Res : Isse T, Oyama T, Kitagawa K, Matsuno K, Matsumoto A, Yoshida A. Diminished alcohol preference in transgenic mice lacking aldehyde dehydrogenase activity. Pharmacogenetics : Kee JY, Kim MO, You IY, Chai JY, Hong ES, An SC. Effects of genetic polymorphisms of ethanol-metabolizing enzymes on alcohol drinking behaviors. Taehan Kan Hakhoe Chi : Kim YD, Oyama T, Isse T, Kim H, Kawamoto T. Expression levels of hepatic cytochrome P450 enzymes in Aldh2-deficient mice following ethanol exposure: a pilot study. Arch Toxicol : Kim YD, Eom SY, Ogawa M, Oyama T, Isse T, Kang JW, Zhang YW, Kawamoto T, Kim H. Ethanol-induced oxidative DNA damage and CYP2E1 expression in liver tissue of Aldh2 knockout mice. J Occup Health : Kitagawa K, Kawamoto T, Kunugita N, Tsukiyama T, Okamoto K, Yoshida A. Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse. FEBS Lett : Lieber CS. Microsomal ethanoloxidizing system (MEOS): the first 30 years ( ) a review. Alcohol Clin Exp Res : Matsumoto A, Ichiba M, Horita M, Yamashita Z, Takahashi T, Isse T, Oyama T, Kawamoto T, Tomokuni K. Lack of aldehyde dehydrogenase ameliorates oxidative stress induced by single-dose ethanol administration in mouse liver. Alcohol : McManus R, Contag AO, Olson RE. Characterization of endogenous ethanol in the mammal. Science : Nordmann R, Ribiere C, Rouach H. Implication of free radical mechanisms in ethanol-induced cellular injury. Free Radic

6 Biol Med : Ogawa M, Isse T, Oyama T, Kunugita N, Yamaguchi T, Kinaga T, Narai R, Matsumoto A, Kim YD, Kim H, Uchiyama I, Kawamoto T. Urinary 8-hydoxydeoxyguanosine (8-OHdG) and plasma malondialdehyde (MDA) levels in Aldh2 knockout mice under acetaldehyde exposure. Ind Health : Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem : Oyama T, Isse T, Kagawa N, Kinaga T, Kim YD, Morita M. Tissue-distribution of aldehyde dehydrogenase 2 and effects of the ALDH2 gene-disruption on the expression of enzymes involved in alcohol metabolism. Front Biosci : Reinke LA, Kotake Y, McCay PB, Janzen EG. Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1- hydroxyethyl radicals with PBN. Free Radic Biol Med : Tokunaga I, Gotohda T, Ishigami A, Kitamura O, Kubo S. Toluene inhalation induced 8-hydroxy-2'-deoxyguanosine formation as the peroxidative degeneration in rat organs. Leg Med (Tokyo) : Yokoyama A, Muramatsu T, Ohmori T, Yokoyama T, Okuyama K, Takahashi H, Hasegawa Y, Higuchi S, Maruyama K, Shirakura K, Ishii H. Alcohol-related cancers and aldehyde dehydrogenase-2 in Japanese alcoholics. Carcinogenesis : Yokoyama A, Muramatsu T, Omori T, Yokoyama T, Matsushita S, Higuchi S, Maruyama K, Ishii H. Alcohol and aldehyde dehydrogenase gene polymorphisms and oropharyngolaryngeal, esophageal and stomach cancers in Japanese alcoholics. Carcinogenesis : Yokoyama A, Watanabe H, Fukuda H, Haneda T, Kato H, Yokoyama T. Multiple cancers associated with esophageal and oropharyngolaryngeal squamous cell carcinoma and the aldehyde dehydrogenase-2 genotype in male Japanese drinkers. Cancer Epidemiol Biomarkers Prev : Zhang XS, Li Y, Brown RA, Ren J. Ethanol and acetaldehyde in alcoholic cardiomyopathy: from bad to ugly en route to oxidative stress. Alcohol :

한국성인에서초기황반변성질환과 연관된위험요인연구

한국성인에서초기황반변성질환과 연관된위험요인연구 한국성인에서초기황반변성질환과 연관된위험요인연구 - - i - - i - - ii - - iii - - iv - χ - v - - vi - - 1 - - 2 - - 3 - - 4 - 그림 1. 연구대상자선정도표 - 5 - - 6 - - 7 - - 8 - 그림 2. 연구의틀 χ - 9 - - 10 - - 11 -