doi:10.1152/ajpgi.00044.2006 291:857-867, 2006. First published Jul 6, 2006;Am J Physiol Gastrointest Liver Physiol

Landar, Kusum K. Kharbanda, Scott Ballinger and Victor Darley-Usmar Ulasova, Melissa Pompilius, Grier P. Page, David Chhieng, Nirag Jhala, Aimee Shannon M. Bailey, Gloria Robinson, Anita Pinner, Laura Chamlee, Elenaliver alcohol-induced mitochondrial dysfunction in the rat

-adenosylmethionine prevents chronicS

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S-adenosylmethionine prevents chronic alcohol-induced mitochondrialdysfunction in the rat liver

Shannon M. Bailey,1 Gloria Robinson,1 Anita Pinner,1 Laura Chamlee,1 Elena Ulasova,2

Melissa Pompilius,2 Grier P. Page,3 David Chhieng,2 Nirag Jhala,2 Aimee Landar,2

Kusum K. Kharbanda,4 Scott Ballinger,2 and Victor Darley-Usmar2

Departments of 1Environmental Health Sciences, 2Pathology, and 3Biostatistics, University of Alabama, Birmingham, Alabama;and 4Veterans Affairs Alcohol Research Center, Department of Veteran Affairs Medical Center, Omaha, Nebraska

Bailey, Shannon M., Gloria Robinson, Anita Pinner, LauraChamlee, Elena Ulasova, Melissa Pompilius, Grier P. Page, DavidChhieng, Nirag Jhala, Aimee Landar, Kusum K. Kharbanda, ScottBallinger, and Victor Darley-Usmar. S-adenosylmethionine preventschronic alcohol-induced mitochondrial dysfunction in the rat liver. Am JPhysiol Gastrointest Liver Physiol 291: G857–G867, 2006. First pub-lished July 6, 2006; doi:10.1152/ajpgi.00044.2006.—An early event thatoccurs in response to alcohol consumption is mitochondrial dysfunction,which is evident in changes to the mitochondrial proteome, respirationdefects, and mitochondrial DNA (mtDNA) damage. S-adenosylmethi-onine (SAM) has emerged as a potential therapeutic for treating alcoholicliver disease through mechanisms that appear to involve decreases inoxidative stress and proinflammatory cytokine production as well as thealleviation of steatosis. Because mitochondria are a source of reactiveoxygen/nitrogen species and a target for oxidative damage, we tested thehypothesis that SAM treatment during alcohol exposure preserves or-ganelle function. Mitochondria were isolated from livers of rats fedcontrol and ethanol diets with and without SAM for 5 wk. Alcoholfeeding caused a significant decrease in state 3 respiration and therespiratory control ratio, whereas SAM administration prevented thesealcohol-mediated defects and preserved hepatic SAM levels. SAM treat-ment prevented alcohol-associated increases in mitochondrial superoxideproduction, mtDNA damage, and inducible nitric oxide synthase induc-tion, without a significant lessening of steatosis. Accompanying theseindexes of oxidant damage, SAM prevented alcohol-mediated losses incytochrome c oxidase subunits as shown using blue native PAGEproteomics and immunoblot analysis, which resulted in partial preserva-tion of complex IV activity. SAM treatment attenuated the upregulationof the mitochondrial stress chaperone prohibitin. Although SAM supple-mentation did not alleviate steatosis by itself, SAM prevented several keyalcohol-mediated defects to the mitochondria genome and proteome thatcontribute to the bioenergetic defect in the liver after alcohol consump-tion. These findings reveal new molecular targets through which SAMmay work to alleviate one critical component of alcohol-induced liverinjury: mitochondria dysfunction.

oxidative stress; proteome; blue native gel electrophoresis; cyto-chrome c oxidase; prohibitin; mitochondrial DNA

CHRONIC ALCOHOL CONSUMPTION causes liver damage by a com-plex process involving oxidative stress, hypoxia, and energymetabolism defects. Early in this process, endotoxin releasedfrom the gut (31) activates Kupffer cells, which in turn leads toan elevated production of cytokines, eicosanoids, and reactiveoxygen/nitrogen species (ROS/RNS). This results in a spec-trum of responses in the hepatocyte including enhanced hyp-oxia (2), mitochondrial damage (5), and increased sensitivity to

nitric oxide (NO)-dependent inhibition of respiration (77). Thisincreased metabolic activity, which includes increased ROS/RNS production, contributes to hepatocyte death (4).

Disruption in oxidative phosphorylation and the inability tomaintain ATP levels are proposed to contribute to the liverdamage from chronic alcohol exposure. Specifically, the in-ability to maintain ATP levels in hepatocytes contributes tonecrotic cell death from exposure to toxicants including etha-nol (32, 36). Chronic alcohol consumption depresses the ac-tivities of all the respiratory complexes except complex II (21).Mitochondrial protein synthesis inhibition due to mitochon-drial DNA (mtDNA) (14, 75) and mitochondrial ribosomedefects (13, 54) are proposed to contribute to the decreasedfunctioning of respiratory complexes and ATP synthesis (67).These disturbances in mitochondria structure and loss of func-tion are also associated with increased mitochondria ROSproduction and oxidative injury to the organelle after chronicalcohol exposure. Because increased mitochondrial oxidantproduction has been identified as one of several key factorsmediating alcoholic liver injury, agents aimed at attenuatingmitochondria ROS production should prevent mitochondrialdysfunction and the progression to permanent liver damage.

One agent that has received considerable attention in recentyears as a potential therapeutic against alcohol-induced liverinjury is S-adenosylmethionine (SAM). SAM is the primarymethyl donor in anabolic metabolism, serves as a precursor forglutathione (GSH), and is synthesized by the enzyme methio-nine adenosyltransferase (MAT) (47). Chronic alcohol con-sumption decreases hepatic SAM levels due to depressedactivity of the hepatocyte-specific isoform of MAT (42). De-pletion of SAM correlates with DNA hypomethylation andstrand breaks (42) as well as increased lipid peroxidation andmitochondrial damage (39). These effects may also contributeto changes in the mitochondrial proteome and modification ofthiols in critical enzymes involved in ethanol metabolismincluding aldehyde dehydrogenase (76). Moreover, MAT1Aknockout mice, which are deficient in SAM, spontaneouslydevelop steatohepatitis and hepatocellular carcinoma, thusdemonstrating that chronic depletion of SAM predisposes theliver to injury (46). Accordingly, the exogenous administrationof SAM has been shown to increase survival in patients withalcoholic cirrhosis (48), presumably through a number ofmechanisms including inhibition of steatosis and inflammationas well as repletion of mitochondrial GSH levels (27, 35, 40,65). Recent studies (1, 25, 59) have indicated that the benefits

Address for reprint requests and other correspondence: S. M. Bailey, Dept.of Environmental Health Sciences, School of Public Health, Univ. of Alabama,1530 3rd Ave. South, Ryals 623, Birmingham, AL 35294 (e-mail:sbailey@uab.edu).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Gastrointest Liver Physiol 291: G857–G867, 2006.First published July 6, 2006; doi:10.1152/ajpgi.00044.2006.

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of SAM might be mediated through effects on mitochondria;however, the specific mechanisms or mitochondria pathwaystargeted by SAM are unknown. Thus, we investigated theability of SAM to prevent chronic alcohol-induced mitochon-drial damage. Studies were carried out to evaluate the effect ofSAM on parameters of mitochondria function as well asmtDNA damage and alterations to the mitochondrial proteomein response to chronic alcohol exposure. Moreover, the abilityof SAM to attenuate the effects of alcohol on mitochondrialfunction will provide insight into whether defects in mitochon-drial function are linked to the development of early stages ofalcohol-mediated liver injury.

MATERIALS AND METHODS

Feeding protocols. Male Sprague-Dawley rats (3 mo of age,Charles River Laboratories) were individually housed and maintainedunder a 12:12-h light-dark cycle for the entire duration of the feedingperiod. Animals had free access to standard laboratory rat chow andwater for at least 1 wk and weighed at least 200 g before beingprovided the liquid diets. Nutritionally adequate control and ethanol-containing liquid diets were formulated by Bio-Serv (Frenchtown,NJ). The ethanol-containing diet provided 36% of the total dailycaloric intake as ethanol, 35% as fat, 18% as protein, and 11% ascarbohydrate. Control animals were pair fed identical diets except thatethanol calories were replaced isocalorically with carbohydrate (dex-trin maltose). A second set of animals were also pair fed the samecontrol and ethanol diets supplemented with SAM (0.8 mg activeSAM/ml diet). SAM was provided as the stable 1,4-butanedisulfonatesalt form (Denosyl SD4, Nutramax Laboratories, Edgewood, MD).SAM was dissolved in the diets 1 h before animals were fed. Animalswere maintained on these feeding protocols for at least 31 days beforebeing used in experiments. These studies were approved by theinstitutional animal care and use committee, and animals receivedhumane care in accordance with the National Institutes of Health(NIH) Guide for the Care and Use of Laboratory Animals (NIHPublication No. 86-23).

Mitochondria isolation. Mitochondria were isolated by differentialcentrifugation of the liver homogenate (6). Mitochondria function wasassessed by measuring state 3 and 4 respiration and the respiratorycontrol ratio (RCR) using glutamate/malate or succinate as substrates.Cytochrome c oxidase and citrate synthase activities were measuredusing standard spectrophotometric measures (63, 80). MitochondrialGSH concentrations were measured using an enzymatic recyclingassay (72). Ethanol and SAM treatments had no effect on mitochon-drial protein yield (control, 23 � 2.0 mg protein/g liver; ethanol, 24 �1.8 mg protein/g liver; control � SAM, 20 � 2.0 mg protein/g liver;and ethanol � SAM, 21 � 0.9 mg protein/g liver, P � 0.9).

Liver histology and biochemical measurements. Liver sectionswere evaluated by two pathologists blinded to the experimentalgroups. Steatosis was assessed by determining the percentages ofhepatocytes containing fat droplets and were graded as 0 (�5%), 1�(6–25%), 2� (26–50%), and 3� (51–75%). Intra-acinar inflamma-tion was scored as the presence of inflammatory cell aggregates:absence of inflammation was scored as 0, one focus was scored as 1,and two to four foci were scored 2 per �10 objective (78). Liversamples were homogenized in 0.5 M perchloric acid, and the filteredextracts were directly subjected to HPLC analysis for the determina-tion of hepatic SAM levels following the procedures of Fu et al. (26)as detailed in Ref. 8. Triglycerides were measured in the whole liverhomogenate as described in Ref. 35.

Mitochondrial superoxide production. Rates of O2�� production in

mitochondria were determined using a chemiluminescent coelentera-zine assay (70). The production of O2

�� was stimulated by the additionof succinate (1 mM) to mitochondria (60 �g) suspended in the samebuffer used to measure rates of respiration. This allowed for the

measurement of O2�� production to be done under the same conditions

used to measure oxygen consumption. Coelenterazine (prepared inethanol) was added to the buffer immediately before the measure-ments to achieve a concentration of 5 �M for assays. Increases inchemiluminescence were monitored for 5 min at 37°C using anAutoLumat plus model 953 luminometer (Berthold, Germany), andrates of O2

�� production were corrected for background rates, i.e.,signals generated solely with reactants. The signal (relative lightunits/s) was integrated over a 30-s period (30–60 s) and used in thefinal calculation.

Blue native gel electrophoresis. Ethanol and/or SAM-mediatedalterations in the levels of proteins that comprise the oxidativephosphorylation system were assessed using blue native (BN)-PAGEproteomics (11, 75). Oxidative phosphorylation complexes were ex-tracted intact from 1 mg of mitochondrial protein using 1% n-dodecyl-�-D-maltoside in 0.75 M aminocaproic acid and 50 mM BisTris (pH7.0). Protein extracts were mixed with 2.5 �l of a 5% (wt/vol)suspension of Coomassie brilliant blue G-250 in 0.5 M aminocaproicacid before being applied to nondenaturing 5–16.5% gradient gels toseparate individual intact oxidative complexes. After native one-dimensional gel electrophoresis was performed, the entire vertical gellane containing all the complexes was cut from the gel and laid on topof a denaturing Tris-tricine-SDS-PAGE gradient gel to resolve theindividual polypeptides of the complexes based on molecular weight.Samples from each pair of untreated or SAM-treated control andethanol-fed animals were resolved on one gel to minimize for intergeldifferences. Gels were stained with Coomassie blue using identicalstaining and destaining conditions. Image analysis on two-dimen-sional BN-PAGE gels was performed using Quantity One software(Bio-Rad).

Immunoblot analysis. For inducible NO synthase (iNOS) andcytochrome P-450 2E1 (CYP2E1) detection, whole liver homogenatewas used, whereas isolated mitochondria were used to detect mito-chondria proteins. Levels of iNOS protein were detected using a1:5,000 dilution of antibody (Chemicon). Cytochrome c oxidasesubunits I, IV, and Vb were detected using 1:10,000, 1:5,000, and1:500 dilutions, respectively (Molecular Probes). Cytochrome c wasdetected using a 1:1,000 dilution (BD Pharmingen). CYP2E1 proteinwas detected using a 1:1,000 dilution (Chemicon). Prohibitin proteinwas detected using a 1:1,000 dilution (Oncogene), whereas BAP37protein was detected using a 1:20,000 dilution of antibody (providedby Dr. Phillip Coates). Mitochondrial heat shock protein (Hsp)70 andHsp60 proteins were detected using 1:10,000 and 1:2,500 dilutions,respectively (StressGen Biotechnologies). After membranes had beenincubated with the appropriate secondary antibodies, proteins werevisualized using ECL, and the intensities of immunoreactive proteinbands were quantified using NIH Image (Bethesda, MD). The proteinconcentration loaded onto gels was within the linear range of the ECLsystem used for all proteins.

Quantitative PCR. Genomic DNA was extracted from livers andquantified using PicoGreen (Molecular Probes), and 15 ng of DNAwere used for quantitative PCR. The sensitivity of the assay isincreased via amplification of large targets, thereby increasing theprobability of encountering a DNA lesion. A 16,059-bp quantitativePCR product that included all but 236 bp of the NADH5/6 genes inthe mtDNA was amplified using a specified primer set (38). Copynumber differences in mtDNA were normalized using a short quan-titative PCR, which yielded products directly related to gene copynumbers. mtDNA damage was quantified by comparing the relativeefficiency of amplification of large (15 kb) fragments of DNA andnormalizing this with gene copy numbers by the amplification ofsmaller (�250 bp) fragments, which have a statistically negligiblelikelihood of containing damaged bases (38, 75).

Statistical analyses. Data represent means � SE for 6 pairs ofanimals/group. Significant differences between treatments were ob-tained using paired t-tests and two-factor ANOVA. P � 0.05 was

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considered as significant. The Mann-Whitney rank sum test was usedfor pathology analyses.

RESULTS

Animals and liver histopathology. There were no significantdifferences in weight gain among all groups (Table 1). Ethanolconsumption in untreated and SAM-treated groups was 10.8 �0.4 and 12.9 � 0.5 g �kg�1 �day�1, respectively, and wasassociated with a significant increase in liver weight and theliver-to-body weight ratio in the ethanol-only group (Table 1).Consumption of ethanol resulted in the development of a mildsteatosis, which was accompanied by increased triglyceridelevels in both untreated and SAM-treated ethanol-fed animalsversus their corresponding controls (Table 1). However, tri-glyceride levels were increased to a lesser extent in the SAM-treated ethanol group compared with the ethanol-only group.Ethanol consumption induced a mild inflammation that wasslightly, but not statistically, decreased in ethanol-fed animalscoadministered SAM (Table 1).

SAM prevents alcohol-mediated decreases in mitochondriarespiration. Mitochondrial dysfunction occurs after chronic alco-hol consumption and is associated with depressed mitochondrialrespiration and ATP synthesis. As mentioned in MATERIALS AND

METHODS, the mitochondria protein yield was equivalent among allgroups. Similarly, there were no differences in citrate synthaseactivity, a commonly used mitochondrial marker enzyme, amongall groups (control, 0.244 � 0.01 �mol �min�1 �mg�1; ethanol,0.273 � 0.02 �mol �min�1 �mg�1; control � SAM, 0.267 � 0.1�mol �min�1 �mg�1; and ethanol � SAM, 0.253 � 0.1�mol �min�1 �mg�1, P � 0.1). To determine whether SAM sup-plementation prevented chronic alcohol-mediated defects in mi-tochondria function, oxygen consumption in the presence ofsubstrates (glutamate/malate or succinate) and ADP was mea-sured in coupled mitochondria isolated from livers of animals fedcontrol and ethanol diets with and without added SAM. State 3respiration (ADP dependent) and the RCR were significantlydecreased in animals fed ethanol compared with controls (Fig. 1,A–D). Chronic alcohol exposure also resulted in a significantdecrease in cytochrome c oxidase activity (Fig. 1E). In contrast,state 4 respiration (ADP independent) was unaffected by ethanolconsumption, suggesting that uncoupling does not contribute tomitochondrial dysfunction (data not shown). State 3 respirationand the RCR were largely preserved in ethanol-fed animalscoadministered SAM (Fig. 1, A–D). Moreover, SAM partiallyprevented the alcohol-dependent loss in cytochrome c oxidaseactivity (Fig. 1E). This alcohol-associated defect in mitochondrialfunction was not accompanied by significant alterations in mito-chondrial GSH levels (control, 6.9 � 1.1 nmol/mg protein; etha-nol, 8.6 � 1.1 nmol/mg protein, control � SAM, 7.1 � 0.7

nmol/mg protein; and ethanol � SAM, 8.5 � 0.5 nmol/mgprotein, P � 0.84). However, the coadministration of SAMprevented the ethanol-associated decrease in hepatic levels ofSAM (control, 2.47 � .026 nmol �liver�1 �g body wt�1; ethanol,1.67 � 0.029 nmol �liver�1 �g body wt�1; control � SAM, 2.81 �0.042 nmol �liver�1 �g body wt�1; and ethanol � SAM, 2.11 �0.039 nmol �liver�1 �g body wt�1, P � 0.03). Taken together,these data demonstrate that SAM supplementation prevents alco-hol-dependent mitochondrial dysfunction because SAM pre-served respiratory rates at control levels.

SAM prevents alcohol-dependent losses in oxidative phos-phorylation proteins. It is possible that SAM maintains mito-chondria respiration through preservation against the alcohol-associated loss in oxidative phosphorylation proteins. Indeed,two-dimension BN-PAGE proteomics supported the hypothe-sis that SAM protects against the loss in both mitochondria-and nuclear-encoded polypeptides that comprise the oxidativephosphorylation system (75). Representative BN-PAGE pro-teomic maps of the oxidative phosphorylation proteins areshown in Fig. 2. From these maps, it was apparent that chronicalcohol consumption caused a loss of respiratory chain proteins(Fig. 2, A and B). This was especially true for the proteins thatcomprise cytochrome c oxidase (circled proteins, Fig. 2, A andB) and, to a lesser extent, those subunits that comprise complexI. Quantification of the band density within each complexrevealed a significant decrease in the overall content of cyto-chrome c oxidase and NADH dehydrogenase (complex I) inresponse to chronic alcohol consumption (Fig. 2E) with onlyminor effects on ubiquinol cytochrome bc1 reductase (complexIII) and ATP synthase (complex V). It is postulated thatdecreases in respiratory complex proteins contribute to thedepressed state 3 respiration and cytochrome c oxidase activityin ethanol-fed animals. Whereas SAM supplementation onlymodestly prevented the alcohol-associated loss in complex Isubunits, SAM prevented a loss in cytochrome c oxidasesubunits in response to chronic alcohol (Fig. 2, C–E). Thus,changes in some of the cytochrome c oxidase subunits wereverified by Western blot analysis. Immunoblot analysis indi-cated a 70% decrease in the level of mitochondria-encodedcytochrome c oxidase subunit I protein in mitochondria fromethanol-fed rats vs. controls, which was partially attenuated bythe coadministration of SAM (Fig. 3A). Similarly, SAM alsoprevented the alcohol-mediated loss in two of the nuclear-encoded subunits, subunits IV and Vb (Fig. 3, C and D). SAMalone had no effect on the expression of these subunits incontrol animals, and ethanol and/or SAM treatment had noeffect on the levels of cytochrome c (Fig. 3B). These dataindicate that SAM protects at the molecular level through

Table 1. Effect of EtOH and SAM on liver parameters

Con EtOH Con � SAM EtOH � SAM

Body weight gain, g 118�12 111�13 115�7.3 99�13Liver weight, g 12.7�0.6 13.9�0.7* 12.2�0.5 13.1�1.0Liver-to-body weight ratio, % 3.3�0.08 3.7�0.12* 3.6�0.12 3.9�0.14Steatosis score 0.0�0.0 1.7�0.43* 0.0�0.0 1.5�0.5Triglycerides, �mol/liver 183�31 947�304* 242�25 804�167†Inflammation score 0.3�0.2 1.2�0.17* 0.16�0.16 0.66�0.3

Data represent means � SE; n � 6 animals/group. Con, control; EtOH, ethanol; SAM, S-adenosylmethionine *P � 0.05 compared with Con; †P � 0.05compared with Con � SAM.

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preservation of the mitochondrial proteome in response tochronic alcohol consumption.

Effect of ethanol and SAM on the mitochondrial chaperoneprohibitin. To date, the impact of chronic alcohol consumptionon the mitochondrial chaperone system is undefined. Theproteins prohibitin and BAP37 together form a complex withinthe mitochondrial inner membrane that functions in the assem-bly of respiratory complexes (50). Studies have shown thatmetabolic stress associated with mitochondrial protein synthe-sis inhibition causes an upregulation of prohibitin protein. It issuggested that this occurs as an adaptive response to helpprotect against a continued loss of mitochondrial gene productsthat might occur in response to the imbalance between thecontent of mitochondria- and nuclear-encoded subunits in theinner membrane (19, 51). Because chronic alcohol consump-tion induces a similar imbalance (75), we chose to examine theeffect of chronic alcohol and SAM on prohibitin. Both prohib-

itin and BAP37 protein were increased by 50% and 40%,respectively, in mitochondria from ethanol-fed rats comparedwith controls (Fig. 4, A and B). In contrast, there were nodifferences in prohibitin and BAP37 levels in mitochondriaisolated from control and ethanol-fed animals in the SAMgroup (Fig. 4, A and B). Chronic alcohol also had no effect onthe levels of two additional mitochondria-associated chaper-ones, Hsp70 and Hsp60 (Fig. 4, C and D).

Effect of SAM on mitochondria O2�� production and iNOS

expression. It is hypothesized that the effect of SAM to preventthe alcohol-associated losses in oxidative phosphorylation pro-teins (Figs. 2 and 3) will also attenuate chronic alcohol-associated increases in mitochondrial O2

�� production. Asshown in Fig. A, O2

�� production was significantly increased inmitochondria isolated from ethanol-fed animals compared withcontrols. Mitochondria from ethanol-fed animals also gener-ated 40% more O2

�� compared with control mitochondria in

Fig. 1. S-adenosylmethionine (SAM) preventschronic alcohol-mediated decreases in mitochon-drial respiration and cytochrome c oxidase (COX)activity. State 3 respiration was measured, and therespiratory control ratio (RCR) was determinedusing either glutamate/malate (Glu/Mal; A and B)or succinate (Suc; C and D) as oxidizable substratesin freshly isolated mitochondria. COX activity (E)was measured in frozen mitochondria samples bymonitoring the rate of oxidation of fully reducedcytochrome c at 550 nm. Data represent means �SE for n � 6 animals/group. aP � 0.05 comparedwith control (Con); bP � 0.05 compared withethanol (EtOH) alone.

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the presence of antimycin (Fig. 5B, inset), which is consistentwith increased O2

�� production related to ethanol-elicited dam-age to the cytochrome bc1 region of complex III (4, 20).Importantly, SAM treatment attenuated the alcohol-dependentincrease in mitochondrial O2

�� production in both the presenceand absence of antimycin (Fig. 5). Another potential source ofO2

�� in the chronic alcohol-exposed liver is CYP2E1 (34).Although CYP2E1 protein was induced by chronic alcoholconsumption, SAM did not prevent this increase (Fig. 6A).These results indicate that the ability of SAM to attenuateoxidant production is largely mediated through its ability topreserve the functioning of the electron transport chain viapreventing losses in proteins that comprise respiratory com-plexes (Fig. 2).

Accompanying the attenuation of mitochondrial O2�� produc-

tion was the concomitant suppression of iNOS protein induc-tion in ethanol-fed animals coadministered SAM. Immunoblotanalysis indicated a 70% increase in the level of iNOS proteinin livers of ethanol-fed animals vs. controls, which was largelyattenuated by the coadministration of SAM (Fig. 6B). It ispostulated that SAM contributes to the preservation of mito-chondria function through attenuation of iNOS, thus prevent-ing oxidative damage to mitochondrial proteins, as well asmtDNA, from alcohol-induced increases in RNS.

SAM attenuates chronic alcohol-mediated mtDNA damage.We (75) have previously shown that chronic alcohol consump-tion induces mtDNA damage in young animals (75), which inturn is postulated to negatively impact mitochondrial protein

Fig. 2. SAM prevents the loss of oxidative phos-phorylation proteins in response chronic alcoholconsumption. Representative 2-dimensional bluenative (BN)-PAGE proteomic maps of mitochon-dria isolated from Con (A), EtOH-treated (B),Con � SAM-treated (C), and EtOH � SAM-treated (D) animals are shown. For these gels, 250�g of mitochondrial protein were subjected to1-dimensional BN-PAGE. The 1-dimensional gelstrips were overlaid across a Tris-tricine-SDS-PAGE gel to resolve the individual polypeptidesthat comprise each oxidative phosphorylation sys-tem (OxPhos) complex. The proteins that com-prise each complex (I, V, III, IV, and II) appear asvertically aligned spots on the 2-dimensional gel.The proteins that comprise COX are circled. E:comparison of the relative quantities of complexesI, V, III, and IV in liver mitochondria from theCon, EtOH, Con � SAM, and EtOH � SAMgroups. Results represent means � SE for 3 pairsof animals/group. aP � 0.05 compared with Con;bP �0.05 compared with EtOH; cP � 0.05 com-pared with Con � SAM.

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synthesis and functioning of the oxidative phosphorylationsystem, and thus leads to increased mitochondrial O2

�� produc-tion. The ability of SAM to prevent chronic alcohol-mediatedmtDNA damage was assessed using quantitative PCR. Theprinciple of this assay is that DNA lesions block the polymer-ase leading to decreased amplification of the target of interest,i.e., the mitochondrial genome. Thus, a decreased PCR productcompared with control indicates mtDNA damage. There was asignificant loss in the PCR product from ethanol-fed animalscompared with controls, which was prevented by the coad-ministration of SAM (Fig. 7). There were no differences inmtDNA copy numbers among all groups (data not shown).These results are consistent with the hypothesis that chronicalcohol consumption induces damage to the mitochondrialgenome and that this effect can be prevented by the coadminis-tration of SAM.

DISCUSSION

As a source of ROS/RNS and a locus of resulting oxidative/nitrative modifications, mitochondria are recognized as criticalcomponents in the cellular stress response to toxic agents.Ethanol consumption causes increased mitochondrial O2

�� pro-duction and iNOS induction, which in turn results in increasedNO generation, an enhanced sensitivity of the respiratory chainto inhibition by NO, and the subsequent production of moretoxic RNS like peroxynitrite (ONOO�). This oxidative/nitra-tive stress contributes to mitochondrial injury, which manifestsas an overall defect in oxidative phosphorylation. This causes

further oxidant production and escalating levels of damage atmultiple sites. The consequence of this mechanism with vari-ous feedback loops is an overall decrease in mitochondrialfunction that places the hepatocyte under a bioenergetic stress.This is important because a loss in the capacity for mainte-nance of hepatic ATP levels will ultimately predispose thetissue to permanent damage due to a depression in anabolicprocesses responsible for replacing damaged and/or lost cellu-lar components. Thus, agents aimed at “short circuiting” theseresponses are predicted to prevent mitochondrial dysfunctionand potentially delay the onset or severity of disease. In thisstudy, we evaluated the ability of SAM to prevent severalindexes of alcohol-induced mitochondrial damage.

Whereas SAM supplementation partially preserved hepaticSAM levels in ethanol-fed animals, it had only a minimaleffect on preventing alcohol-induced steatosis (7, 33, 40).Because SAM did not significantly impact steatosis, it ispossible that the concentration of SAM used in the presentstudy was insufficient to alleviate the inhibitory effect ofS-adenosylhomocysteine (SAH) on methyltransferase activity.SAH is formed as a by-product of methyltransferase reactionsinvolving SAM and is increased after alcohol consumption dueto the impaired activity of methionine synthase and subsequentremoval of homocysteine (8, 28). It is proposed that SAHinhibits the methyltransferase that converts phosphatidyleth-anolamine to phosphatidylcholine. Duce and colleagues (24)demonstrated a reduction in phospholipid methyltransferaseactivity in human cirrhotic patients, which was also observed

Fig. 3. SAM prevents the loss of COX subunits inmitochondria from EtOH-fed rats. Top, representativeimmunoblots for COX subunits I (A), IV (C), and Vb(D) as well as for cytochrome c (B) for one pair ofCon and EtOH-treated animals and one pair of ani-mals fed diets supplemented with SAM. Bottom,quantification of protein. Results represent means �SE for n � 4–6 pairs of animals/group. aP � 0.05compared with Con, bP � 0.05 compared with EtOH;cP � 0.05 compared with Con � SAM.

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in baboons fed ethanol chronically (41). Because phosphati-dylcholine is required for the synthesis and secretion of very-low-density lipoprotein, a defect in its synthesis could beexpected to lead to the accumulation of triglyceride in the liver

(52). It is predicted that an increased dose of SAM might havebeen more effective in reducing triglycerides levels via nor-malization of the SAM-to-SAH ratio in the liver. This is likelybecause betaine, a choline metabolite that remethylates homo-cysteine to methionine, prevents alcohol-induced steatosis inrodents when administered in much higher concentrations thanwere used in the present study (33).

In contrast, SAM had a profound effect to attenuate ethanol-elicited defects in the mitochondrial respiratory chain. Thus,studies were focused on elucidating the potential mitochondrialtargets and pathways through which SAM works to preventalcohol-induced mitochondrial dysfunction. An early study(27) has demonstrated that the coadministration of SAM duringchronic alcohol feeding maintained mitochondrial GSH levelsand preserved mitochondria functions. These results suggestedthat SAM protects mitochondria from ethanol-associated oxi-dative damage by maintaining GSH. However, several recentstudies (6, 22, 45, 53, 57, 60) have demonstrated that mito-chondria GSH depletion may not be a consistent feature ofchronic alcohol exposure. We observed a small increase inmitochondrial GSH levels in response to alcohol that mayrepresent an adaptive response to counteract the mild to mod-erate oxidative stress induced by chronic alcohol consumption(43). Similar adaptive increases in GSH have been shown inother systems of mild oxidant exposure (23). Thus, our resultssuggest that the protective effects of SAM on mitochondriamight be mediated through effects on other mitochondrialtargets, such as preservation of the proteins that comprise theoxidative phosphorylation system.

Indeed, recent evidence suggests the involvement of SAM inthe regulation of mitochondrial proteins. It is possible that

Fig. 4. Effect of EtOH and SAM on mitochondrialchaperones. Top, representative immunoblots forprohibitin (A), BAP37 (B), heat shock protein(Hsp)70 (C), and Hsp60 (D) protein from one pairof Con and EtOH-treated animals and one pair ofanimals fed diets supplemented with SAM. Bottom,quantification of proteins. Results representmeans � SE for n � 4–6 pairs of animals/group.aP � 0.05 compared with Con.

Fig. 5. SAM attenuates chronic alcohol-induced increases in mitochondrialsuperoxide (O2

��) production [in relative light units (RLU)/s]. Mitochondriawere isolated from livers of rats fed Con and EtOH-containing diets supple-mented with and without SAM and incubated with colenterazine (5 �M) tomonitor O2

�� production (A). To ascertain the extent of alcohol-induced damageto complex III, mitochondria were incubated with antimycin (20 �g/ml), andrates of O2

�� production were measured using the coelenterazine assay (B,inset). Antimycin results are reported as percentages of Con due to the largedegree of variability in the stimulation of O2

�� levels in response to antimycin.aP � 0.05 compared with Con; bP � 0.05 compared with EtOH.

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SAM maintains mitochondrial respiration by preventing alco-hol-associated losses in the polypeptides that comprise therespiratory complexes (75). BN-PAGE proteomics clearlydemonstrates the preservation in respiratory chain proteins inSAM-treated ethanol animals compared with the low levelsdetected in animals exposed to ethanol alone. This is particu-larly true for cytochrome c oxidase, whose activity and subunitcomposition were partially preserved in ethanol-fed animalscoadministered SAM. Whereas the mechanism responsible forthe ethanol-related decrease in mitochondria-encoded subunitshas been attributed to lesions in the mitochondrial ribosome(12, 54), the decrease in nuclear-encoded subunits has onlyrecently been recognized, and the mechanism is undefined(75). Recent work has demonstrated a coordinated downregu-lation of both mitochondria- and nuclear-encoded subunits ofcytochrome c oxidase, specifically subunits IV and Vb, inresponse to hypoxia due to reduced transcription (79). Simi-larly, Chandel et al. (17) proposed that alterations in cyto-chrome c oxidase activity occur in response to hypoxia andplay a role in oxygen sensing. Our findings demonstrating thepreservation of these hypoxia-sensitive subunits in ethanol-fed

animals coadministered SAM suggests that SAM preventsalcohol-associated mitochondria dysfunction in part throughinteractions with pathways involved in the cellular response tohypoxic stress.

Our results are also consistent with those demonstratingdisrupted mitochondrial function in the MAT1A knockoutmouse, which is deficient in SAM (59). Depletion of hepaticSAM in these mice results in decreased levels of mitochondria-encoded subunits of cytochrome c oxidase, reduced membranepotential, and alterations in prohibitin levels (59). As discussedearlier, the best-described function of the prohibitin complex isas a chaperone within the inner membrane (50). This complexstabilizes mitochondrial translation products by binding tothem before the subunits are transferred to the newly assem-bled complex (51). According to this concept, an alteration inthe level or functioning of the prohibitin complex may impactthe assembly and activity of the oxidative phosphorylationsystem. While prohibitin levels are depressed in the MAT1Aknockout mouse, we observed an upregulation in prohibitinand its binding partner BAP37 in response to chronic alcoholconsumption, which was attenuated by SAM. This responseappears to be specific for inner membrane chaperones becausechronic alcohol consumption had no effect on the classic “heatshock” chaperones Hsp70 and Hsp60, which are associatedwith mitochondria (29). The upregulation of prohibitin andBAP37 suggests an attempt of the hepatocyte to “adapt” to themetabolic stress induced by chronic alcohol consumption bytrying to minimize losses in mitochondria-encoded polypep-tides and thus preserve mitochondrial function. Similar adap-tive increases in prohibitin have been found using other modelsof metabolic stress (19, 51) and apoptosis (71). It is possiblethat the decrease in prohibitin levels observed in the MAT1Aknockout mouse (59) reflects a failure of this adaptive responsein more severe models of liver disease (46). Clearly, the effectof chronic alcohol and SAM on the function of prohibitinwarrants further investigation.

It is well established that there are chronic alcohol-induceddefects in complexes I, III, and IV due to losses in several ofthe polypeptides and redox centers that comprise these enzymecomplexes. These defects are the result of alcohol-induceddamage to the mitochondrial genome and ribosomes (12, 13,54, 75). Decreases in the content of proteins downstream fromthe redox active centers of complex I and III, as well as a lossin complex IV subunits, impedes electron flow within therespiratory chain. As a consequence, the respiratory complexes

Fig. 6. Effect of EtOH and SAM on cytochromeP-450 2E1 (CYP2E1) and inducible nitric oxidesynthase (iNOS) protein levels. Top, representativeimmunoblots of CYP2E1 (A) and iNOS (B) for onepair of Con and EtOH-treated animals and one pairof animals fed diets supplemented with SAM. Bot-tom, quantification of protein. Results representmeans � SE for n � 3–6 pairs of animals/group.aP � 0.01 compared with Con; cP � 0.05 comparedwith Con � SAM.

Fig. 7. Chronic alcohol-mediated mitochondrial DNA (mtDNA) damage isprevented by coadministration of SAM. Genomic DNA was prepared fromlivers, and mtDNA damage was assessed using quantitative PCR (QPCR). Top,representative amounts of QPCR products generated from the liver tissue ofCon-, EtOH-, Con � SAM-, and EtOH � SAM-treated animals. Less QPCRproduct indicates increased mtDNA damage. Bottom, bar graph indicating therelative amount of mtDNA damage (lesions/16.2 kb) in livers from EtOH-fedanimals compared with the corresponding Con animals. Con values are set at0 lesions/16.2 kb. aP � 0.02 compared with Con; bP � 0.004 compared withEtOH.

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are in a more reduced state, which facilitates electron transferto molecular oxygen to generate O2

��. This occurs at complexesI and III because they contain stable semiquinone intermediatesas redox components. Thus, disruption of the electron transportchain in response to chronic alcohol consumption results inincreased mitochondrial O2

�� production. Recent data by Cahilland colleagues have demonstrated that the coadministration ofSAM during ethanol exposure prevents the ethanol-relateddissociation of mitochondrial ribosomes (69), presumablythrough preserving the methylation status of both tRNA andrRNA (30, 61, 62). This would have the effect of preservingmitochondrial protein synthesis and thus maintaining the in-tegrity of oxidative phosphorylation system complexes, whichwas verified in our study using BN-PAGE proteomics (Fig. 2).Moreover, we observed that SAM prevented the hepaticmtDNA damage caused by chronic alcohol consumption.These results are significant because they indicate that theeffect of SAM to prevent oxidative damage to mtDNA willalso contribute to the preservation of the electron transportchain, which will have the ultimate effect of inhibiting furtherincreases in mitochondrial O2

�� generation. Thus, a mechanismlinking the preservation of the electron transport chain systemto attenuation of mitochondrial oxidant production by SAMcan be proposed. In contrast, SAM had no effect on CYP2E1levels, another source of oxidants after chronic alcohol feeding.Although a study (15) has demonstrated that SAM can inhibitCYP2E1 activity at high concentrations in vitro, it is highlyunlikely that these concentrations were achieved in our study,where animals consumed 100–150 �mol SAM/day.CYP2E1 activity was also unaffected by SAM administrationin an acute model of alcohol hepatotoxicity (66). These datasupport the hypothesis that alcohol consumption initiates mo-lecular alterations to liver mitochondria, specifically the elec-tron transport chain, that lead to the increased production ofO2

��, which is attenuated by SAM.NO and its metabolites have also been linked to mechanisms

of mitochondria damage (58), including that observed afterchronic alcohol exposure (49, 77, 78). In the present study,chronic alcohol consumption induced iNOS protein, which isin agreement with previous work (49, 78). Furthermore, astudy (44) has indicated that the protective effects of SAM maybe due in part through inhibition of NO and RNS formation inthe liver via the suppression of TNF-�-mediated iNOS induc-tion. Similarly, we observed an attenuation of iNOS inductionin ethanol-fed animals coadministered SAM. Although theinhibition of NO production has been shown to exert bothdetrimental and beneficial effects against liver toxicity, it isgenerally accepted that the generation of ONOO� duringconditions of excessive NO and O2

�� production, as in the caseof chronic alcohol exposure, would cause mitochondrial dam-age through the posttranslational modification of proteins (10).The interaction of NO and ONOO� with the respiratory chainmay also play a role in predisposing hepatocytes to hypoxicinjury during ethanol metabolism (64, 77, 78). These conceptsprovide a mechanism for the RNS-dependent amplification ofmitochondrial dysfunction at multiple levels that contribute tochronic alcohol-associated losses in hepatocyte viability. Thus,the ability of SAM to downregulate iNOS in the alcohol-exposed liver would be expected to prevent organelle damageby modulating the interaction of NO and other RNS at the levelof the mitochondrion. Specifically, it is postulated that SAM

treatment may prevent the alcohol-associated increased sensi-tivity of mitochondrial respiration to inhibition NO through theattenuation of iNOS induction.

Although experiments were not conducted in the presentstudy to determine the mechanism by which SAM attenuatesiNOS induction by ethanol, it is predicted that SAM modulatesiNOS expression via downregulation of TNF-� expression (74)and effects on the NF-�B system (44). Moreover, it is nowrecognized that iNOS expression is induced by hypoxia (37,73), which occurs from both acute and chronic alcohol expo-sure (2, 3). It is possible that in our model SAM inhibited thealcohol-mediated increase in iNOS by modulation of hypoxiain the liver. This is in part supported by the observation thatSAM treatment prevented the alcohol-associated decrease incytochrome c oxidase subunits IV and Vb, whose expression isknown to be regulated by oxygen tension (17, 79). Furtherstudies are necessary to determine the mechanism throughwhich SAM may modulate hypoxia or hypoxia-sensitive path-ways in the liver, particularly in the context of alcohol expo-sure.

Interestingly, these findings indicate that SAM may befunctioning to preserve mitochondrial function through mech-anisms independent of oxidant production. As mentionedabove, it is likely that SAM supplementation is preservingtransmethylation reactions of nucleic acids, proteins, and lip-ids. This is important because methylation of tRNA and rRNAis known to be crucial for synthesis of mitochondrial proteins.Moreover, Walker and colleagues (16, 18) have identifiedseveral respiratory chain subunits that are methylated post-translationally. Whether SAM deficiency in response tochronic alcohol alters the methylation status of these proteinsand impacts mitochondrial functioning is not known at present.Another study (9) has also indicated arginine methylation as acrucial posttranslational modification of proteins. SAM is alsorequired for the stabilization of cystathionine �-synthase (55),thus identifying novel impacts on the transsulfuration pathwayand the formation of hydrogen sulfide, which is now recog-nized as an important signaling molecule in mammalian sys-tems (56, 68).

In summary, we have demonstrated that SAM preventsalcohol-dependent mitochondrial dysfunction via the preserva-tion of mitochondrial respiration, attenuation of mitochondrialO2

�� production, and maintenance of the integrity of themtDNA. Even though the coadministration of SAM did notprevent steatosis per se, SAM had a profound effect to atten-uate ethanol-elicited defects in the mitochondrial respiratorychain. These seemingly disparate results highlight the impor-tant concept that alcohol-elicited mechanisms that impair thefunction of the mitochondrial electron transport chain andthose that initiate steatosis may not be the same, overlap, orinteract. Similarly, our results underscore that SAM almostcertainly works through ROS/RNS-dependent and -indepen-dent mechanisms at multiple levels and sites of action toimprove mitochondria functionality. Specifically, this workreveals that SAM prevents alterations to both the mitochondrialgenome and proteome induced by chronic alcohol consumptionand lays the groundwork to identify novel mechanisms thatlink SAM-mediated protection to effects on mitochondrialfunction. These results predict that SAM will preserve thecapacity of mitochondria to maintain ATP levels and sustainthe energy-requiring processes needed to replace nucleic acids,

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structural lipids, and proteins damaged in response to contin-ued alcohol exposure. As long as ATP levels remain normal,much of the damage elicited by ethanol can be repaired, andhepatocyte necrosis will not occur. Thus, the ability of SAM topreserve mitochondrial function after early exposure to alcoholmay prevent the progression to more permanent liver injuryand cell death in the chronic alcohol consumer when used incombination with other therapeutic agents or strategies.

ACKNOWLEDGMENTS

The authors thank Dr. Phillip J. Coates (University of Dundee, Dundee,UK) for kindly providing the BAP37 antiserum and Dr. Shaonin Ji, TracyWarden, Julie Kiessling, and Terry Lewis for technical assistance.

GRANTS

This work was funded by National Institute on Alcohol Abuse and Alco-holism Grants AA-15172 (to S. M. Bailey) and AA-13395 (to V. Darley-Usmar).

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