j pharmacol exp ther 2001 mansouri 737 43

8/12/2019 J Pharmacol Exp Ther 2001 Mansouri 737 43

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Acute Ethanol Administration Oxidatively Damages andDepletes Mitochondrial DNA in Mouse Liver, Brain, Heart, andSkeletal Muscles: Protective Effects of Antioxidants

ABDELLAH MANSOURI, CHRISTINE DEMEILLIERS, SABINE AMSELLEM, DOMINIQUE PESSAYRE, andBERNARD FROMENTY

Institut National de la Sante et de la Recherche Medicale Unite 481 and Centre de Recherches de lAssociation Claude Bernard sur lesHepatites Virales, Hopital Beaujon, Clichy, France

Received January 2, 2001; accepted April 30, 2001 This paper is available online at http://jpet.aspetjournals.org

ABSTRACT

Ethanol metabolism causes oxidative stress and lipid peroxi-dation not only in liver but also in extra-hepatic tissues. Ethanoladministration has been shown to cause oxidative degradationand depletion of hepatic mitochondrial DNA (mtDNA) in ro-dents, but its in vivo effects on the mtDNA of extra-hepatictissues have not been assessed. We studied the effects of anacute intragastric ethanol administration (5 g/kg) on brain,heart, skeletal muscle, and liver mtDNA in mice. Ethanol ad-ministration caused mtDNA depletion and replacement of itssupercoiled form by linearized forms in all tissues examined.Maximal mtDNA depletion was about similar (ca. 50%) in allorgans studied. It occurred 2 h after ethanol administration inheart, skeletal muscle, and liver but after 10 h in brain. This

mtDNA depletion was followed by increased mtDNA synthesis.A secondary, transient increase in mtDNA levels occurred 24 hafter ethanol administration in all organs. In hepatic or extra-hepatic tissues, mtDNA degradation and depletion were pre-vented by 4-methylpyrazole, an inhibitor of ethanol metabolism,and attenuated by vitamin E, melatonin, or coenzyme Q, threeantioxidants. In conclusion, our study shows for the first timethat ethanol metabolism also causes oxidative degradation ofthe mitochondrial genome in brain, heart, and skeletal muscles.These effects could contribute to the development of (cardio-

)myopathy and brain injury in some alcoholic patients. Antioxi-dants prevent these effects in mice and could be useful inpersevering drinkers.

Alcoholic patients can develop not only liver lesions butalso pancreatitis, brain damage, peripheral neuropathy, car-diomyopathy, and skeletal muscle myopathy (Fromenty andPessayre, 1995; Neiman, 1998). Reactive oxygen species(ROS) and free radicals are generated during ethanol metab-olism, causing oxidative stress and lipid peroxidation in liver(Fromenty and Pessayre, 1995; Kurose et al., 1996), brain(Calabrese et al., 1998), heart (Nordmann et al., 1992), andskeletal muscles (Adachi et al., 2000).

In hepatocytes, ethanol-induced free radical and ROS gen-eration involves mitochondria, microsomal cytochrome P4502E1 (CYP2E1), and ferrous iron and to a lesser extent, per-

oxisomes and cytosolic xanthine and aldehyde oxidases (Kuk-ielka and Cederbaum, 1992; Fromenty and Pessayre, 1995;Tsukamoto, 2000). Macrophagic NADPH oxidase is anotherimportant source of ROS in the liver of alcoholized animals(Kono et al., 2000).

Mitochondria are major targets for ethanol toxicity in liver,brain, heart, skeletal muscles, and exocrine pancreas (Fro-

menty and Pessayre, 1995; Baker and Kramer, 1999; Cahillet al., 1999). In the liver, acute and chronic ethanol intoxica-tion causes oxidative damage to mitochondrial proteins,phospholipids (cardiolipin), and mitochondrial DNA(mtDNA) (Fromenty and Pessayre, 1995; Wieland and Lau-terburg, 1995; Cahill et al., 1999; Cederbaum, 1999). Acuteintragastric ethanol administration (5 g/kg) causes oxidativedegradation and depletion of hepatic mtDNA in mice (Man-souri et al., 1999), and the prevalence of hepatic mtDNAdeletions is increased in alcoholic patients (Fromenty et al.,1995; Mansouri et al., 1997). However, in contrast to hepaticmtDNA, there is no information on the possible in vivo effects

of ethanol on the mtDNA of extra-hepatic tissues.In the present study, we show that the oxidative stress due

to ethanol metabolism also causes extensive degradation anddepletion of brain, heart, and skeletal muscle mtDNA inmice. Interestingly, the extent of mtDNA depletion in theseextra-hepatic tissues is about the same as in the liver, sug-gesting that the ethanol-induced free radical and/or ROSformation could be as high in brain and muscles as in theliver. Importantly, we also show that the ethanol-inducedmtDNA alterations can be prevented by several antioxidants

This work was supported in part by the Institut de Recherches Scientifiquessur les Boissons (IREB). A.M. was a recipient of a fellowship from the Associ-ation Francaise pour lEtude du Foie (AFEF).

ABBREVIATIONS:ROS, reactive oxygen species; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; CYP2E1, cytochrome P4502E1; kb, kilobase.

0022-3565/01/2982-737743$3.00THEJOURNAL OF PHARMACOLOGY ANDEXPERIMENTAL THERAPEUTICS Vol. 298, No. 2Copyright 2001 by The American Society for Pharmacology and Experimental Therapeutics 3733/918890JPET 298:737743, 2001 Printed in U.S.A.

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synthesis. To assess this mechanism, [3H]thymidine was ad-ministered 10 or 18 h after alcohol administration, and its in

vivo incorporation into DNA was studied for 2 h. [3H]thymi-dine incorporation into mtDNA was increased 10 h afteralcohol administration in liver, skeletal muscle, and heartand 18 h after ethanol administration in brain (Table 1). Incontrast, [3H]thymidine incorporation into nuclear DNA waseither unchanged or slightly decreased (Table 1), indicating

that the increased DNA synthesis only affected the mitochon-drial genome.

Prevention of mtDNA Depletion by 4-Methypyrazole

and Antioxidants.In a previous study, the ethanol-inducedhepatic mtDNA depletion was prevented by 4-methylpyra-

zole, an inhibitor of ethanol metabolism, or melatonin, anantioxidant (Mansouri et al., 1999). In the present study, wetested the preventive effects of these compounds and alsoseveral others against ethanol-induced mtDNA depletion inliver, skeletal muscles, heart, and brain. Among the 11 com-pounds tested, only vitamin E, melatonin, 4-methylpyrazole(Fig. 3), coenzyme Q10 (Table 2) and -lipoic acid (data notshown), had protective effects in the liver, whereas cyana-mide, silymarin,S-adenosylmethionine,N-acetylcysteine, vi-tamin C, or dehydroepiandrosterone pretreatments had noeffect (data not shown). Interestingly, vitamin E, melatonin,or 4-methylpyrazole also protected against ethanol-mediatedmtDNA depletion in skeletal muscles, heart, and brain (Fig.3), with 4-methylpyrazole being the most active compound in

Fig. 2. Ethanol-induced mtDNA depletion in skeletal muscles, heart, andbrain. Total DNA was extracted from skeletal muscles and heart 2 h after

ethanol and/or water administration or from brain 10 h after the intoxi-cation. Slot blot hybridization was performed with two probes recognizingmtDNA and nDNA, respectively. Slot blots performed with the nDNAprobe ensured that the amount of deposited DNA was about similar foreach mouse. A representative experiment with five control mice and fiveintoxicated mice is shown. Quantitative data are given in Fig. 1.

TABLE 1

[3H]Thymidine incorporation into mtDNA and nDNA after ethanoladministration[3H]Thymidine was administered 10 h after ethanol and/or water administration forstudies performed in liver, skeletal muscles, or heart or 18 hours after ethanoladministration for studies in brain. Its incorporation into mtDNA and nDNA wasstudied for an additional 2 h. Results are means S.E.M. for the number ofdeterminations specified in parentheses.

Incorporationinto mtDNA

Incorporationinto nDNA

cpm/g DNA

LiverControl (n 5) 1.8 0.3 3.8 0.1Ethanol (n 5) 4.9 0.2* 3.2 0.3

Skeletal musclesControl (n 8) 2.1 0.7 3.8 0.5Ethanol (n 8) 5.5 0.8* 2.7 0.2*

HeartControl (n 5) 2.7 0.3 3.5 0.3Ethanol (n 6) 8.0 1.3* 4.3 0.4

BrainControl (n 4) 0.3 0.1 2.1 0.2Ethanol (n 4) 4.4 0.4* 2.1 0.4

* Different from control mice, P 0.05.

Fig. 3.Prevention of ethanol-mediated mtDNA depletion in liver, skele-tal muscles, heart, and brain by vitamin E and melatonin, two antioxi-dants, or by 4-methylpyrazole, an inhibitor of ethanol metabolism. Micewere pretreated as described under Materials and Methods. The mtDNA/nDNA ratio (mean S.E.M. for 9 14 treated mice) was assessed by slotblot hybridization 2 h after ethanol and/or water administration in liver,skeletal muscles, and heart and after 10 h in brain. Results (mean S.E.M. for 1020 treated mice) are expressed as percentages of control

values (10 30 mice). E, ethanol; Vit E, vitamin E; 4-MP, 4-methylpyra-zole. *Different from control mice, P 0.05. , different from unprotectedethanol-intoxicated mice, P 0.05.

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all tissues (Fig. 3). Finally, coenzyme Q10 also afforded asignificant, albeit partial, protection against ethanol-inducedmtDNA depletion in heart (Table 2) and brain (data notshown). It was noteworthy that treatment of mice with vita-

min E, melatonin, 4-methylpyrazole, or coenzyme Q did notsignificantly change mtDNA levels in the different tissues(data not shown), thus suggesting that their protective effectagainst ethanol-induced mtDNA depletion cannot be ex-plained by an intrinsic ability to increase tissue mtDNAlevels in mice.

Partial Prevention of Hepatic mtDNA Overshoot by

4-Methypyrazole.Since 4-methylpyrazole almost fully pro-tected mice against ethanol-induced mtDNA depletion, wealso assessed its ability to prevent the overshoot in mtDNAlevels that followed the depletion. Mice were thus treatedwith either ethanol or with 4-methylpyrazole plus ethanoland were killed 24 h later for hepatic mtDNA analysis. Ourresults showed that whereas mtDNA levels in intoxicated

mice (n 12) were significantly increased by 42% comparedwith the controls (n 7), mtDNA levels in alcoholized micepretreated with 4-methylpyrazole (n 11) were increased byonly 25% (data not shown). Therefore, our data suggestedthat ethanol-induced mtDNA depletion cannot fully accountfor the overshoot of mtDNA resynthesis.

Effects on mtDNA Forms and Prevention of These

Effects by 4-Methypyrazole and Antioxidants. We haveshown previously that ethanol administration modifies therepartition of the different hepatic mtDNA forms as a conse-quence of mtDNA strand breaks (Mansouri et al., 1999).Present results indicate similar effects in skeletal muscles,heart, and brain (Figs. 4 and 5). Two hours after alcoholadministration, the supercoiled form of liver, skeletal muscle,and heart mtDNA (i.e., an intact and native form) was de-creased, whereas its abnormal linearized form was increased(Figs. 4 and 5). A similar pattern was observed in brain 10 hafter ethanol intoxication (Fig. 5).

Present results also show for the first time that 4-meth-ylpyrazole or melatonin (Fig. 5) and also coenzyme Q10(Table2) prevent ethanol-induced effects on hepatic mtDNA forms.Interestingly, melatonin or 4-methylpyrazole also efficientlyprotected mice against ethanol-induced loss of supercoiledmtDNA in skeletal muscles, heart, and brain (Figs. 4 and 5).We also verified that the coenzyme Q10 pretreatment alsoprevented ethanol-induced mtDNA strand breaks in the

heart (Table 2). Finally, further experiments with micetreated with 4-methylpyrazole or melatonin alone showedthat these derivatives did not increase the proportion ofsupercoiled mtDNA in the liver (data not shown). Theseresults suggested that increased supercoiled mtDNA in in-toxicated mice pretreated with either melatonin or 4-meth-ylpyrazole could not be ascribed to the sole effect of theseprotective drugs.

Discussion

This study shows for the first time that an alcoholic bingecauses extensive mtDNA degradation and mtDNA depletionin mouse heart, skeletal muscles, and brain, followed byincreased mtDNA replication and increased mtDNA levels(Figs. 15). These data extend a previous study showinghepatic mtDNA degradation and recovery in the same model(Mansouri et al., 1999). Importantly, the maximal serumethanol concentrations achieved in this model are ca. 4 g/li-ter, a concentration that can occur in severely intoxicatedpatients (Mansouri et al., 1999).

In all tissues, ethanol-mediated mtDNA degradation andmtDNA depletion were fully prevented by 4-methylpyrazole,an inhibitor of both alcohol dehydrogenase and CYP2E1(Figs. 3 and 5), suggesting that ethanol metabolism plays amajor role in ethanol-mediated mtDNA damage. Ethanol

TABLE 2

Coenzyme Q10

protects against ethanol-induced mtDNA depletion and degradation in liver and heartWater, ethanol, and coenzyme Q10were administered to mice as described under Materials and Methods, and total DNA was extracted from liver or heart 2 h after ethanolor water administration. The mtDNA/nDNA ratio and the percentage of the three main mtDNA forms (supercoiled, circular, and linear) were assessed by slot blot andSouthern blot hybridization, respectively. Results are means S.E.M. for five to fifteen determinations.

mtDNA/nDNAmtDNA

Supercoiled Circular Linear

% control % mtDNA

LiverControl 100 11 26 9 62 7 12 4Ethanol 44 10* 9 5 63 4 28 5*Ethanol CoQ 75 6 50 9 40 4*, 10 6

HeartControl 100 5 25 8 63 8 12 4Ethanol 50 5* 2 1* 67 5 31 4*Ethanol CoQ 83 8 67 14*, 15 9*, 18 8

CoQ, coenzyme Q10.* Different from control mice, P 0.05. Different from unprotected ethanol-intoxicated mice, P 0.05.

Fig. 4. Ethanol-induced changes in mtDNA forms in skeletal muscles andprevention of these changes by melatonin. Melatonin was administeredas described underMaterials and Methods, and total DNA was extractedfrom skeletal muscles 2 h after ethanol and/or water administration.Southern blots of total DNA (1.5 g in control mice, 5 g in ethanol-treated mice and, 1.5 g in melatonin-protected, ethanol-treated mice)were hybridized with an mtDNA probe. Note the loss of supercoiledmtDNA in ethanol-intoxicated mice and the prevention of this loss inmice also treated with melatonin.

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metabolism causes acetaldehyde formation, CYP2E1-medi-ated 1-hydroxyethyl radical, and ROS formation, and alsoincreases the NADH/NAD ratio, which causes reduction offerric iron to ferrous iron, a potent generator of the hydroxylradical, which can then cause lipid peroxidation, releasingreactive aldehydes such as malondialdehyde and 4-hy-droxynonenal. Acetaldehyde, ROS, the 1-hydroxyethyl radi-cal, and lipid peroxidation products may all damage DNA(Brooks, 1997; Mansouri et al., 1999). Cyanamide, an inhib-itor of acetaldehyde metabolism (Efthivoulou and Berry,1997), was not protective in our study. Although a failure ofour pretreatment protocol to confer significant protection tomice is conceivable, this result may also be due to an antag-onistic effect of cyanamide, which increases levels of acetal-dehyde (that may damage mtDNA) while decreasing levels ofNADH (that may decrease ROS production). Clearly, further

investigations are needed to determine the molecular speciesthat trigger mtDNA depletion after an alcoholic binge.

In all tissues, ethanol-mediated mtDNA depletion wasmostly prevented by vitamin E and melatonin (Figs. 35).These powerful antioxidants would not be expected to protectagainst acetaldehyde toxicity, but they can act on both ROSand ROS-mediated lipid peroxidation. Collectively, these re-sults suggest that the increased ROS formation triggered byethanol metabolism plays a major role in ethanol-inducedmtDNA depletion. As the extent of ethanol-induced mtDNAdepletion was essentially similar in liver, heart, skeletalmuscles, and brain (Fig. 1), the overall formation of thesemtDNA-damaging agents may be about similar in these dif-ferent tissues. Because extra-hepatic tissues contain low lev-els of alcohol dehydrogenase and microsomal CYP2E1 (Kerret al., 1989; Thum and Borlak, 2000; Voirol et al., 2000),other cellular components, such as mitochondria, may bemainly involved in ROS generation during ethanol intoxica-tion. CYP2E1 is highly expressed in brain mitochondria(Bhagwat et al., 1995) and could contribute, in part, to eth-anol-induced ROS formation in these organelles.

In organs with large amounts of lipids, such as the brain,

ROS could oxidize unsaturated lipids causing lipid peroxida-tion, which in turn releases 4-hydroxynonenal and malondi-aldehyde, both of which react with respiratory chain polypep-tides (Chen et al., 2000), thus blocking the flow of electronsalong the respiratory chain, which may increase mitochon-drial ROS formation (Pessayre et al., 2000). This secondarycause of sustained ROS generation might explain the moreprolonged mtDNA depletion in brain rather than in organswith lower amounts of lipids, such as liver, heart, or skeletalmuscles.

In the present study, mtDNA depletion in the diverse tis-sues was associated with major changes in the repartition ofmtDNA forms. The normal, supercoiled form of mtDNA hadalmost disappeared, with a corresponding increase in linear-

ized mtDNA forms (Figs. 4 and 5), showing that ethanolintoxication causes mtDNA strand breaks. Ethanol intoxica-tion also causes the formation of oxidized DNA bases andabasic (apyrimidinic/apurinic) sites in hepatic DNA (Wielandand Lauterburg, 1995; Mansouri et al., 1999). The DNArepair machinery is incomplete in the mitochondria, andmtDNA molecules harboring too many lesions are destroyedby mitochondrial nucleases instead of being repaired (Cro-teau et al., 1999; Mansouri et al., 1999), thus causing tran-sient mtDNA depletion (Mansouri et al., 1999).

As mtDNA levels are tightly regulated (Tang et al., 2000),this mtDNA depletion triggers an adaptive increase inmtDNA synthesis (Mansouri et al., 1999; Holt et al., 2000).[3H]Thymidine incorporation into mtDNA is increased inliver and extra-hepatic organs (Table 1) causing prompt res-toration of mtDNA levels and then increased mtDNA levelsat 24 h (Fig. 1). It is noteworthy that mtDNA first tended todecrease and then significantly increase in cardiac myocytescultured with 200 mM ethanol (Kennedy, 1997). However,our data point to additional mechanism(s) involved inmtDNA resynthesis, since 4-methylpyrazole only partiallyprevented the late mtDNA overshoot while almost fully pro-tecting against ethanol-induced mtDNA depletion. Interest-ingly, recent data suggest that mtDNA levels can increase inresponse to endogenous or exogenous oxidative stress (Lee etal., 2000).

Fig. 5.Ethanol-induced changes in mtDNA forms in liver, skeletal mus-cles, heart, and brain and prevention of these changes by melatonin or4-methylpyrazole. Protective substances or saline were administered asdescribed under Materials and Methods. Percentages of supercoiled (S),circular (C), and full-length linear (L) mtDNA forms were determined bydensitometric analysis of Southern blot autoradiographs, 2 h after intra-gastric ethanol and/or water administration in liver, skeletal muscles,and heart, and 10 h after ethanol administration in brain. Results aremeans S.E.M. for 8 to 18 control mice and 5 to 14 treated mice. E,ethanol; 4-MP, 4-methylpyrazole. , different from control mice,P 0.05., different from unprotected ethanol-intoxicated mice, P 0.05.



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Tsukamoto H (2000) CYP2E1 and ALD. Hepatology 32:154155.Voirol P, Jonzier-Perey M, Porchet F, Reymond M, Janzer RC, Bouras C, Strobel

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Address correspondence to: Dr. Bernard Fromenty, INSERM U-481,Hopital Beaujon, 100 Bd du General Leclerc, 92 118 Clichy Cedex, Fra nce.E-mail: [email protected]


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