Effect of ethanol on pro-apoptotic mechanisms in polarized hepatic cells

oNatalia A Osna, MD, PhD, Series EditorEffect of ethanol on pro-apoptotic mechanisms in polarizedhepatic cellsBenita L McVicker, Dean Tuma, Carol A CaseyBenita L McVicker, Dean J Tuma, Carol A Casey, Liver Key words: Hepatoma hybrid cells; Ethanol; Apoptosis;Study Unit, Department of Veterans Affairs Medical Center; and Fas receptor; CaspaseDepartments of Intermal Medicine and Biochemistry moleculariology, University of Nebraska Medical Center, Omaha, NE MCVicker BL, Tuma D), Casey CA. Effect of ethanol on pro-Supported by the National Institute on Alcohol Abuse and apoptotic mechanisms in polarized hepatic cells. World JAlcoholism and the Departinent of Veterans AffairsGastroenterol2007;13(37):4960-4966Correspondence to: Benita L McVicker, PhD, veterans AffairsMedical Center, Research Service (151), 4101 Woolworth Avenue,http:!www.wignet.com100927131908Omaha, NE 68105, United States. bmevicker @ unmc.eduTelephone:+1-402-3468800-3547Fax:+1-402-4490604Received: June 30. 2007Revised: July 18, 2007INTRODUCTIONIt has been well established that chronic ethanol consump-Abstraction can lead to a variety of pathological consequences(ethanol-relateed liver injuries such as hepatomegaly,Chronic ethanol consumption is associated with serious fatty liver, alcoholic hepatitis and cirrhosis), yet theand potentially fatal alcohol-related liver injuries such as mechanism(s)by which ethanol causes hepatotoxicityhepatomegaly,alcoholic hepatitis and cirrhosis. Moreover, requires further clarification From several years oft has been docume nted that the clinical progression investigation, certain factors have been identified as havingwith an increase in hepatocellular death that involves a role in ethanol-induced toxicity, such as changes inapoptotic mechanisms. Although much informationredox status(NAD/NADH ratio), the accumulation ofhas been learned about the clinical manifestations acetaldehyde(a product of ethanol oxidation), depletionassociated with alcohol-related diseases, the search of antioxidants such as glutathione, and the generation ofcontinues for a better understanding of the molecular reactive oxygen species(roS). As a consequence, it hasind or cellular mechanisms by which ethanol exerts itsen demonstrated that amongst the various deleteriousdeleterious effects such as the induction of pro-apoptoticeffects that can result from ethanol administration, it hasmechanisms and related cell damaging events. As part been shown that alcohol-induced diseases are accompaniedof the effort to enhance our understanding of those by morphological liver changes that include the increasedparticular cellular pathways and mechanisms associated production of apoptotic cells". More specifically, itvith ethanol toxicity, researchers over the years have has been demonstrated that ethanol administration isutilized a variety of model systems. Recently, work linked to hepatocyte apoptosis" 4 and that the numberhas come forth demonstrating the utility of a hybrid of apoptotic cells detected in the liver correlates withstudy of alcohol-associated alterations in hepatocellularchanisms Success with such emerging modelApoptosis is a regulated mode of cell death, which issystems could aid in the development of potential chatacterized by specific biochemical and morphologicaltherapeutic treatments for the prevention of alcoholchanges in the cell. Morphologically, during inductioninduced apoptotic cell death that may ultimately serve of apoptosis, the affected cells shrink, lose cytoskeletalas a significant target in delaying the onset and/orprogression of clinical symptoms of alcohol-mediated contacts and undergo chromatin condensation. duringliver disease. This review article summarizes the currentunderstanding of ethanol-mediated modifications in cellsurvival and thus the promotion of pro-apoptotic eventswith emphasis on analyses made in various experimentalCTH中国煤化工 d and eliminatedCNMH Gg cells!. The searchodel systems, particularly the more recentlyical anu signal transduction pathwayscharacterized WIF-B cell systeminvolved in producing the characteristic apoptoticmorphological cellular changes has been unde@2007 The WG Press. All rights reservedseveral years. The current dogma is that apoptosis isw而点教播McVicker BL et al Ethanol-mediated apoptosis in hepatic cellsthought to occur by two main pathways involving either an cthanol, apoptosis is the preferential mode of injuryextrinsic route(which utilizes death receptors), and/or an as death was found to be triggered by the Fas receptorintrinsic pathway that involves mitochondrial intracellular system". 'Thus, ethanol administration is related to cellularstress signals". Once triggered, the apoptotic machinery injury mechanisms, yet a complete understanding of theis set into motion through the activation of a family of presumably intertwined factors that can be involved inintracellular cysteine proteases known as caspases, which hepatotoxicity is still sought. The following is a briefamplify appropriate signals by acting as initiators as well as revicw of current knowledge concerning the factorsexecutioners in the death program. For instance, there are identified in alcohol-mediated apoptotic liver damagedownstream executioner enzymes(caspases 3, 6, and 7)that are activated by upstream initiator caspases(caspases 8, Oxidative stress and alcohol-induced liver apoptosis2,9, and 10) that are in turn regulated by specific protein- Following the recognition that ethanol and its metabolitesprotein interactions. As an example, caspases may be induce the formation of reactive oxygen species(ROS)invated in an extrinsic manner by membrane signaling liver cells; studies were able to link the ethanol-mediatedevents(death domain transmembrane receptors)and/ or induction of oxidative stress to the observed enhancementby intracellular intrinsic changes resulting in the release of of apoptosis. Specifically, it has been demonstratedspecific proteins from the mitochondria. Intertwined in that one of the mechanisms responsible for ethanolthese pathways are the proteins that control the intrinsic or induced hepatotoxicity appears to involve the inductionextrinsic routes of apoptosis that often belong to families of intracellular enzymes, such as alcohol dehydrogenaseat have specific domains that mediate their action. Some (ADH)and the induction of CYP2E1, that oxidize ethanolof the regulatory proteins that have been identified include to reactive metabolites, producing reactive oxygen speciesthose of the Bcl-2 family, which possess anti-apoptotic as and lipid peroxides 2o). In linking the ethanol-inducedwell as pro-apoptotic activity tia the Bcl2 and Bax geneskidative stress with liver cell apoptosis, it was determinedctIvethat rOS cause damage to the mitochondria by alteringNormally, in a healthy individual, the highly regulated mitochondrial membrane potential and/or membraneapoptotic system is counterbalanced by cytoprotective permeability. This in turn can initiate the release of prosignals that maintain tissue homeostasis. However, when apoptotic factors, such as cytochrome c, thus activatingan organism is subjected to repeated or exacerbated the caspase cascade25-271. Identified targets in this processinsults of a pathological stimulus such as alcohol, pro- include mitochondrial DNA and specific proteins that areapoptotic death factors can be inappropriately expressed promoters of apoptosis(Bax, Bad, and Bid),which areshifting the balance towards enhancement of the apoptotic related to the Bcl-2 oncogene familymachinery and subsequent deleterious effects. Hence, itEthanol treatment was also shown to impair antioxidanthas become clinically relevant that a better understanding levels in hepatocytes, resulting in ROS generation andof the mechanisms and factors involved in apoptosis becreased oxidative stress. For example, the balance ofconcerning the role of apoptosis in the initiation and glutathione is impaired by ethanol tteatment and wasascertained which has lead to an emergence of studiesfound to be involved in the regulation of mitochondrialprogression of alcohol-induced liver injuryfunction in alcohol-associated apoptosis". In particular,was determined that the mitochondrial glutathione(mGETHANOL-MEDIATED PATHOLOGICALFACTORS AND APOPTOSISOver the past decade, significant progress has been made administration results in enhanced oxidative stress; thatconcerning the identification of contributing factors that mitochondria are intimately involved in the survival ofare involved in the initiation and progression of apoptosis the cell as both a producer and target of ROS; and thatin both clinical and experimental alcoholic liver disease apoptotic death can be the consequential end of thesestates. In genetal, it has been shown that alcohol-mediated changes.oxidative stress mechanisms an; (2) the effects of various The role of cytokines in ethanol-induced apoptosiscytokines, particularly TNF-a and TGF-B 222;(3)the Cytokines are produced by multiple cell types in the liverinvolvement of death receptor pathways (TNF-reccptor and are integral in cellular signaling prormma1 and Fas/CD95).2.How and when these factors liver cells use well-developed defense mechanisms aselicit hepatotoxic cffccts is not completely understood, protection against cytokine-meciiated damage. Howevcr,however it has been noted that a direct correlation exists when toxins such as ethanol stress the cells, they lose thcirbetween the ethanol concentration and time of exposure survival protection and become susceptible to the effectsin the ability of these factors to promote apoptotic and/ of the various cvtokinesor necrotic cell death. For instance, it has been shown中国煤化工 as the ethanol-relatcdthat higher amounts of alcohol resulted in a decline in releaspoptosis with an increase in promotion of necrotic cell ( TNCNMHGIL6. I8.L10.anddeath, presumably from the induction of microsomal transforming growth factor-beta 1, TGF-B1] is associatedcytochrome components, specifically cytochrome P450 with the promotion of pro-apoptotic mechanisms.3212E1(CYP2E1). Conversely, at lowcr concentrations of Furthcrmorc, TNF-a and TGF-B in particular have4962 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol October 7, 2007 Volume 13 Number 37been strongly implicated in ethanol-induced apoptosis. Involvement of the Fas/as ligand system in ethanol-pecifically, it has been well documented that the chronic induced liver cell apoptosisexposure of ethanol can result in an elevation of hepatic Of the death receptor-mediated pathways,the involvementTNF-a, which in turn has been related to the activation of the Fas/Fas ligand system has proven to play aof caspases and the apoptotic program.. One pathway significant role in alcohol-related apoptosis in the liverthe interaction with the death receptor family, Fas ligand/ surface protein) is similar to other death receptors in thatFas, and TNF/TNF receptor(TNFR) systems. Within a specific interaction of the oligomerized receptor withthis family are three characterized receptors(TNFRl, an associated ligand (i.e. Fas ligand) or antibody stimulatesTNFR2, and the Fas antigen, which are structurally rclated the recruitment of the cytoplasmic adapter protein(Faswith shared homology for a death domain sequence, associatcd death domain, FADD) that mediates caspasea C-terminal intracellular domain that is involved in activation and the signaling of the apoptotic deathapoptosis pathways. In another mechanism of TNF-a- program. In the liver, increases in the expression ofinduced cell death, the role of TNF-a and mitochondrial mcmbrane- bound Fas as well as the levels of soluble FasGSH depletion in ethanol-related apoptosis was further and Fas ligand have been associated with pathologicaldefined. As mentioned above, ethanol impairs the transport conditions associated with alcoholic hepatitis".. Also,of cellular glutathione(particularly mGSH) resulting in its the up-regulation of Fas ligand in hepatocytes followingdepletion and the subsequent sensitization of the cell to cthanol treatment is thought to induce apoptotic deathundergo an apoptotic death. This ethanol-related depletion of neighboring cells by interacting with the Fas receptorof mGSH was also found to be associated with TNF- on the surface of those cells". Overall, it is thought thatmediated cell death in hepatocytes, as the cells bccame the activation of Fas results in the promotion of theapoptotic as a result of TNF-related events(such as the apoptotic program that can include the induction of thegeneration of hydrogen peroxide). Validation of this caspase cascade and permeabilization of the mitochondrialassociation came through studies demonstrating that TNF- membrane. Moreover, the importance of the Fas/Fasa-induced oxidative stress and subsequent apoptosis could ligand system in caspase 3 activation and apoptosis in thebe prevented by incubating hepatocytes with antioxidants liver following ethanol treatment was substantiated as theas well as S-adenosyl-L-methionine, a substance thatIministration of a caspase 3 inhibitor was shown to blockincreases m GSH levels 55. Overall, the data obtained ethanol-induced caspase 3 activity along with apoptosissfrom numerous studies has demonstrated that ethanol Thus, there is a growing amount of evidence that theexacerbatesTNF-related hepatotoxicity, in part, through Fas/Fas ligand system is a critical element in the activationthe induction and perpetuation of apoptotic cell death of the caspase cascade and the subsequent demise of liverpathways.Participation of TGF-B1 in ethanol-induced apoptosishas also been widely studied. TGF-Bl is the prototype MECHANISMS OF ALCOHOL-INDUCEDmember of the TGF-B family that is invoived in a diversearray of biological activities including development,APOPTOSISdifferentiation, tissue remodeling, and apoptosisThe enhancement of our understanding of theSeveral liver cells(including hepatocytes and hepatic mechanisms involved in the apoptotic cascade isstellate cells)can produce TGF-B1 in response to alcohol becoming exceedingly important and relevant to diseasetoxicity, and this production is considered to have a states such as ALD. However, studies concerning thesignificant impact in the initiation and progression of effect of ethanol on cellular processes such as apoptosisalcoholic liver disease 58.39). Researchers have identified are often limited due to the applicability of the modelsome of the pro-apoptotic pathways that have been system. As an example, the dosc required for ethanolinduced by the up-regulation of TGF-B1 in response to induced hepatotoxicity in vitro has been found to varethanol treatment. It has been demonstrated utilizing a between cell strains presumably due to the differencefetal hepatocyte model that TGF-B1 induces apoptosis in the metabolism of ethanol by ADH and CYP2E/Bby producing oxidative stress in the cells by increasing Also, the use of freshly isolated hepatocyte models wereROS production and decreasing the level of a natural found to produce confounding results when searching foantioxidant,glutathione) 4. In addition, the increase mechanistic parameters as the cells dediffcrcntiatc (i.loscin TGF-Bl-mediated signaling, resulting in caspase cell polarity and many liver-specific characteristics suchactivation, can be associated with the enhanced cleavage as the ability to metabolize ethanol) within a few hoursof the caspase substrate PARP [poly(ADP)-ribose after isolation, making the cells useful for short-tetmolymerase]-. It has also been identified that TGF-B1 culture conditions only. Despite these lincan activate the caspase cascade by either of the two studies have been performed yielding useful informationprimary pro-apoptotic mechanisms(death receptor- conce中国煤化工 ms that are inducedmediated pathway or intracellular stress-signaled pathway followinvolving mitochondrial changes and the releasc ofCNMHGst began to develofcytochrome c), and either pathway can involve the concerning the relevance of apoptotic cell death information of an apoptosome complex that activates the liver pathology with particular interest in hepatocellularproteolysis of the cell)injury associated with ethanol toxicity. Since theMcVicker BL et a/ Ethanol-mediated apoptosis in hepatic cells4963acceptance that apoptosis may play a significant role in fed animal models as sources of isolated hepatocytesthe development of liver injury, studies have searched in order to provide more compatible models to humanfor the potential mechanisms and pathways that are pathology than most cell culture systems provideinvolved. In animal models. it was demonstrated that However, limitations were often observed in the modelethanol feeding resuled in an increase in apoptotic cells systems used. For example, the study of protein traffickingin the liver" that was subsequently shown to be related events in cultured systems has been hampered by theto the induction of reactive oxygen species(ROS)as lack of a well-polarized cell that adequately mimics thea result of ethanol metabolism and the generation of complexity of in wto hepatocyte protein delivery systemsacetaldehyde( 5. Furthermore, it was concluded that the (i.c. the indirect transport of apical membrane proteins)ethanol-induced ROS Production was driven by redox Recently, our labotatory has demonstrated that theanges that ultimately lead to mitochondrial dysfunction use of the WIF-B hepatoma hybrid cell line(in whichand caspase activation. These intrinsic events were not, polarity is a stable and dominant trait) 63, 6 is an idealhowever, the only mechanism by which ethanol was found in witro model for studying the effects of ethanol on cellularto induce apoptosis. Specifically, it was determined that processes. Specifically, the WIF-B cells were found tocthanol can also induce apoptosis through thc involvement exhibit alcohol dehydrogenase(ADH) activity allowingof the extrinsic death receptor pathway via the Fas/CD95for the efficientolism of ethanol. alsoreceptorwere observed in cellular triglyceride levels in the WIF-BIn other works, acute studies involving ethanol cells following ethanol treatment, similar to the reportedtreatment to cells isolated from animals also demonstrated fat accumulation that is observed in human alcoholic liverthat oxidative stress was involved in mitochondrial injury. In addition, treatment of the WIF-B cells withmembrane changes that were found to result in cytochrome alcohol resulted in morphological changes in the cells asc release and caspase activation>25. Additionally, studies demonstrated by decreases in bile canalicular formationsing developed recombinant cell lines demonstrated the and ccll-cell contacts. Thus, the use of the WIF-B cellrole ethanol-inducible CYP2E1 has in alcohol-mediated line to study ethanol-induced cellular mechanisms is quiteapoptosis. Particularly, it was shown that ethanol-induced appropriate since the cells present a unique culture systempoptosis was related to oxidative stress and subsequent that has the ability to metabolize ethanol and is a systemlipid peroxidation in CYP2E1 over expressing cells 54, that more closely resembles human morphology thanconfirming the central role ROS and CYP2E1 have inother known cell linesethanol-mediated cell death as hypothesized in previousThe WIF-B hybrid cell iscross between a humanworks25355. Additional evaluations demonstrated that fibroblast(WI 38)and a Fao rat hepatoma cell line,8)ach activation of intrinsic apoptotic pathways could be This clone represents a polarized and differentiated ccllrelated to alcohol-mediated glutathione depletion which of hepatic origin that exhibits long-term viability inenhances the sensitivity of the cell to succumb to death, culture, develops a hepatocellular-polarized phenotypeespecially when faced with additional insults(e.g. increases and expresses human genes coding for liver-specificin TGF-B expression or the presence of Hepatitis proteins (albumin, fibrinogen, and ADH). One ofvirus).o. Furthermore, continued searches for prothe unique characteristics of the WIFneapoptotic mechanisms involved in cthanol-mediated cell the cells grow in monolayers and acquire a polarizedjury have provided additional information regardingphenotype as the cells form bile canalicular-likethe potential contribution regulatory mechanisms have in(BC) that have concentrated apical membrane proteins.hepatocellular apoptosis, such as the role of NF-KB and Therefore, these cells are useful for the functional studiesethanol-mediated alterations in proteosome function t1 a ; of hepatocyte-specific properties such as the transport ofwhether the processes are p53 dependent(63, 6); and what membrane proteins. Indeed, several studies have utilizedrole pro-apoptotic Bcl-2 family proteins have in mediating the advantages of the maintained polarized cell to studymitochondrial permeability and apoptosis during alcohol intercellular communication, bile acid transport systems,and membrane trafficking pathwaysOverall, substantial information has been gainedIn recent studies using this hepatic model, it waslinking oxidative stress from alcohol metabolism mia adh demonstrated that ethanol treatment induces apoptosis viaand CYP2E1 to the induction of several pro-apoptotsignals emanating from the pro-apoptotic death reccptormechanisms involving both intrinsic and extrinsic systems (i.e. Fas/CD95)as well as from intrinsic signalsapoptosis pathways. However, the development and ustthat resulted in mitochondrial changes. Specifically,of additional model systems, such as a recently described it was demonstrated that ethanol treatment of WIF-Bpolarized hepatic ccll line, may significantly contributecells resulted in a significant increase in apoptotic-rclateour understanding of pro-apoptotic mechanisms induced morphological changes(cells that contained condensedas a consequence of cthanol administrationchromatin) that were also associated with pro-apoptoticfeatures ( indicated by the induction ofcas中国煤化工 hown that caspase-3ETHANOL-INDUCED APOPTOSIS IN AactIvCNMHGB cells was related toPOLARIZED LIVER CELL MODEL(MPT) as ethanoltreatment resulted in cytochrome c release that was foundMany of the studies performed previously concerning the to be sensitive to cyclosporine A (an inhibitor of MPT)effect of ethanol on cellular processes have used alcohol- Additionally, it was determined that in the cthanol-induced4964 ISSN 1007-9327 CN 14-1219/R Worid J Gastroenteral October 7, 2007 Volume 13 Number 37EthanolInvolving ethanol abusesights into the cellularmechanisms involved in the initiation and propagation ofNFas/translocation ADHCYPZEsignificantly impact our understanding ofalcohol-induced liver disease and may lead to the potentialCaspase 8development of therapeutic interventions. The use ofan emerging model system, polarized hepatic WIF-Bcells, could significantly impact the study of alcoholCaspase 3related hepatocellular injury, especially concerning thedelineation of mechanisms involved in cthanol-inducedcell death. In addition, polarized hepatic cell culturesmay aid in theApoptotic cell deatcquisition of translational informthe WIF-B cells offer a more compatible model systemthat better correlates to human pathology for analysis ofinitiation and propagation of ethanol-induced apoptosis. Ethanol metabolism and Potential therapeutic targets that could modulapopoutconsequential generation of reactive oxygen species are implicated in themechanisms induced by ethanol.activation of pro-apoptotic mechanisms via death receptor (ie. Fas/CD95)as weilas intrinsic apoptotic pathwaysREFERENCESDufour MC, Stinson F5, Caces MF. Trends in cirrhosisapoptosis in WIF-B cells, the activities of upstreammorbidity and mortality: United States, 1979-1988. 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Biochem Pharmacol 2004; 67: 2167-2174selectively sort apical Proteins from cell surface to a novel68 Decaens C, Rodriguez P, Bouchaud C, Cassio D Establishmentcomabut lack apical retention mechanisms. Mo? Biolof hepatic cell polarity in the rat hepatoma-huell2002;13:3400-3415hybrid WIF-B9. A biphasic phenomenon goin73 McVicker BL, Tuma Daseypithelial polarized phenotype to an hepaticEthanol-induced apoptosisrized hepatic cells possiblyCell Sc1996;109:1623-1635throug69 Bender V, Bravo P, Decaens C, Cassio D. The structural and006:30:1906-1915s-Editor Ma N L Editor Alpini GD E-Editor Yin DH中国煤化工CNMHG万数据