Byoung J. (B.J.) Song, PhD, Unit Chief
National Institute on Alcohol Abuse and Alcoholism
National Institutes of Health

5625 Fishers Lane, Room 2S30:MSC 9410
Bethesda MD 20892-9410
telephone: +1 301 496 3985

fax: +1 301 594 3113
e-mail: bjs@mail.nih.gov

 

Alcohol metabolism, functional consequence, and signaling mechanism

In my laboratory, we study the functional role of two enzymes involved in ethanol and acetaldehyde metabolism, in increased oxidative damage and the signaling mechanism during cell death upon exposure to potentially toxic compounds including ethanol. The two enzymes we have studied are ethanol-inducible cytochrome P450 2E1 (CYP2E1) and mitochondrial aldehyde dehydrogenase (ALDH2), which is also involved in the metabolism of toxic lipid peroxides. In the past, we have cloned the cDNAs for rat and human CYP2E1 and showed at least seven distinct means of regulation of CYP2E1 protein. Unlike other P450 gene families, most of which are transcriptionally activated by their respective inducers, CYP2E1 gene is not transcriptionally activated by its inducers such as ethanol and acetone. In fact, we demonstrated that the CYP2E1 activity and protein level are elevated through protein stabilization after exposure to various CYP2E1 inducers (Song et al., J Biol Chem 1986, 1989 and 1995). In the past 8 – 9 years, we have re-focused our research projects on the functional role of CYP2E1 in increased levels of oxidative stress and the cell death signaling pathway. In particular, we are studying the role of the increased CYP2E1 activity with the reduced activity of ALDH2 in alcohol-mediated cellular and tissue damage. Our studies are likely to provide similar mechanisms of oxidative damage in different tissues under many pathological conditions where increased oxidative and nitrosative stress is implicated.

 

Characterization of oxidized proteins in alcohol-exposed animal and human tissues Song, Moon, Abdelmegeed, Henderson in collaboration with Salem, Pacher, Hood, Veenstra

Increased oxidative/nitrosative stress is one of the major contributing factors in ethanol (alcohol) mediated cell and tissue damage. The majority of reactive oxygen and nitrogen species (ROS/RNS) in alcohol-exposed cells and tissues is being produced through direct inhibition of the mitochondrial respiratory chain and induction (activation) of CYP2E 1, inducible nitric oxide synthase, and NADPH-oxidase (Song et al., Alcohol Clin Exp Res 20:138A-146A, 1996). Despite the well-established roles of ROS/RNS in alcohol-induced cellular dysfunction and injury, it is poorly understood which proteins are oxidatively-modified by elevated ROS/RNS and whether their functions are altered. To address these questions, we recently developed a sensitive method of using biotin-N-maleimide (biotin-NM) as a specific probe to positively identify oxidized proteins in alcohol-exposed hepatoma cells and animal tissues (Suh et al., Proteomics 4:3401-12, 2004). In the past 4 years, we have applied this targeted proteomics approach to identify oxidized proteins in cytosolic and mitochondrial fractions of alcohol- exposed mouse or rat livers. The biotin-NM labeled oxidized proteins were purified with streptavidin-agarose beads and resolved by 2-D gel electrophoresis. Silver-stained protein spots,

that displayed differential abundances in alcohol-fed mouse or rat livers compared to those in the pair-fed controls, were excised from the 2-D gels, in-gel digested with trypsin and subjected to mass spectrometry. Mass spectrometric data revealed that many cytosolic proteins involved in chaperone activities, anti-oxidant defense, intermediary metabolism including the transmethylation pathway and cytoskeletal proteins were oxidized in alcohol-fed mouse livers. Our current results are likely to explain the underlying mechanisms for the inactivation of some of these enzymes leading to the reduced levels of antioxidants such as S-adenosylmethionine and glutathione with the increased levels of homocysteine observed in alcohol-exposed animal tissues and alcoholic human subjects. Oxidative inactivation of anti-oxidant enzymes such as catalase and peroxiredoxin through sulfinic/sulfonic acid formation of its active site Cys may contribute to the elevated levels of peroxides observed in CYP2E1-containing E47 HepG2 hepatoma cells and alcohol-exposed animals (Kim et al., Proteomics 6: 1250-60, 2006).

Having established a sensitive method, we extended our studies to identify oxidatively-modified proteins in animal models of alcoholic and nonalcoholic fatty liver with inflammatory injury (ASH and NASH) to investigate the underlying mechanisms of mitochondrial dysfunction and apoptosis. First of all, we have analyzed mitochondrial proteins in alcohol-exposed rat livers and pair-fed controls (Moon et al., Hepatology 44:1218-30, 2006). Many mitochondrial proteins involved in mitochondrial electron transfer, energy production, beta-oxidation of fatty acids, and chaperone activities were also oxidatively-modified in alcohol-exposed rat livers. For instance, mitochondrial 3-ketoacyl-CoA thiolase involved in the beta-oxidation of fatty acids was oxidatively-modified and inactivated in alcohol-fed rats, compared to that in the pair-fed control rats. Inhibition of 3-ketoacyl-CoA thiolase and three other enzymes in the mitochondrial beta- oxidation pathway is consistent with increased fat accumulation determined by biochemical and histological methods. Our immunoblot analysis with the antibody against 3-nitroTyr also showed that tyrosine (Tyr) residues of mitochondrial ATP synthase (complex V) were nitrated in alcohol-exposed animals, resulting in its inhibition and subsequently reduced ATP production.

Nitration of the active site Tyr residues of ATP synthase was further confirmed by mass spectral analysis. Furthermore, ALDH2 involved in the metabolism of acetaldehyde and toxic lipid peroxides were oxidized, leading to inhibition of its activity. To directly demonstrate oxidative modification of ALDH2 in alcohol-fed rats, we immunopurified the mitochondrial ALDH2 protein from pair-fed control rat livers or alcohol-fed rats. One immunopurified ALDH2 protein was identified for both groups. However, S-nitrosylated protein, detected with the anti-S-nitrosylated-Cys antibody, was observed only in the alcohol-fed rats but not in control animals. Addition of a reducing agent such as DTT caused disappearance of the S-nitrosylated-Cys band in alcohol-fed rats. These data strongly suggest that the active site Cys302 and other Cys residues of ALDH2 could be reversibly S-nitrosylated in alcohol-fed rats. Similar results about oxidative modification of ALDH2 and other ALDH isozymes were also observed in ethanol-exposed animals (Moon et al., FEBS Lett 581:3967-72, 2007). Based on our results, inactivation of many of these oxidized mitochondrial proteins likely contributes to alcohol-induced mitochondrial dysfunction and increased sensitivity toward ethanol-mediated oxidative tissue injury. In collaboration with Dr. Norman Salem, we have studied the beneficial effects of dietary intake of polyunsaturated fatty acids (PUFA) such as arachidonic (AA,20:4n6,w-6) and docosahexaenoic (DHA,22:6n3, w-3) acids against alcoholic fatty liver and mitochondrial dysfunction in ethanol- exposed Long Evans rats. Our result showed that chronic administration of an ethanol-liquid diet containing low but adequate levels of linoleic and linolenic acids without AA and DHA promotes fatty liver. Fat accumulation was accompanied by increased oxidative/nitrosative stress through elevated levels of ethanol-inducible CYP2E 1, iNOS, nitrite and mitochondrial hydrogen peroxide. However, these increments and alcoholic fatty liver were normalized in rats fed the alcohol-DHA/AA-supplemented diet. The number of oxidatively-modified mitochondrial proteins displayed on 2-D gels was markedly increased following alcohol exposure but significantly reduced in rats fed the alcohol-DHA/AA-supplemented diet. The suppressed activities of ALDH2, ATP synthase, and 3-ketoacyl-CoA thiolase in ethanol-exposed rats also recovered in animals fed the ethanol-DHA/AA-supplemented diet. These results suggest that physiologically relevant levels of DHA-containing PUFA are protective against alcohol- mediated mitochondrial dysfunction and fatty liver through decreasing the levels of ROS/RNS.

We also collaborated with Dr. Pal Pacher, LPS, NIAAA, to identify the oxidized proteins to study the mechanism of mitochondrial dysfunction and injury following hepatic ischemia-reperfusion (I/R) as a mouse model of NASH in the absence and presence of a peroxynitrite scavenger MnTMPyP. Liver histology and plasma transaminase activity data showed that mouse livers were severely damaged following the I/R procedure (1-h ischemia followed by reperfusion for 2-h, 10-h, or 24-h) in the absence of MnTMPyP. These changes were accompanied with elevated levels of nitrite, 3-nitrotyrosine (3-NT), and iNOS compared to those in sham-operated controls. Pretreatment with MnTMTyP fully restored liver histology with normalized levels of plasma transaminases, nitrite, 3-NT, and iNOS. Comparative 2-D gel analysis revealed markedly increased numbers of oxidized and S-nitrosylated mitochondrial proteins following hepatic I/R injury. Many key mitochondrial enzymes involved in cellular defense, fat metabolism, energy supply, and chaperones were oxidatively-modified. MnTMPyP pretreatment decreased the number of oxidatively-modified proteins and restored the suppressed activities of mitochondrial ALDH2, 3-ketoacyl-CoA thiolases, and ATP synthase following I/R injury. These results strongly suggest that increased nitrosative stress is critically important in promoting S¬nitrosylation and nitration of various mitochondrial proteins, leading to their inactivation, contributing to mitochondrial dysfunction with decreased energy supply and increased hepatic injury. Based on these results, we feel that this approach can be used in future studies to study the mechanism of tissue damage and to identify another beneficial agent against oxidative tissue injury in many other organs such as brain, heart, and kidney.

In collaboration with Drs. Natalie D. Eddington and James Lee at the University of Maryland, we also studied the mechanism of mitochondrial dysfunction and nonalcoholic liver damage caused by acute exposure to MDMA (3,4-methylenedioxymethamphetamine, ecstasy). We hypothesized that key mitochondrial proteins are oxidatively-modified and inactivated, contributing to liver damage in MDMA-exposed tissues. MDMA-treated rats showed abnormal liver histology with significant elevations of plasma transaminases, iNOS, and the level of hydrogen peroxide. Comparative 2-D gel analysis revealed markedly increased levels of oxidatively-modified proteins labeled with the biotin-NM probe in MDMA-exposed rats compared to control rats. Mass spectrometric analysis identified oxidatively-modified mitochondrial proteins involved in energy supply, fat metabolism, antioxidant defense, and chaperone activities. Consequently, the activities of ALDH2, 3-ketoacyl-CoA thiolases, and ATP synthase were significantly inhibited following MDMA exposure. Our data show for the first time that MDMA causes oxidative inactivation of key mitochondrial enzymes which most likely contributes to mitochondrial dysfunction and subsequent liver damage in MDMA-exposed animals.

Finally, we plan to study the mechanisms of oxidative modifications of mitochondrial proteins and inactivation (dysfunction) prior to fat accumulation with potential inflammation in CYP2E1 - null mice or peroxisomal proliferator-activated receptor α (PPARα)-null mice compared to wild type mice treated with an ethanol-liquid diet or a high fat diet as a mouse model of ASH or NASH.

 

Publications 2007-2008:

Moon KH, Abdelmegeed MA, and Song BJ. Inactivation of cytosolic aldehyde dehydrogenase via S-nitrosylation in ethanol-exposed rat liver.

FEBS Lett 581:3967-72, 2007.

Jo SA, Kim EK, Park MH, Han C, Park HY, Jang Y, Song BJ, Jo I. A Glu487Lys polymorphism in the gene for mitochondrial aldehyde dehydrogenase 2 is associated with myocardial infarction in elderly Korean men.

Clin Chim Acta 3 82:43-7, 2007.

Moon KH, Kim BJ, Wan J, Lee YM, Song BJ. Reversible inactivation of mitochondrial aldehyde dehydrogenase by translational modifications in hepatoma cells and alcohol-fed rat livers. (In: Weiner H, Plapp BV, Lindahl R, and Maser E. ed.) Enzymology and Molecular Biology of Carbonyl Metabolism vol. 13, pp 22-32, Purdue University Press, West Lafayette, Indiana, 2007.

Moon KH, Hood BL, Mukhopadhyay P, Kwon YI, Conrads TP, Veenstra TD, Song BJ and Pacher P. Oxidative inactivation of many mitochondrial proteins leads to dysfunction and injury in hepatic ischemia reperfusion.

Gastroententerology 135:1344-57, 2008.

Moon KH, Upreti VV, Yu L-R, Lee IJ, Ye X, Eddington ND, Veenstra TD, and Song BJ. Mechanism of 3 ,4-methylenedioxymethamphetamine (MDMA, Ecstasy)-induced mitochon-drial dysfunction in rat liver.

Proteomics 8: 3906-18, 2008.

Song BJ, Moon KH, Olson NU, and Salem Jr N. Prevention of alcoholic fatty liver and mito- chondrial dysfunction in the rat by long-chain polyunsaturated fatty acids.

J Hepatol. 49: 262-73, 2008.

Donelson E, Chen L, Zhang X, Goswami P, Vernell R, Song BJ, and Hardwick JP. Genomic structure and regulation of the rat hepatic leukotriene B4-hydroxylase CYP4F1 gene by peroxisome proliferators.

Arch Biochem Biophys 472:1-16, 2008.

Choi HJ, Song BJ, Gong Y-D, Gwak WJ, and Soh Y. Rapid degradation of hypoxia-inducible factor-1a by KRH 102053, a new activator of prolyl hydroxylase 2.

Brit J Pharmacol 154:114-25, 2008.

Purohit V, Gao B, and Song BJ. Molecular mechanisms of alcoholic fatty liver.
Alcohol Clin Exp Res 2008, in press.


Prior key references:

Kim BJ, Hood BL, Aragon RA, Hardwick JP, Conrads TP, Veenstra TD, Song BJ. Increased oxi- dation and degradation of cytosolic proteins in alcohol-exposed mouse liver and hepatoma cells.

Proteomics 2006; 6: 1250-60.

Moon KH, Hood BL, Kim BJ, Hardwick JP, Conrads TP, Veenstra TD, Song BJ. Inactivation of oxidized and S-nitrosylated mitochondrial proteins in alcoholic fatty liver in rats.

Hepatology 2006 44: 1218-30.

 

Signaling mechanism during cell death caused by ethanol and other toxic compounds
Song, Abdelmegeed, Moon, Henderson in collaboration with Gonzalez, Shields

In addition, we have investigated the signaling mechanism during cellular damage caused by many toxic compounds including ethanol. Our earlier results showed selective and persistent activation of c-Jun N-terminal protein kinase (JNK) by many toxic substrates of CYP2E1 such as acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, and long chain fatty acids (Soh et al., Mol Pharmacol 58:535, 2000; Song et al., Chem Biol Interact 130-133: 943, 2001; Bae et al., Mol Pharmacol 60: 847, 2001). In contrast, we observed that ethanol, another substrate of CYP2E1, and non-CYP2E1 substrates such as troglitazone (Bae and Song, Mol Pharmacol 63: 401, 2003; Bae et al., Tox Lett 139: 67, 2003), hydrogen peroxide, etoposide, and staurosporine (STS) activated JNK and p38 protein kinase (p38 kinase) simultaneously. Our results also showed that all these compounds caused translocation of proapoptotic Bax from cytoplasm to mitochondria in a time-dependent manner.

Since the mechanism of Bax activation and its mitochondrial translocation prior to apoptosis was unknown, we further investigated the mechanism for Bax activation after cells were treated with various cell death stimulants. Our results showed that various toxic compounds potently activate the stress-activated protein kinases (e.g., JNK and p38 kinase), which subsequently phosphorylate Bax before it was translocated to mitochondria to initiate actual cell death observed at later time points. Phosphorylation of Bax was demonstrated by the shift of the pI value of 4.0 (phosphorylated Bax) from pI 5.1 (non-phosphorylated Bax) on 2-D gels and confirmed by metabolic labeling with 32P-inorganic phosphate. Important roles of JNK and/or p38 kinase in mitochondrial translocation of Bax and apoptosis were demonstrated by using a specific inhibitor of JNK or p38 kinase and a specific siRNA to MAPKK4, the upstream kinases of JNK and/or p38 kinase. Pretreatment with each agent significantly reduced the activity of each protein kinase and the rates of mitochondrial translocation of Bax and apoptosis. To determine the phosphorylated amino acid, several Bax mutants (Ser87Ala, Thr167Ala and others) were prepared, based on the fact that JNK and p38 kinase are proline-directed protein kinases. Critical roles of phosphorylation of Bax in its mitochondrial translocation were further confirmed by confocal microscopy of various Bax mutants and transfection into Bax/Bak double knockout mouse embryonic fibroblast cells. Our confocal microscopic results with various Bax mutants clearly show that Thr1 67 is a critical amino acid which is phosphorylated by stress-activated protein kinases. Taken together, our results suggest that JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation, which might disrupt the previous interaction between the N-terminal domain and the C-terminal transmembrane domain of Bax. Exposure of the C-terminal transmembrane domain is likely to lead to translocation of Bax to mitochondria to initiate mitochondria-dependent apoptosis through changing the mitochondrial permeability. Our results demonstrate for the first time that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to its translocation to mitochondria prior to apoptosis (Kim et al., J Biol Chem 281: 2 1256-65, 2006). Our unpublished results also show that ethanol alone can activate both JNK and p38 kinase, which promote phosphorylation of Bax prior to its translocation to mitochondria to initiate mitochondria-dependent apoptosis in cultured colon cells. Taken together, our results are likely to explain the underlying mechanisms for the positive relationship between activation of JNK or p38 kinase and apoptosis caused by many distinct cell death stimulants or pathological conditions, as previously reported by many scientists.

 

Publication 2007:

Lee MS, Bae MA. Docosahexaenoic acid induces apoptosis in CYP2E1-containing HepG2 cells by activating the c-Jun N-terminal protein kinase related mitochondrial damage.

J Nutr Biochem 18: 348-54, 2007.

 

Prior key reference:

Kim BJ, Ryu SW, Song BJ. JNK- and p38 kinase mediated phosphorylation of Bax leads to its activation, mitochondrial translocation and apoptosis of human hepatoma HepG2 cells.

J Biol Chem 281: 21256-65, 2006.