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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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NEUROREGULATION AND MOTILITY

S-adenosylmethionine prevents chronic alcohol-induced mitochondrial dysfunction 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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
An early event that occurs in response to alcohol consumption is mitochondrial dysfunction, which is evident in changes to the mitochondrial proteome, respiration defects, and mitochondrial DNA (mtDNA) damage. S-adenosylmethionine (SAM) has emerged as a potential therapeutic for treating alcoholic liver disease through mechanisms that appear to involve decreases in oxidative stress and proinflammatory cytokine production as well as the alleviation of steatosis. Because mitochondria are a source of reactive oxygen/nitrogen species and a target for oxidative damage, we tested the hypothesis that SAM treatment during alcohol exposure preserves organelle function. Mitochondria were isolated from livers of rats fed control and ethanol diets with and without SAM for 5 wk. Alcohol feeding caused a significant decrease in state 3 respiration and the respiratory control ratio, whereas SAM administration prevented these alcohol-mediated defects and preserved hepatic SAM levels. SAM treatment prevented alcohol-associated increases in mitochondrial superoxide production, mtDNA damage, and inducible nitric oxide synthase induction, without a significant lessening of steatosis. Accompanying these indexes of oxidant damage, SAM prevented alcohol-mediated losses in cytochrome c oxidase subunits as shown using blue native PAGE proteomics and immunoblot analysis, which resulted in partial preservation of complex IV activity. SAM treatment attenuated the upregulation of the mitochondrial stress chaperone prohibitin. Although SAM supplementation did not alleviate steatosis by itself, SAM prevented several key alcohol-mediated defects to the mitochondria genome and proteome that contribute to the bioenergetic defect in the liver after alcohol consumption. These findings reveal new molecular targets through which SAM may work to alleviate one critical component of alcohol-induced liver injury: mitochondria dysfunction.

oxidative stress; proteome; blue native gel electrophoresis; cytochrome c oxidase; prohibitin; mitochondrial DNA

CHRONIC ALCOHOL CONSUMPTION causes liver damage by a complex process involving oxidative stress, hypoxia, and energy metabolism defects. Early in this process, endotoxin released from the gut (31) activates Kupffer cells, which in turn leads to an elevated production of cytokines, eicosanoids, and reactive oxygen/nitrogen species (ROS/RNS). This results in a spectrum of responses in the hepatocyte including enhanced hypoxia (2), mitochondrial damage (5), and increased sensitivity to nitric oxide (NO)-dependent inhibition of respiration (77). This increased metabolic activity, which includes increased ROS/RNS production, contributes to hepatocyte death (4).

Disruption in oxidative phosphorylation and the inability to maintain ATP levels are proposed to contribute to the liver damage from chronic alcohol exposure. Specifically, the inability to maintain ATP levels in hepatocytes contributes to necrotic cell death from exposure to toxicants including ethanol (32, 36). Chronic alcohol consumption depresses the activities of all the respiratory complexes except complex II (21). Mitochondrial protein synthesis inhibition due to mitochondrial DNA (mtDNA) (14, 75) and mitochondrial ribosome defects (13, 54) are proposed to contribute to the decreased functioning of respiratory complexes and ATP synthesis (67). These disturbances in mitochondria structure and loss of function are also associated with increased mitochondria ROS production and oxidative injury to the organelle after chronic alcohol exposure. Because increased mitochondrial oxidant production has been identified as one of several key factors mediating alcoholic liver injury, agents aimed at attenuating mitochondria ROS production should prevent mitochondrial dysfunction and the progression to permanent liver damage.

One agent that has received considerable attention in recent years as a potential therapeutic against alcohol-induced liver injury is S-adenosylmethionine (SAM). SAM is the primary methyl donor in anabolic metabolism, serves as a precursor for glutathione (GSH), and is synthesized by the enzyme methionine adenosyltransferase (MAT) (47). Chronic alcohol consumption decreases hepatic SAM levels due to depressed activity of the hepatocyte-specific isoform of MAT (42). Depletion of SAM correlates with DNA hypomethylation and strand breaks (42) as well as increased lipid peroxidation and mitochondrial damage (39). These effects may also contribute to changes in the mitochondrial proteome and modification of thiols in critical enzymes involved in ethanol metabolism including aldehyde dehydrogenase (76). Moreover, MAT1A knockout mice, which are deficient in SAM, spontaneously develop steatohepatitis and hepatocellular carcinoma, thus demonstrating that chronic depletion of SAM predisposes the liver to injury (46). Accordingly, the exogenous administration of SAM has been shown to increase survival in patients with alcoholic cirrhosis (48), presumably through a number of mechanisms including inhibition of steatosis and inflammation as well as repletion of mitochondrial GSH levels (27, 35, 40, 65). Recent studies (1, 25, 59) have indicated that the benefits of SAM might be mediated through effects on mitochondria; however, the specific mechanisms or mitochondria pathways targeted by SAM are unknown. Thus, we investigated the ability of SAM to prevent chronic alcohol-induced mitochondrial damage. Studies were carried out to evaluate the effect of SAM on parameters of mitochondria function as well as mtDNA damage and alterations to the mitochondrial proteome in response to chronic alcohol exposure. Moreover, the ability of SAM to attenuate the effects of alcohol on mitochondrial function will provide insight into whether defects in mitochondrial function are linked to the development of early stages of alcohol-mediated liver injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Feeding protocols. Male Sprague-Dawley rats (3 mo of age, Charles River Laboratories) were individually housed and maintained under a 12:12-h light-dark cycle for the entire duration of the feeding period. Animals had free access to standard laboratory rat chow and water for at least 1 wk and weighed at least 200 g before being provided 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 daily caloric intake as ethanol, 35% as fat, 18% as protein, and 11% as carbohydrate. Control animals were pair fed identical diets except that ethanol calories were replaced isocalorically with carbohydrate (dextrin maltose). A second set of animals were also pair fed the same control and ethanol diets supplemented with SAM (0.8 mg active SAM/ml diet). SAM was provided as the stable 1,4-butanedisulfonate salt form (Denosyl SD4, Nutramax Laboratories, Edgewood, MD). SAM was dissolved in the diets 1 h before animals were fed. Animals were maintained on these feeding protocols for at least 31 days before being used in experiments. These studies were approved by the institutional animal care and use committee, and animals received humane care in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23).

Mitochondria isolation. Mitochondria were isolated by differential centrifugation of the liver homogenate (6). Mitochondria function was assessed by measuring state 3 and 4 respiration and the respiratory control ratio (RCR) using glutamate/malate or succinate as substrates. Cytochrome c oxidase and citrate synthase activities were measured using standard spectrophotometric measures (63, 80). Mitochondrial GSH concentrations were measured using an enzymatic recycling assay (72). Ethanol and SAM treatments had no effect on mitochondrial 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 sections were evaluated by two pathologists blinded to the experimental groups. Steatosis was assessed by determining the percentages of hepatocytes containing fat droplets and were graded as 0 (<5%), 1+ (6–25%), 2+ (26–50%), and 3+ (51–75%). Intra-acinar inflammation 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 x10 objective (78). Liver samples were homogenized in 0.5 M perchloric acid, and the filtered extracts were directly subjected to HPLC analysis for the determination of hepatic SAM levels following the procedures of Fu et al. (26) as detailed in Ref. 8. Triglycerides were measured in the whole liver homogenate as described in Ref. 35.

Mitochondrial superoxide production. Rates of O2·– production in mitochondria were determined using a chemiluminescent coelenterazine assay (70). The production of O2·– was stimulated by the addition of succinate (1 mM) to mitochondria (60 µg) suspended in the same buffer 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 in ethanol) was added to the buffer immediately before the measurements to achieve a concentration of 5 µM for assays. Increases in chemiluminescence were monitored for 5 min at 37°C using an AutoLumat plus model 953 luminometer (Berthold, Germany), and rates of O2·– production were corrected for background rates, i.e., signals generated solely with reactants. The signal (relative light units/s) was integrated over a 30-s period (30–60 s) and used in the final calculation.

Blue native gel electrophoresis. Ethanol and/or SAM-mediated alterations in the levels of proteins that comprise the oxidative phosphorylation system were assessed using blue native (BN)-PAGE proteomics (11, 75). Oxidative phosphorylation complexes were extracted intact from 1 mg of mitochondrial protein using 1% n-dodecyl-beta-D-maltoside in 0.75 M aminocaproic acid and 50 mM BisTris (pH 7.0). Protein extracts were mixed with 2.5 µl of a 5% (wt/vol) suspension of Coomassie brilliant blue G-250 in 0.5 M aminocaproic acid before being applied to nondenaturing 5–16.5% gradient gels to separate individual intact oxidative complexes. After native one-dimensional gel electrophoresis was performed, the entire vertical gel lane containing all the complexes was cut from the gel and laid on top of a denaturing Tris-tricine-SDS-PAGE gradient gel to resolve the individual polypeptides of the complexes based on molecular weight. Samples from each pair of untreated or SAM-treated control and ethanol-fed animals were resolved on one gel to minimize for intergel differences. Gels were stained with Coomassie blue using identical staining and destaining conditions. Image analysis on two-dimensional BN-PAGE gels was performed using Quantity One software (Bio-Rad).

Immunoblot analysis. For inducible NO synthase (iNOS) and cytochrome P-450 2E1 (CYP2E1) detection, whole liver homogenate was used, whereas isolated mitochondria were used to detect mitochondria proteins. Levels of iNOS protein were detected using a 1:5,000 dilution of antibody (Chemicon). Cytochrome c oxidase subunits I, IV, and Vb were detected using 1:10,000, 1:5,000, and 1:500 dilutions, respectively (Molecular Probes). Cytochrome c was detected using a 1:1,000 dilution (BD Pharmingen). CYP2E1 protein was detected using a 1:1,000 dilution (Chemicon). Prohibitin protein was detected using a 1:1,000 dilution (Oncogene), whereas BAP37 protein was detected using a 1:20,000 dilution of antibody (provided by Dr. Phillip Coates). Mitochondrial heat shock protein (Hsp)70 and Hsp60 proteins were detected using 1:10,000 and 1:2,500 dilutions, respectively (StressGen Biotechnologies). After membranes had been incubated with the appropriate secondary antibodies, proteins were visualized using ECL, and the intensities of immunoreactive protein bands were quantified using NIH Image (Bethesda, MD). The protein concentration loaded onto gels was within the linear range of the ECL system used for all proteins.

Quantitative PCR. Genomic DNA was extracted from livers and quantified using PicoGreen (Molecular Probes), and 15 ng of DNA were used for quantitative PCR. The sensitivity of the assay is increased via amplification of large targets, thereby increasing the probability of encountering a DNA lesion. A 16,059-bp quantitative PCR product that included all but 236 bp of the NADH5/6 genes in the mtDNA was amplified using a specified primer set (38). Copy number differences in mtDNA were normalized using a short quantitative PCR, which yielded products directly related to gene copy numbers. mtDNA damage was quantified by comparing the relative efficiency of amplification of large (>15 kb) fragments of DNA and normalizing this with gene copy numbers by the amplification of smaller (<250 bp) fragments, which have a statistically negligible likelihood of containing damaged bases (38, 75).

Statistical analyses. Data represent means ± SE for 6 pairs of animals/group. Significant differences between treatments were obtained using paired t-tests and two-factor ANOVA. P < 0.05 was considered as significant. The Mann-Whitney rank sum test was used for pathology analyses.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Animals and liver histopathology. There were no significant differences in weight gain among all groups (Table 1). Ethanol consumption in untreated and SAM-treated groups was 10.8 ± 0.4 and 12.9 ± 0.5 g·kg–1·day–1, respectively, and was associated with a significant increase in liver weight and the liver-to-body weight ratio in the ethanol-only group (Table 1). Consumption of ethanol resulted in the development of a mild steatosis, which was accompanied by increased triglyceride levels in both untreated and SAM-treated ethanol-fed animals versus their corresponding controls (Table 1). However, triglyceride 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 was slightly, but not statistically, decreased in ethanol-fed animals coadministered SAM (Table 1).


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  		Table 1. Effect of EtOH and SAM on liver parameters

 
SAM prevents alcohol-mediated decreases in mitochondria respiration. Mitochondrial dysfunction occurs after chronic alcohol consumption and is associated with depressed mitochondrial respiration and ATP synthesis. As mentioned in MATERIALS AND METHODS, the mitochondria protein yield was equivalent among all groups. Similarly, there were no differences in citrate synthase activity, a commonly used mitochondrial marker enzyme, among all groups (control, 0.244 ± 0.01 µmol·min–1·mg–1; ethanol, 0.273 ± 0.02 µmol·min–1·mg–1; control + SAM, 0.267 ± 0.1 µmol·min–1·mg–1; and ethanol + SAM, 0.253 ± 0.1 µmol·min–1·mg–1, P = 0.1). To determine whether SAM supplementation prevented chronic alcohol-mediated defects in mitochondria function, oxygen consumption in the presence of substrates (glutamate/malate or succinate) and ADP was measured in coupled mitochondria isolated from livers of animals fed control and ethanol diets with and without added SAM. State 3 respiration (ADP dependent) and the RCR were significantly decreased in animals fed ethanol compared with controls (Fig. 1, A–D). Chronic alcohol exposure also resulted in a significant decrease in cytochrome c oxidase activity (Fig. 1E). In contrast, state 4 respiration (ADP independent) was unaffected by ethanol consumption, suggesting that uncoupling does not contribute to mitochondrial dysfunction (data not shown). State 3 respiration and the RCR were largely preserved in ethanol-fed animals coadministered SAM (Fig. 1, A–D). Moreover, SAM partially prevented the alcohol-dependent loss in cytochrome c oxidase activity (Fig. 1E). This alcohol-associated defect in mitochondrial function was not accompanied by significant alterations in mitochondrial GSH levels (control, 6.9 ± 1.1 nmol/mg protein; ethanol, 8.6 ± 1.1 nmol/mg protein, control + SAM, 7.1 ± 0.7 nmol/mg protein; and ethanol + SAM, 8.5 ± 0.5 nmol/mg protein, P = 0.84). However, the coadministration of SAM prevented the ethanol-associated decrease in hepatic levels of SAM (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 alcohol-dependent mitochondrial dysfunction because SAM preserved respiratory rates at control levels.


Figure 1
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  		Fig. 1. S-adenosylmethionine (SAM) prevents chronic alcohol-mediated decreases in mitochondrial respiration and cytochrome c oxidase (COX) activity. State 3 respiration was measured, and the respiratory control ratio (RCR) was determined using either glutamate/malate (Glu/Mal; A and B) or succinate (Suc; C and D) as oxidizable substrates in freshly isolated mitochondria. COX activity (E) was measured in frozen mitochondria samples by monitoring the rate of oxidation of fully reduced cytochrome c at 550 nm. Data represent means ± SE for n = 6 animals/group. aP < 0.05 compared with control (Con); bP < 0.05 compared with ethanol (EtOH) alone.

 
SAM prevents alcohol-dependent losses in oxidative phosphorylation proteins. It is possible that SAM maintains mitochondria respiration through preservation against the alcohol-associated loss in oxidative phosphorylation proteins. Indeed, two-dimension BN-PAGE proteomics supported the hypothesis that SAM protects against the loss in both mitochondria- and nuclear-encoded polypeptides that comprise the oxidative phosphorylation system (75). Representative BN-PAGE proteomic maps of the oxidative phosphorylation proteins are shown in Fig. 2. From these maps, it was apparent that chronic alcohol consumption caused a loss of respiratory chain proteins (Fig. 2, A and B). This was especially true for the proteins that comprise cytochrome c oxidase (circled proteins, Fig. 2, A and B) and, to a lesser extent, those subunits that comprise complex I. Quantification of the band density within each complex revealed a significant decrease in the overall content of cytochrome c oxidase and NADH dehydrogenase (complex I) in response to chronic alcohol consumption (Fig. 2E) with only minor effects on ubiquinol cytochrome bc1 reductase (complex III) and ATP synthase (complex V). It is postulated that decreases in respiratory complex proteins contribute to the depressed state 3 respiration and cytochrome c oxidase activity in ethanol-fed animals. Whereas SAM supplementation only modestly prevented the alcohol-associated loss in complex I subunits, SAM prevented a loss in cytochrome c oxidase subunits in response to chronic alcohol (Fig. 2, C–E). Thus, changes in some of the cytochrome c oxidase subunits were verified by Western blot analysis. Immunoblot analysis indicated a 70% decrease in the level of mitochondria-encoded cytochrome c oxidase subunit I protein in mitochondria from ethanol-fed rats vs. controls, which was partially attenuated by the coadministration of SAM (Fig. 3A). Similarly, SAM also prevented the alcohol-mediated loss in two of the nuclear-encoded subunits, subunits IV and Vb (Fig. 3, C and D). SAM alone had no effect on the expression of these subunits in control animals, and ethanol and/or SAM treatment had no effect on the levels of cytochrome c (Fig. 3B). These data indicate that SAM protects at the molecular level through preservation of the mitochondrial proteome in response to chronic alcohol consumption.


Figure 2
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  		Fig. 2. SAM prevents the loss of oxidative phosphorylation proteins in response chronic alcohol consumption. Representative 2-dimensional blue native (BN)-PAGE proteomic maps of mitochondria isolated from Con (A), EtOH-treated (B), Con + SAM-treated (C), and EtOH + SAM-treated (D) animals are shown. For these gels, 250 µg of mitochondrial protein were subjected to 1-dimensional BN-PAGE. The 1-dimensional gel strips were overlaid across a Tris-tricine-SDS-PAGE gel to resolve the individual polypeptides that comprise each oxidative phosphorylation system (OxPhos) complex. The proteins that comprise each complex (I, V, III, IV, and II) appear as vertically aligned spots on the 2-dimensional gel. The proteins that comprise COX are circled. E: comparison of the relative quantities of complexes I, V, III, and IV in liver mitochondria from the Con, EtOH, Con + SAM, and EtOH + SAM groups. Results represent means ± SE for 3 pairs of animals/group. aP < 0.05 compared with Con; bP <0.05 compared with EtOH; cP < 0.05 compared with Con + SAM.

 

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

 
Effect of ethanol and SAM on the mitochondrial chaperone prohibitin. To date, the impact of chronic alcohol consumption on the mitochondrial chaperone system is undefined. The proteins prohibitin and BAP37 together form a complex within the mitochondrial inner membrane that functions in the assembly of respiratory complexes (50). Studies have shown that metabolic stress associated with mitochondrial protein synthesis inhibition causes an upregulation of prohibitin protein. It is suggested that this occurs as an adaptive response to help protect against a continued loss of mitochondrial gene products that might occur in response to the imbalance between the content of mitochondria- and nuclear-encoded subunits in the inner membrane (19, 51). Because chronic alcohol consumption induces a similar imbalance (75), we chose to examine the effect of chronic alcohol and SAM on prohibitin. Both prohibitin and BAP37 protein were increased by 50% and 40%, respectively, in mitochondria from ethanol-fed rats compared with controls (Fig. 4, A and B). In contrast, there were no differences in prohibitin and BAP37 levels in mitochondria isolated from control and ethanol-fed animals in the SAM group (Fig. 4, A and B). Chronic alcohol also had no effect on the levels of two additional mitochondria-associated chaperones, Hsp70 and Hsp60 (Fig. 4, C and D).


Figure 4
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  		Fig. 4. Effect of EtOH and SAM on mitochondrial chaperones. Top, representative immunoblots for prohibitin (A), BAP37 (B), heat shock protein (Hsp)70 (C), and Hsp60 (D) protein from one pair of Con and EtOH-treated animals and one pair of animals fed diets supplemented with SAM. Bottom, quantification of proteins. Results represent means ± SE for n = 4–6 pairs of animals/group. aP < 0.05 compared with Con.

 
Effect of SAM on mitochondria O2·– production and iNOS expression. It is hypothesized that the effect of SAM to prevent the alcohol-associated losses in oxidative phosphorylation proteins (Figs. 2 and 3) will also attenuate chronic alcohol-associated increases in mitochondrial O2·– production. As shown in Fig. A, O2·– production was significantly increased in mitochondria isolated from ethanol-fed animals compared with controls. Mitochondria from ethanol-fed animals also generated 40% more O2·– compared with control mitochondria in the presence of antimycin (Fig. 5B, inset), which is consistent with increased O2·– production related to ethanol-elicited damage to the cytochrome bc1 region of complex III (4, 20). Importantly, SAM treatment attenuated the alcohol-dependent increase in mitochondrial O2·– production in both the presence and absence of antimycin (Fig. 5). Another potential source of O2·– in the chronic alcohol-exposed liver is CYP2E1 (34). Although CYP2E1 protein was induced by chronic alcohol consumption, SAM did not prevent this increase (Fig. 6A). These results indicate that the ability of SAM to attenuate oxidant production is largely mediated through its ability to preserve the functioning of the electron transport chain via preventing losses in proteins that comprise respiratory complexes (Fig. 2).


Figure 5
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  		Fig. 5. SAM attenuates chronic alcohol-induced increases in mitochondrial superoxide (O2·–) production [in relative light units (RLU)/s]. Mitochondria were isolated from livers of rats fed Con and EtOH-containing diets supplemented with and without SAM and incubated with colenterazine (5 µM) to monitor O2·– production (A). To ascertain the extent of alcohol-induced damage to complex III, mitochondria were incubated with antimycin (20 µg/ml), and rates of O2·– production were measured using the coelenterazine assay (B, inset). Antimycin results are reported as percentages of Con due to the large degree of variability in the stimulation of O2·– levels in response to antimycin. aP < 0.05 compared with Con; bP < 0.05 compared with EtOH.

 

Figure 6
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  		Fig. 6. Effect of EtOH and SAM on cytochrome P-450 2E1 (CYP2E1) and inducible nitric oxide synthase (iNOS) protein levels. Top, representative immunoblots of CYP2E1 (A) and iNOS (B) for one pair of Con and EtOH-treated animals and one pair of animals fed diets supplemented with SAM. Bottom, quantification of protein. Results represent means ± SE for n = 3–6 pairs of animals/group. aP < 0.01 compared with Con; cP < 0.05 compared with Con + SAM.

 
Accompanying the attenuation of mitochondrial O2·– production was the concomitant suppression of iNOS protein induction in ethanol-fed animals coadministered SAM. Immunoblot analysis indicated a 70% increase in the level of iNOS protein in livers of ethanol-fed animals vs. controls, which was largely attenuated by the coadministration of SAM (Fig. 6B). It is postulated that SAM contributes to the preservation of mitochondria function through attenuation of iNOS, thus preventing oxidative damage to mitochondrial proteins, as well as mtDNA, from alcohol-induced increases in RNS.

SAM attenuates chronic alcohol-mediated mtDNA damage. We (75) have previously shown that chronic alcohol consumption induces mtDNA damage in young animals (75), which in turn is postulated to negatively impact mitochondrial protein synthesis and functioning of the oxidative phosphorylation system, and thus leads to increased mitochondrial O2·– production. The ability of SAM to prevent chronic alcohol-mediated mtDNA damage was assessed using quantitative PCR. The principle of this assay is that DNA lesions block the polymerase leading to decreased amplification of the target of interest, i.e., the mitochondrial genome. Thus, a decreased PCR product compared with control indicates mtDNA damage. There was a significant loss in the PCR product from ethanol-fed animals compared with controls, which was prevented by the coadministration of SAM (Fig. 7). There were no differences in mtDNA copy numbers among all groups (data not shown). These results are consistent with the hypothesis that chronic alcohol consumption induces damage to the mitochondrial genome and that this effect can be prevented by the coadministration of SAM.


Figure 7
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  		Fig. 7. Chronic alcohol-mediated mitochondrial DNA (mtDNA) damage is prevented by coadministration of SAM. Genomic DNA was prepared from livers, and mtDNA damage was assessed using quantitative PCR (QPCR). Top, representative amounts of QPCR products generated from the liver tissue of Con-, EtOH-, Con + SAM-, and EtOH + SAM-treated animals. Less QPCR product indicates increased mtDNA damage. Bottom, bar graph indicating the relative amount of mtDNA damage (lesions/16.2 kb) in livers from EtOH-fed animals compared with the corresponding Con animals. Con values are set at 0 lesions/16.2 kb. aP = 0.02 compared with Con; bP = 0.004 compared with EtOH.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
As a source of ROS/RNS and a locus of resulting oxidative/nitrative modifications, mitochondria are recognized as critical components in the cellular stress response to toxic agents. Ethanol consumption causes increased mitochondrial O2·– production and iNOS induction, which in turn results in increased NO generation, an enhanced sensitivity of the respiratory chain to inhibition by NO, and the subsequent production of more toxic RNS like peroxynitrite (ONOO–). This oxidative/nitrative stress contributes to mitochondrial injury, which manifests as an overall defect in oxidative phosphorylation. This causes further oxidant production and escalating levels of damage at multiple sites. The consequence of this mechanism with various feedback loops is an overall decrease in mitochondrial function that places the hepatocyte under a bioenergetic stress. This is important because a loss in the capacity for maintenance of hepatic ATP levels will ultimately predispose the tissue to permanent damage due to a depression in anabolic processes responsible for replacing damaged and/or lost cellular components. Thus, agents aimed at "short circuiting" these responses are predicted to prevent mitochondrial dysfunction and potentially delay the onset or severity of disease. In this study, we evaluated the ability of SAM to prevent several indexes of alcohol-induced mitochondrial damage.

Whereas SAM supplementation partially preserved hepatic SAM levels in ethanol-fed animals, it had only a minimal effect on preventing alcohol-induced steatosis (7, 33, 40). Because SAM did not significantly impact steatosis, it is possible that the concentration of SAM used in the present study was insufficient to alleviate the inhibitory effect of S-adenosylhomocysteine (SAH) on methyltransferase activity. SAH is formed as a by-product of methyltransferase reactions involving SAM and is increased after alcohol consumption due to the impaired activity of methionine synthase and subsequent removal of homocysteine (8, 28). It is proposed that SAH inhibits the methyltransferase that converts phosphatidylethanolamine to phosphatidylcholine. Duce and colleagues (24) demonstrated a reduction in phospholipid methyltransferase activity in human cirrhotic patients, which was also observedin baboons fed ethanol chronically (41). Because phosphatidylcholine is required for the synthesis and secretion of very-low-density lipoprotein, a defect in its synthesis could be expected to lead to the accumulation of triglyceride in the liver (52). It is predicted that an increased dose of SAM might have been more effective in reducing triglycerides levels via normalization of the SAM-to-SAH ratio in the liver. This is likely because betaine, a choline metabolite that remethylates homocysteine to methionine, prevents alcohol-induced steatosis in rodents when administered in much higher concentrations than were 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 mitochondrial targets and pathways through which SAM works to prevent alcohol-induced mitochondrial dysfunction. An early study (27) has demonstrated that the coadministration of SAM during chronic alcohol feeding maintained mitochondrial GSH levels and preserved mitochondria functions. These results suggested that SAM protects mitochondria from ethanol-associated oxidative damage by maintaining GSH. However, several recent studies (6, 22, 45, 53, 57, 60) have demonstrated that mitochondria GSH depletion may not be a consistent feature of chronic alcohol exposure. We observed a small increase in mitochondrial GSH levels in response to alcohol that may represent an adaptive response to counteract the mild to moderate oxidative stress induced by chronic alcohol consumption (43). Similar adaptive increases in GSH have been shown in other systems of mild oxidant exposure (23). Thus, our results suggest that the protective effects of SAM on mitochondria might be mediated through effects on other mitochondrial targets, such as preservation of the proteins that comprise the oxidative phosphorylation system.

Indeed, recent evidence suggests the involvement of SAM in the regulation of mitochondrial proteins. It is possible that SAM maintains mitochondrial respiration by preventing alcohol-associated losses in the polypeptides that comprise the respiratory complexes (75). BN-PAGE proteomics clearly demonstrates the preservation in respiratory chain proteins in SAM-treated ethanol animals compared with the low levels detected in animals exposed to ethanol alone. This is particularly true for cytochrome c oxidase, whose activity and subunit composition were partially preserved in ethanol-fed animals coadministered SAM. Whereas the mechanism responsible for the ethanol-related decrease in mitochondria-encoded subunits has been attributed to lesions in the mitochondrial ribosome (12, 54), the decrease in nuclear-encoded subunits has only recently been recognized, and the mechanism is undefined (75). Recent work has demonstrated a coordinated downregulation of both mitochondria- and nuclear-encoded subunits of cytochrome c oxidase, specifically subunits IV and Vb, in response to hypoxia due to reduced transcription (79). Similarly, Chandel et al. (17) proposed that alterations in cytochrome c oxidase activity occur in response to hypoxia and play a role in oxygen sensing. Our findings demonstrating the preservation of these hypoxia-sensitive subunits in ethanol-fed animals coadministered SAM suggests that SAM prevents alcohol-associated mitochondria dysfunction in part through interactions with pathways involved in the cellular response to hypoxic stress.

Our results are also consistent with those demonstrating disrupted mitochondrial function in the MAT1A knockout mouse, which is deficient in SAM (59). Depletion of hepatic SAM in these mice results in decreased levels of mitochondria-encoded subunits of cytochrome c oxidase, reduced membrane potential, and alterations in prohibitin levels (59). As discussed earlier, the best-described function of the prohibitin complex is as a chaperone within the inner membrane (50). This complex stabilizes mitochondrial translation products by binding to them before the subunits are transferred to the newly assembled complex (51). According to this concept, an alteration in the level or functioning of the prohibitin complex may impact the assembly and activity of the oxidative phosphorylation system. While prohibitin levels are depressed in the MAT1A knockout mouse, we observed an upregulation in prohibitin and its binding partner BAP37 in response to chronic alcohol consumption, which was attenuated by SAM. This response appears to be specific for inner membrane chaperones because chronic alcohol consumption had no effect on the classic "heat shock" chaperones Hsp70 and Hsp60, which are associated with mitochondria (29). The upregulation of prohibitin and BAP37 suggests an attempt of the hepatocyte to "adapt" to the metabolic stress induced by chronic alcohol consumption by trying to minimize losses in mitochondria-encoded polypeptides and thus preserve mitochondrial function. Similar adaptive increases in prohibitin have been found using other models of metabolic stress (19, 51) and apoptosis (71). It is possible that the decrease in prohibitin levels observed in the MAT1A knockout mouse (59) reflects a failure of this adaptive response in more severe models of liver disease (46). Clearly, the effect of chronic alcohol and SAM on the function of prohibitin warrants further investigation.

It is well established that there are chronic alcohol-induced defects in complexes I, III, and IV due to losses in several of the polypeptides and redox centers that comprise these enzyme complexes. These defects are the result of alcohol-induced damage to the mitochondrial genome and ribosomes (12, 13, 54, 75). Decreases in the content of proteins downstream from the redox active centers of complex I and III, as well as a loss in complex IV subunits, impedes electron flow within the respiratory chain. As a consequence, the respiratory complexes are in a more reduced state, which facilitates electron transfer to molecular oxygen to generate O2·–. This occurs at complexes I and III because they contain stable semiquinone intermediates as redox components. Thus, disruption of the electron transport chain in response to chronic alcohol consumption results in increased mitochondrial O2·– production. Recent data by Cahill and colleagues have demonstrated that the coadministration of SAM during ethanol exposure prevents the ethanol-related dissociation of mitochondrial ribosomes (69), presumably through preserving the methylation status of both tRNA and rRNA (30, 61, 62). This would have the effect of preserving mitochondrial protein synthesis and thus maintaining the integrity of oxidative phosphorylation system complexes, which was verified in our study using BN-PAGE proteomics (Fig. 2). Moreover, we observed that SAM prevented the hepatic mtDNA damage caused by chronic alcohol consumption. These results are significant because they indicate that the effect of SAM to prevent oxidative damage to mtDNA will also contribute to the preservation of the electron transport chain, which will have the ultimate effect of inhibiting further increases in mitochondrial O2·– generation. Thus, a mechanism linking the preservation of the electron transport chain system to attenuation of mitochondrial oxidant production by SAM can be proposed. In contrast, SAM had no effect on CYP2E1 levels, another source of oxidants after chronic alcohol feeding. Although a study (15) has demonstrated that SAM can inhibit CYP2E1 activity at high concentrations in vitro, it is highly unlikely that these concentrations were achieved in our study, where animals consumed ~100–150 µmol SAM/day. CYP2E1 activity was also unaffected by SAM administration in an acute model of alcohol hepatotoxicity (66). These data support the hypothesis that alcohol consumption initiates molecular alterations to liver mitochondria, specifically the electron transport chain, that lead to the increased production of O2·–, which is attenuated by SAM.

NO and its metabolites have also been linked to mechanisms of mitochondria damage (58), including that observed after chronic alcohol exposure (49, 77, 78). In the present study, chronic alcohol consumption induced iNOS protein, which is in agreement with previous work (49, 78). Furthermore, a study (44) has indicated that the protective effects of SAM may be due in part through inhibition of NO and RNS formation in the liver via the suppression of TNF-{alpha}-mediated iNOS induction. Similarly, we observed an attenuation of iNOS induction in ethanol-fed animals coadministered SAM. Although the inhibition of NO production has been shown to exert both detrimental and beneficial effects against liver toxicity, it is generally accepted that the generation of ONOO– during conditions of excessive NO and O2–· production, as in the case of chronic alcohol exposure, would cause mitochondrial damage through the posttranslational modification of proteins (10). The interaction of NO and ONOO– with the respiratory chain may also play a role in predisposing hepatocytes to hypoxic injury during ethanol metabolism (64, 77, 78). These concepts provide a mechanism for the RNS-dependent amplification of mitochondrial dysfunction at multiple levels that contribute to chronic 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 damage by modulating the interaction of NO and other RNS at the level of the mitochondrion. Specifically, it is postulated that SAM treatment may prevent the alcohol-associated increased sensitivity of mitochondrial respiration to inhibition NO through the attenuation of iNOS induction.

Although experiments were not conducted in the present study to determine the mechanism by which SAM attenuates iNOS induction by ethanol, it is predicted that SAM modulates iNOS expression via downregulation of TNF-{alpha} expression (74) and effects on the NF-{kappa}B system (44). Moreover, it is now recognized that iNOS expression is induced by hypoxia (37, 73), which occurs from both acute and chronic alcohol exposure (2, 3). It is possible that in our model SAM inhibited the alcohol-mediated increase in iNOS by modulation of hypoxia in the liver. This is in part supported by the observation that SAM treatment prevented the alcohol-associated decrease in cytochrome c oxidase subunits IV and Vb, whose expression is known to be regulated by oxygen tension (17, 79). Further studies are necessary to determine the mechanism through which SAM may modulate hypoxia or hypoxia-sensitive pathways in the liver, particularly in the context of alcohol exposure.

Interestingly, these findings indicate that SAM may be functioning to preserve mitochondrial function through mechanisms independent of oxidant production. As mentioned above, it is likely that SAM supplementation is preserving transmethylation reactions of nucleic acids, proteins, and lipids. This is important because methylation of tRNA and rRNA is known to be crucial for synthesis of mitochondrial proteins. Moreover, Walker and colleagues (16, 18) have identified several respiratory chain subunits that are methylated posttranslationally. Whether SAM deficiency in response to chronic alcohol alters the methylation status of these proteins and impacts mitochondrial functioning is not known at present. Another study (9) has also indicated arginine methylation as a crucial posttranslational modification of proteins. SAM is also required for the stabilization of cystathionine beta-synthase (55), thus identifying novel impacts on the transsulfuration pathway and the formation of hydrogen sulfide, which is now recognized as an important signaling molecule in mammalian systems (56, 68).

In summary, we have demonstrated that SAM prevents alcohol-dependent mitochondrial dysfunction via the preservation of mitochondrial respiration, attenuation of mitochondrial O2·– production, and maintenance of the integrity of the mtDNA. Even though the coadministration of SAM did not prevent steatosis per se, SAM had a profound effect to attenuate ethanol-elicited defects in the mitochondrial respiratory chain. These seemingly disparate results highlight the important concept that alcohol-elicited mechanisms that impair the function of the mitochondrial electron transport chain and those that initiate steatosis may not be the same, overlap, or interact. Similarly, our results underscore that SAM almost certainly works through ROS/RNS-dependent and -independent mechanisms at multiple levels and sites of action to improve mitochondria functionality. Specifically, this work reveals that SAM prevents alterations to both the mitochondrial genome and proteome induced by chronic alcohol consumption and lays the groundwork to identify novel mechanisms that link SAM-mediated protection to effects on mitochondrial function. These results predict that SAM will preserve the capacity of mitochondria to maintain ATP levels and sustain the energy-requiring processes needed to replace nucleic acids, structural lipids, and proteins damaged in response to continued alcohol exposure. As long as ATP levels remain normal, much of the damage elicited by ethanol can be repaired, and hepatocyte necrosis will not occur. Thus, the ability of SAM to preserve mitochondrial function after early exposure to alcohol may prevent the progression to more permanent liver injury and cell death in the chronic alcohol consumer when used in combination with other therapeutic agents or strategies.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by National Institute on Alcohol Abuse and Alcoholism Grants AA-15172 (to S. M. Bailey) and AA-13395 (to V. Darley-Usmar).


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


   FOOTNOTES
 

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{at}uab.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Agrimi G, Di Noia MA, Marobbio CM, Fiermonte G, Lasorsa FM, and Palmieri F. Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution. Biochem J 379: 183–190, 2004.[CrossRef][ISI][Medline]
  2. Arteel GE, Iimuro Y, Yin M, Raleigh JA, and Thurman RG. Chronic enteral ethanol treatment causes hypoxia in rat liver tissue in vivo. Hepatology 25: 920–926, 1997.[CrossRef][ISI][Medline]
  3. Arteel GE, Raleigh JA, Bradford BU, and Thurman RG. Acute alcohol produces hypoxia directly in rat liver tissue in vivo: role of Kupffer cells. Am J Physiol Gastrointest Liver Physiol 271: G494–G500, 1996.[Abstract/Free Full Text]
  4. Bailey SM and Cunningham CC. Acute and chronic ethanol increases reactive oxygen species generation and decreases viability in fresh, isolated rat hepatocytes. Hepatology 28: 1318–1326, 1998.[CrossRef][ISI][Medline]
  5. Bailey SM and Cunningham CC. Contribution of mitochondria to oxidative stress associated with alcoholic liver disease. Free Radic Biol Med 32: 11–16, 2002.[CrossRef][ISI][Medline]
  6. Bailey SM, Patel VB, Young TA, Asayama K, and Cunningham CC. Chronic ethanol consumption alters the glutathione/glutathione peroxidase-1 system and protein oxidation status in rat liver. Alcohol Clin Exp Res 25: 726–733, 2001.[CrossRef][ISI][Medline]
  7. Barak AJ, Beckenhauer HC, Badakhsh S, and Tuma DJ. The effect of betaine in reversing alcoholic steatosis. Alcohol Clin Exp Res 21: 1100–1102, 1997.[CrossRef][ISI][Medline]
  8. Barak AJ, Beckenhauer HC, Mailliard ME, Kharbanda KK, and Tuma DJ. Betaine lowers elevated S-adenosylhomocysteine levels in hepatocytes from ethanol-fed rats. J Nutr 133: 2845–2848, 2003.[Abstract/Free Full Text]
  9. Bedford MT and Richard S. Arginine methylation an emerging regulator of protein function. Mol Cell 18: 263–272, 2005.[CrossRef][ISI][Medline]
  10. Brookes PS, Kraus DW, Shiva S, Doeller JE, Barone MC, Patel RP, Lancaster JR Jr, and Darley-Usmar V. Control of mitochondrial respiration by NO*, effects of low oxygen and respiratory state. J Biol Chem 278: 31603–31609, 2003.[Abstract/Free Full Text]
  11. Brookes PS, Pinner A, Ramachandran A, Coward L, Barnes S, Kim H, and Darley-Usmar VM. High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes. Proteomics 2: 969–977, 2002.[CrossRef][ISI][Medline]
  12. Cahill A, Baio DL, Ivester PI, and Cunningham CC. Differential effects of chronic ethanol consumption on hepatic mitochondrial and cytoplasmic ribosomes. Alcohol Clin Exp Res 20: 1362–1367, 1996.[CrossRef][ISI][Medline]
  13. Cahill A and Cunningham CC. Effects of chronic ethanol feeding on the protein composition of mitochondrial ribosomes. Electrophoresis 21: 3420–3426, 2000.[CrossRef][ISI][Medline]
  14. Cahill A, Wang X, and Hoek JB. Increased oxidative damage to mitochondrial DNA following chronic ethanol consumption. Biochem Biophys Res Commun 235: 286–290, 1997.[CrossRef][ISI][Medline]
  15. Caro AA and Cederbaum AI. Inhibition of CYP2E1 catalytic activity in vitro by S-adenosyl-L-methionine. Biochem Pharmacol 69: 1081–1093, 2005.[CrossRef][ISI][Medline]
  16. Carroll J, Fearnley IM, Skehel JM, Runswick MJ, Shannon RJ, Hirst J, and Walker JE. The post-translational modifications of the nuclear encoded subunits of complex I from bovine heart mitochondria. Mol Cell Proteomics 4: 693–699, 2005.[Abstract/Free Full Text]
  17. Chandel NS, Budinger GR, Choe SH, and Schumacker PT. Cellular respiration during hypoxia. Role of cytochrome oxidase as the oxygen sensor in hepatocytes. J Biol Chem 272: 18808–18816, 1997.[Abstract/Free Full Text]
  18. Chen R, Fearnley IM, Palmer DN, and Walker JE. Lysine 43 is trimethylated in subunit C from bovine mitochondrial ATP synthase and in storage bodies associated with batten disease. J Biol Chem 279: 21883–21887, 2004.[Abstract/Free Full Text]
  19. Coates PJ, Nenutil R, McGregor A, Picksley SM, Crouch DH, Hall PA, and Wright EG. Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence. Exp Cell Res 265: 262–273, 2001.[CrossRef][ISI][Medline]
  20. Coleman WB and Cunningham CC. Effects of chronic ethanol consumption on the synthesis of polypeptides encoded by the hepatic mitochondrial genome. Biochim Biophys Acta 1019: 142–150, 1990.[Medline]
  21. Cunningham CC, Coleman WB, and Spach PI. The effects of chronic ethanol consumption on hepatic mitochondrial energy metabolism. Alcohol Alcohol 25: 127–136, 1990.[Abstract/Free Full Text]
  22. Deaciuc IV, Fortunato F, D'Souza NB, Hill DB, Schmidt J, Lee EY, and McClain CJ. Modulation of capase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide. Alcohol Clin Exp Res 23: 349–356, 1999.[CrossRef][ISI][Medline]
  23. Dickinson DA, Moellering DR, Iles KE, Patel RP, Levonen AL, Wigley A, Darley-Usmar VM, and Forman HJ. Cytoprotection against oxidative stress and the regulation of glutathione synthesis. Biol Chem 384: 527–537, 2003.[CrossRef][ISI][Medline]
  24. Duce AM, Ortiz P, Cabrero C, and Mato JM. S-adenosyl-L-methionine synthetase and phospholipid methyltransferase are inhibited in human cirrhosis. Hepatology 8: 65–68, 1988.[ISI][Medline]
  25. Fernandez-Checa JC, Colell A, and Garcia-Ruiz C. S-adenosyl-L-methionine and mitochondrial reduced glutathione depletion in alcoholic liver disease. Alcohol 27: 179–183, 2002.[CrossRef][ISI][Medline]
  26. Fu W, Dudman NP, Perry MA, Young K, and Wang XL. Interrelations between plasma homocysteine and intracellular S-adenosylhomocysteine. Biochem Biophys Res Commun 271: 47–53, 2000.[CrossRef][ISI][Medline]
  27. Garcia-Ruiz C, Morales A, Colell A, Ballesta A, Rodes J, Kaplowitz N, and Fernandez-Checa JC. Feeding S-adenosyl-L-methionine attenuates both ethanol-induced depletion of mitochondrial glutathione and mitochondrial dysfunction in periportal and perivenous rat hepatocytes. Hepatology 21: 207–214, 1995.[CrossRef][ISI][Medline]
  28. Halsted CH, Villanueva JA, Devlin AM, Niemela O, Parkkila S, Garrow TA, Wallock LM, Shigenaga MK, Melnyk S, and James SJ. Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig. Proc Natl Acad Sci USA 99: 10072–10077, 2002.[Abstract/Free Full Text]
  29. He L and Lemasters JJ. Heat shock suppresses the permeability transition in rat liver mitochondria. J Biol Chem 278: 16755–16760, 2003.[Abstract/Free Full Text]
  30. Helm M, Brule H, Degoul F, Cepanec C, Leroux JP, Giege R, and Florentz C. The presence of modified nucleotides is required for cloverleaf folding of a human mitochondrial tRNA. Nucleic Acids Res 26: 1636–1643, 1998.[Abstract/Free Full Text]
  31. Hines IN and Wheeler MD. Recent advances in alcoholic liver disease. III. Role of the innate immune response in alcoholic hepatitis. Am J Physiol Gastrointest Liver Physiol 287: G310–G314, 2004.[Abstract/Free Full Text]
  32. Ivester P, Lide MJ, and Cunningham CC. Effect of chronic ethanol consumption on the energy state and structural stability of periportal and perivenous hepatocytes. Arch Biochem Biophys 322: 14–21, 1995.[CrossRef][ISI][Medline]
  33. Ji C and Kaplowitz N. Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice. Gastroenterology 124: 1488–1499, 2003.[CrossRef][ISI][Medline]
  34. Kessova I and Cederbaum AI. CYP2E1: biochemistry, toxicology, regulation and function in ethanol-induced liver injury. Curr Mol Med 3: 509–518, 2003.[CrossRef][ISI][Medline]
  35. Kharbanda KK, Rogers DD, 2nd Mailliard ME, Siford GL, Barak AJ, Beckenhauer HC, Sorrell MF, and Tuma DJ. A comparison of the effects of betaine and S-adenosylmethionine on ethanol-induced changes in methionine metabolism and steatosis in rat hepatocytes. J Nutr 135: 519–524, 2005.[Abstract/Free Full Text]
  36. Kim JS, He L, and Lemasters JJ. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun 304: 463–470, 2003.[CrossRef][ISI][Medline]
  37. Kleinert H, Pautz A, Linker K, and Schwarz PM. Regulation of the expression of inducible nitric oxide synthase. Eur J Pharmacol 500: 255–266, 2004.[CrossRef][ISI][Medline]
  38. Knight-Lozano CA, Young CG, Burow DL, Hu ZY, Uyeminami D, Pinkerton KE, Ischiropoulos H, and Ballinger SW. Cigarette smoke exposure and hypercholesterolemia increase mitochondrial damage in cardiovascular tissues. Circulation 105: 849–854, 2002.[Abstract/Free Full Text]
  39. Lieber CS. S-adenosyl-L-methionine and alcoholic liver disease in animal models: implications for early intervention in human beings. Alcohol 27: 173–177, 2002.[CrossRef][ISI][Medline]
  40. Lieber CS, Casini A, DeCarli LM, Kim CI, Lowe N, Sasaki R, and Leo MA. S-adenosyl-L-methionine attenuates alcohol-induced liver injury in the baboon. Hepatology 11: 165–172, 1990.[CrossRef][ISI][Medline]
  41. Lieber CS, Robins SJ, and Leo MA. Hepatic phosphatidylethanolamine methyltransferase activity is decreased by ethanol and increased by phosphatidylcholine. Alcohol Clin Exp Res 18: 592–595, 1994.[CrossRef][ISI][Medline]
  42. Lu SC, Huang ZZ, Yang H, Mato JM, Avila MA, and Tsukamoto H. Changes in methionine adenosyltransferase and S-adenosylmethionine homeostasis in alcoholic rat liver. Am J Physiol Gastrointest Liver Physiol 279: G178–G185, 2000.[Abstract/Free Full Text]
  43. Lu SC, Huang ZZ, Yang JM, and Tsukamoto H. Effect of ethanol and high-fat feeding on hepatic gamma-glutamylcysteine synthetase subunit expression in the rat. Hepatology 30: 209–214, 1999.[CrossRef][ISI][Medline]
  44. Majano PL, Garcia-Monzon C, Garcia-Trevijano ER, Corrales FJ, Camara J, Ortiz P, Mato JM, Avila MA, and Moreno-Otero R. S-adenosylmethionine modulates inducible nitric oxide synthase gene expression in rat liver and isolated hepatocytes. J Hepatol 35: 692–699, 2001.[CrossRef][ISI][Medline]
  45. Mari M and Cederbaum AI. CYP2E1 overexpression in HepG2 cells induces glutathione synthesis by transcriptional activation of gamma-glutamylcysteine synthetase. J Biol Chem 275: 15563–15571, 2000.[Abstract/Free Full Text]
  46. Martinez-Chantar ML, Corrales FJ, Martinez-Cruz LA, Garcia-Trevijano ER, Huang ZZ, Chen L, Kanel G, Avila MA, Mato JM, and Lu SC. Spontaneous oxidative stress and liver tumors in mice lacking methionine adenosyltransferase 1A. FASEB J 16: 1292–1294, 2002.[Abstract/Free Full Text]
  47. Mato JM, Alvarez L, Ortiz P, and Pajares MA. S-adenosylmethionine synthesis: molecular mechanisms and clinical implications. Pharmacol Ther 73: 265–280, 1997.[CrossRef][ISI][Medline]
  48. Mato JM, Camara J, Fernandez de Paz J, Caballeria L, Coll S, Caballero A, Garcia-Buey L, Beltran J, Benita V, Caballeria J, Sola R, Moreno-Otero R, Barrao F, Martin-Duce A, Correa JA, Pares A, Barrao E, Garcia-Magaz I, Puerta JL, Moreno J, Boissard G, Ortiz P, and Rodes J. S-adenosylmethionine in alcoholic liver cirrhosis: a randomized, placebo-controlled, double-blind, multicenter clinical trial. J Hepatol 30: 1081–1089, 1999.[CrossRef][ISI][Medline]
  49. McKim SE, Gabele E, Isayama F, Lambert JC, Tucker LM, Wheeler MD, Connor HD, Mason RP, Doll MA, Hein DW, and Arteel GE. Inducible nitric oxide synthase is required in alcohol-induced liver injury: studies with knockout mice. Gastroenterology 125: 1834–1844, 2003.[CrossRef][ISI][Medline]
  50. Mishra S, Murphy LC, Nyomba BL, and Murphy LJ. Prohibitin: a potential target for new therapeutics. Trends Mol Med 11: 192–197, 2005.[CrossRef][ISI][Medline]
  51. Nijtmans LG, de Jong L, Artal Sanz M, Coates PJ, Berden JA, Back JW, Muijsers AO, van der Spek H, and Grivell LA. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins. EMBO J 19: 2444–2451, 2000.[CrossRef][ISI][Medline]
  52. Noga AA, Zhao Y, and Vance DE. An unexpected requirement for phosphatidylethanolamine N-methyltransferase in the secretion of very low density lipoproteins. J Biol Chem 277: 42358–42365, 2002.[Abstract/Free Full Text]
  53. Oh SI, Kim CI, Chun HJ, and Park SC. Chronic ethanol consumption affects glutathione status in rat liver. J Nutr 128: 758–763, 1998.[Abstract/Free Full Text]
  54. Patel VB and Cunningham CC. Altered hepatic mitochondrial ribosome structure following chronic ethanol consumption. Arch Biochem Biophys 398: 41–50, 2002.[CrossRef][ISI][Medline]
  55. Prudova A, Bauman Z, Braun A, Vitvitsky V, Lu SC, and Banerjee R. S-adenosylmethionine stabilizes cystathionine beta-synthase and modulates redox capacity. Proc Natl Acad Sci USA 103: 6489–6494, 2006.[Abstract/Free Full Text]
  56. Pryor WA, Houk KN, Foote CS, Fukuto JM, Ignarro LJ, Squadrito GL, and Davies KJ. Free radical biology and medicine: it's a gas, man! Am J Physiol Regul Integr Comp Physiol 291: R491–R511, 2006.[Abstract/Free Full Text]
  57. Puzziferri I, Signorile A, Guerrieri F, Papa S, Cuomo V, and Steardo L. Chronic low dose ethanol intake: biochemical characterization of liver mitochondria in rats. Life Sci 66: 477–484, 2000.[CrossRef][ISI][Medline]
  58. Radi R, Cassina A, Hodara R, Quijano C, and Castro L. Peroxynitrite reactions and formation in mitochondria. Free Radic Biol Med 33: 1451–1464, 2002.[CrossRef][ISI][Medline]
  59. Santamaria E, Avila MA, Latasa MU, Rubio A, Martin-Duce A, Lu SC, Mato JM, and Corrales FJ. Functional proteomics of nonalcoholic steatohepatitis: mitochondrial proteins as targets of S-adenosylmethionine. Proc Natl Acad Sci USA 100: 3065–3070, 2003.[Abstract/Free Full Text]
  60. Sastre J, Minana JB, Alguacil P, Malet G, Gomez-Cambronero L, Marin JA, Vina JR, and Vina J. Chronic ethanol feeding causes oxidative stress in rat liver mitochondria. Prevention by S-adenosyl methionine. Free Radic Res 30: 325–327, 1999.[CrossRef]
  61. Seidel-Rogol BL, McCulloch V, and Shadel GS. Human mitochondrial transcription factor B1 methylates ribosomal RNA at a conserved stem-loop. Nat Genet 33: 23–24, 2003.[CrossRef][ISI][Medline]
  62. Shadel GS. Coupling the mitochondrial transcription machinery to human disease. Trends Genet 20: 513–519, 2004.[CrossRef][ISI][Medline]
  63. Shepherd JA and Garland GP. Citrate synthase from rat liver. Methods Enzymol 13: 11–19, 1969.