Overexpression of CYP2E1 in Mitochondria Sensitizes HepG2 Cells to the Toxicity Caused by Depletion of Glutathione

Abstract

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease. Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease. Generation of reactive oxidative species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; BSO, buthionine-(S,R)-sulfoximine; CYP2E1, cytochrome P450 2E1; mtCYP2E1, mitochondrial cytochrome P450 2E1; Mn-SOD, manganese-superoxide dismutase; DCF-DA, 2′-7′-dichlorofluorescein diacetate; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 3-NT, 3-nitrotyrosine; 4-HNE, 4-hydroxynonenal; ER, endoplasmic reticulum; Adx, adrenodoxin; AdR, adrenodoxin reductase; TCE, total cell extract; HPLC, high pressure liquid chromatography. is one of the mechanisms by which ethanol is toxic to the liver and other tissues. Induction of CYP2E1 by ethanol appears to be one of the central pathways by which ethanol is believed to generate a state of oxidative stress. CYP2E1 is active in oxidizing ethanol to acetaldehyde and in oxidizing many agents to reactive metabolites that are hepatotoxic (1Lieber C.S. Physiol. Rev. 1997; 77: 517-544Crossref PubMed Scopus (787) Google Scholar). CYP2E1 is also reactive in the production of O.2- and H2O2 during microsomal mixed function oxidase activity (2Gorsky L.D. Koop D.R. Coon M.J. J. Biol. Chem. 1984; 259: 6812-6817Abstract Full Text PDF PubMed Google Scholar, 3Ekstrom G. Ingelman-Sundberg M. Biochem. Pharmacol. 1989; 38: 1313-1319Crossref PubMed Scopus (498) Google Scholar). Correlations between induction of CYP2E1, lipid peroxidation, and ethanol-induced liver injury have been found in the intragastric infusion model of ethanol-induced liver injury (4Castillo T. Koop D.R. Kamimura S. Triadafilopoulos G. Tsukamoto H. Hepatology. 1992; 16: 992-996Crossref PubMed Scopus (191) Google Scholar, 5Ingelman-Sundberg M. Johansson I. Yin H. Terelius Y. Eliasson E. Clot P. Albano E. Alcohol. 1993; 10: 447-452Crossref PubMed Scopus (213) Google Scholar, 6Morimoto M. Zern M.A. Hagbjork A.L. Ingelman-Sundberg M. French S.W. Proc. Soc. Exp. Biol. Med. 1994; 207: 197-205Crossref PubMed Scopus (157) Google Scholar, 7Nanji A.A. Zhao S. Sadrzadeh S.M. Dannenberg A.J. Tahan S.R. Waxman D.J. Alcohol Clin. Exp. Res. 1994; 18: 1280-1285Crossref PubMed Scopus (236) Google Scholar, 8Sadrzadeh S.M. Nanji A.A. Price P.L. J. Pharmacol. Exp. Ther. 1994; 269: 632-636PubMed Google Scholar, 9Tsukamoto H. Horne W. Kamimura S. Niemela O. Parkkila S. Yla-Herttuala S. Brittenham G.M. J. Clin. Investig. 1995; 96: 620-630Crossref PubMed Scopus (452) Google Scholar, 10Fang C. Lindros K.O. Badger T.M. Ronis M.J. Ingelman-Sundberg M. Hepatology. 1998; 27: 1304-1310Crossref PubMed Scopus (78) Google Scholar), and inhibitors of CYP2E1 partially prevent the injury (6Morimoto M. Zern M.A. Hagbjork A.L. Ingelman-Sundberg M. French S.W. Proc. Soc. Exp. Biol. Med. 1994; 207: 197-205Crossref PubMed Scopus (157) Google Scholar, 10Fang C. Lindros K.O. Badger T.M. Ronis M.J. Ingelman-Sundberg M. Hepatology. 1998; 27: 1304-1310Crossref PubMed Scopus (78) Google Scholar). The addition of ethanol, iron, or arachidonic acid to HepG2 cell lines that overexpress human CYP2E1 (E9 cells, E47 cells) decreased cell viability and caused apoptosis (11Chen M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. 1993; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Although CYP2E1 is predominantly located in the membrane of the ER, it also been to be in M.J. Johansson I. J. H. Ingelman-Sundberg M. J. Biochem. PubMed Scopus Google Scholar), I. Biol. Med. 1997; PubMed Scopus Google Scholar), Eliasson E. M.A. Albano E. Ingelman-Sundberg M. Biochem. PubMed Scopus Google Scholar), and the of the membrane E. Pharmacol. Google Scholar, Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A study that CYP2E1 is also in mitochondria of rat liver and that it be induced by M.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that an NH2-terminally truncated form of CYP2E1 was targeted to the mitochondria Ingelman-Sundberg M. PubMed Scopus Google Scholar). was that form of CYP2E1 was an NH2-terminally truncated form of CYP2E1 of which was to be catalytically and in the mitochondrial Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In truncated form of CYP2E1 was in levels in in mitochondria from rat liver Ingelman-Sundberg M. PubMed Scopus Google Scholar). In the study, found that chronic alcohol consumption could induce rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and damage to mitochondria, HepG2 cell lines overexpressing CYP2E1 in mitochondria were and the role of mitochondrial targeted CYP2E1 in oxidative stress and damage to mitochondria and was and were from in human CYP2E1 was from M. as a was from BSO, 2′-7′-dichlorofluorescein and were from of and of were in a by The of and were with ethanol and that were and the with to of and protein of were with were as J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of liver was in of and a The was The were The was in a and were and The of the between the and was in and The was in of and rat liver mitochondrial CYP2E1 Western blot the mitochondria were the as M.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the mitochondria were with the a of and with a of of were with the and in containing and mitochondria from cells in the mitochondrial was in of the and used Western blot or aconitase activity were from the by The was with and used Western blot analysis. cDNA of CYP2E1 lacking the sequence was by and plasmid containing human CYP2E1 cDNA as The cDNA was with and in the of the The sequence of the was by Cell and cells were from the and were in containing and in a in HepG2 cells were and to The plasmid and containing the human CYP2E1 cDNA lacking the sequence were transfected into HepG2 cells the cells were and cell in containing the were to and to CYP2E1 in was by Western cell lines with of mitochondrial CYP2E1, as as cells transfected with the empty were and in containing Western protein of mitochondria or cell was and with were with CYP2E1 as followed by with to as the the was followed by to were to Mn-SOD, and and protein adducts and activity of was by the of as Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). from and mE27 cells were and by The of of mitochondria, an generating and of adrenodoxin and of adrenodoxin by of and in a of the was by the addition of the were by with an and mE27 cells as as E47 cells HepG2 cell overexpressing CYP2E1 in the were in containing and were with in were in in and in followed by in were with a of a CYP2E1 and a of followed by in and with a of a with and a of a with in PBS, cells were with followed by an in The were and by GSH and mitochondrial GSH levels were as in Biochem. PubMed Scopus Google with the and mE27 cells were treated with or BSO. of the total and mitochondrial were and the protein were of containing of protein was mixed with an of of acid and were followed by The were used The of of and of the containing of The of of GSH was used a and the was to a was with the The GSH was by with the GSH and as of as a of of the cells with or BSO, an of that GSH was a of and were in the were with PBS, and in of PBS, and was in a and with a of and from cells were are as of of of BSO to and mE27 cells was by the were a and in The was the Cell of was to the of an of was to an The of the was The was used as a The was as the of cell viability. The from the of cells was used as the viability The of viability was by the mitochondrial aconitase activity was by the and the from the The aconitase activity is to the of the of which is by the in of the in the mitochondrial membrane potential were by the cells with A.L. 1997; PubMed Scopus Google Scholar). were the cells were with containing were by and in of The of was by to and are from were were compared by were Induction of CYP2E1 by in been to levels of CYP2E1 in the (1Lieber C.S. Physiol. Rev. 1997; 77: 517-544Crossref PubMed Scopus (787) Google Scholar). CYP2E1 is in rat liver mitochondria and ethanol mitochondrial CYP2E1. from with ethanol by or by were and the mitochondria and as as total cell were The liver mitochondrial were by as M.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the activity in the mitochondrial compartment was of the mitochondria by The induction of CYP2E1 by ethanol consumption was by of a from or rat in ethanol induced the of CYP2E1 in total cell and in In the liver of the CYP2E1 in the mitochondrial compartment was detected in of that in was induced by chronic consumption of ethanol To the of the mitochondria, the membrane was with to the protein and cytochrome by and The results that protein was detected in and was in the mitochondrial cytochrome and were detected in the mitochondrial These results the of CYP2E1 in the mitochondria M.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Ingelman-Sundberg M. PubMed Scopus Google Scholar, Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and that be induced by of CYP2E1 in HepG2 cell lines overexpressing mitochondrial CYP2E1 (mE10 and and one cell transfected with empty vector were and used in the Western blot CYP2E1 in TCE, from and mitochondria in these HepG2 cell lines as as E47 cells, which CYP2E1 in the Pharmacol. 1998; PubMed Scopus Google Scholar). In TCE, CYP2E1 was in the E47 cells with a of truncated form of CYP2E1 was a 40-kDa was in mE10 and mE27 cells with the CYP2E1 and form was The 40-kDa was in the E47 cells or in the transfected HepG2 of the of The 40-kDa in mE10 and mE27 cells were in the is that these 40-kDa were from the of mitochondria in the and mtCYP2E1, which is into the mitochondria, mitochondrial were also detected in the of of these cells These results that the 40-kDa was located in the in the truncated C.S. Physiol. Rev. 1997; 77: 517-544Crossref PubMed Scopus (787) Google Scholar, L.D. Koop D.R. Coon M.J. J. Biol. Chem. 1984; 259: 6812-6817Abstract Full Text PDF PubMed Google Scholar, 3Ekstrom G. Ingelman-Sundberg M. Biochem. Pharmacol. 1989; 38: 1313-1319Crossref PubMed Scopus (498) Google Scholar, T. Koop D.R. Kamimura S. Triadafilopoulos G. Tsukamoto H. Hepatology. 1992; 16: 992-996Crossref PubMed Scopus (191) Google Scholar, 5Ingelman-Sundberg M. Johansson I. Yin H. Terelius Y. Eliasson E. Clot P. Albano E. Alcohol. 1993; 10: 447-452Crossref PubMed Scopus (213) Google Scholar, 6Morimoto M. Zern M.A. Hagbjork A.L. Ingelman-Sundberg M. French S.W. Proc. Soc. Exp. Biol. Med. 1994; 207: 197-205Crossref PubMed Scopus (157) Google Scholar, 7Nanji A.A. Zhao S. Sadrzadeh S.M. Dannenberg A.J. Tahan S.R. Waxman D.J. Alcohol Clin. Exp. Res. 1994; 18: 1280-1285Crossref PubMed Scopus (236) Google Scholar, 8Sadrzadeh S.M. Nanji A.A. Price P.L. J. Pharmacol. Exp. Ther. 1994; 269: 632-636PubMed Google Scholar, 9Tsukamoto H. Horne W. Kamimura S. Niemela O. Parkkila S. Yla-Herttuala S. Brittenham G.M. J. Clin. Investig. 1995; 96: 620-630Crossref PubMed Scopus (452) Google Scholar, 10Fang C. Lindros K.O. Badger T.M. Ronis M.J. Ingelman-Sundberg M. Hepatology. 1998; 27: 1304-1310Crossref PubMed Scopus (78) Google Scholar, M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. 1993; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. Johansson I. J. H. Ingelman-Sundberg M. J. Biochem. PubMed Scopus Google Scholar, I. Biol. Med. 1997; PubMed Scopus Google Scholar, Eliasson E. M.A. Albano E. Ingelman-Sundberg M. Biochem. PubMed Scopus Google Scholar, E. Pharmacol. Google Scholar, Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Ingelman-Sundberg M. PubMed Scopus Google Scholar, Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, A.L. 1997; PubMed Scopus Google Scholar, Pharmacol. 1998; PubMed Scopus Google Scholar, Alcohol Clin. Exp. Res. PubMed Scopus Google Scholar, I. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. S. 1997; PubMed Scopus Google Scholar, P. D.R. J. 1994; PubMed Scopus Google Scholar, M. I. 1995; PubMed Scopus Google Scholar, M. Ingelman-Sundberg M. PubMed Scopus Google Scholar, M.A. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. M.A. PubMed Scopus Google in these transfected cell it could be detected in transfected cells might be to the of of the to mitochondria or an in of in these cell lines as compared with the cells and was in the mitochondria from mE10 and mE27 cells, in mitochondria from cells, the mitochondrial was in mitochondrial The CYP2E1 protein in the mitochondrial compartment of mE10 or mE27 cells was of the CYP2E1 levels in the liver mitochondria of the and of that in the liver mitochondria of with ethanol of Western CYP2E1 in mitochondria from the mE27 cells was activity In the of and AdR, mitochondria from mE27 cells activity with activity that of mitochondria from cells transfected with empty plasmid (pCI-neo). in the of and AdR, in activity was that the truncated and as as a mitochondrial was in and mE27 cells with CYP2E1 and followed by the addition of and were by is in the of cells as is Although CYP2E1 and are in E47 cells, is between the CYP2E1 and the of these CYP2E1 in the and in the mitochondria. CYP2E1 and other in the mE10 and mE27 cells, that the in the mitochondrial compartment was that with the Mn-SOD, the of from the CYP2E1 HepG2 to have that depletion of GSH with BSO causes a loss of viability in E47 cells as compared with HepG2 cells Alcohol Clin. Exp. Res. PubMed Scopus Google Scholar). To of mitochondrial CYP2E1 the of HepG2 cells to GSH and mE27 cells were treated with BSO and cell viability was of the mE10 and mE27 cells of the cell To that is to the GSH or were into the the with BSO, and cell viability was In mE10 and mE27 cells, induced by BSO be by of GSH or that stress a role in the toxicity. The of and mE27 cells or with with BSO or BSO GSH or was by cells a mE10 and mE27 cells treated with BSO, cells treated with BSO as as mE10 and mE27 cells treated with BSO GSH or and The of and mE27 cells or treated with BSO or BSO GSH or was by cells a with BSO and GSH in mE27 CYP2E1 and the of BSO the depletion of and mitochondrial and mE27 cells were treated with or BSO GSH levels in total cell or mitochondrial were that and mitochondrial GSH levels were decreased to of protein to of was found in and mitochondrial GSH levels between cells and mE27 the of mE27 cells to is a of in or mitochondrial GSH levels or in the of or mitochondrial GSH as a of the BSO CYP2E1 and levels in and mE27 cells were by the was to a in mE10 and mE27 cells and E47 cells as compared with cells with BSO followed by with The in found with E47 cells as compared with mE10 and mE27 cells that the is as as the microsomal CYP2E1 in generating To the mitochondria were to levels of protein adducts and lipid adducts were and mE27 cells were with or BSO were and Western blot was with the and protein adducts were in mE10 and mE27 cells, with or BSO as compared with cells protein and mE27 cells were treated with or BSO were and Western blot was with and protein The increased and protein adducts in mE10 and mE27 cells are to by The are one of CYP2E1 is an and is a of in cells to the loss of from the and the and aconitase is a of oxidative damage I. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. S. 1997; PubMed Scopus Google Scholar). In the of BSO, mitochondrial aconitase activity in mE10 and mE27 cells was that in with BSO, the mitochondrial aconitase activity in mE10 and mE27 cells was as compared with cells These results that mitochondrial CYP2E1 ROS, which causes mitochondrial oxidative stress protein and in aconitase and depletion of GSH oxidative stress. the in mitochondrial oxidative stress mitochondrial mitochondrial membrane potential was by with to the mitochondria is to the mitochondrial membrane potential A.L. 1997; PubMed Scopus Google Scholar). a of in and mE27 cells or treated with BSO, BSO GSH or BSO In to a of cells with which a in mitochondrial membrane BSO the mitochondrial membrane potential in the by BSO were found with the mE10 and mE27 cells, as of mE10 cells and of mE27 cells with BSO, as compared with of cells in the GSH and protect mE10 and mE27 from the loss of mitochondrial membrane potential induced by BSO, as and of mE10 cells and and mE27 cells to the with BSO GSH and A cyclosporine A the mitochondrial membrane and prevent apoptosis or from P. D.R. J. 1994; PubMed Scopus Google Scholar, M. I. 1995; PubMed Scopus Google Scholar). one important of the membrane is the mitochondrial membrane potential, which of the mE10 and mE27 cells with BSO the that a membrane a role in the was by the of cyclosporine and mE27 cells were with BSO in the or of cyclosporine A, and the cell viability was by with BSO, of mE10 and mE27 cells cell of cyclosporine A these cells from BSO induced with cells viability CYP2E1 an important role in alcohol liver and in CYP2E1 is in the endoplasmic reticulum it was also detected in mitochondria M.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Ingelman-Sundberg M. PubMed Scopus Google Scholar, Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Ingelman-Sundberg M. PubMed Scopus Google Scholar, M.A. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. M.A. PubMed Scopus Google Scholar). M.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that the mitochondrial CYP2E1 in rat liver was of the of the microsomal CYP2E1 and of the of the microsomal CYP2E1 In a microsomal CYP2E1 and mitochondrial CYP2E1 H. M.A. PubMed Scopus Google mitochondrial CYP2E1 protein and activity was that of microsomal CYP2E1 with These were with mitochondria and the of high of CYP2E1 in the CYP2E1 is by and In the current study, CYP2E1 was detected in mitochondria of rat and it was induced by chronic consumption of is important to that the of CYP2E1 in the mitochondria a in mitochondrial CYP2E1 an to that the mitochondria from the chronic and the CYP2E1, mitochondrial as cytochrome and Mn-SOD, the microsomal also activity of the microsomal P450 from these CYP2E1, the microsomal of cytochrome and to mitochondrial of the the CYP2E1 in these mitochondria mitochondrial CYP2E1 and CYP2E1 from also that CYP2E1 was in the of the mitochondria of the rat liver results with Western The mechanism by which CYP2E1 protein is to mitochondria is of CYP2E1 are in the mitochondria, a form protein A M.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. M.A. PubMed Scopus Google and a 40-kDa form that the and that be NH2-terminally truncated to a mitochondrial form of CYP2E1 lacking Ingelman-Sundberg M. PubMed Scopus Google Scholar, Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Ingelman-Sundberg M. PubMed Scopus Google Scholar). The and the are to cause and with and signal and to the CYP2E1 to the mitochondria Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of these of mitochondrial CYP2E1 are catalytically active with CYP2E1 as other mitochondrial adrenodoxin adrenodoxin as and are with P450 M.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Ingelman-Sundberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the current study, HepG2 cell lines overexpressing mitochondrial CYP2E1 were established by of plasmid containing human CYP2E1 cDNA lacking the hydrophobic targeting signal The of a 40-kDa mitochondrial CYP2E1 was detected by Western blot and by truncated mitochondrial CYP2E1 activity in the of and These results that the hydrophobic of CYP2E1 is the targeting of the protein to the membrane of the and that of the hydrophobic targeting signal results in targeting of truncated protein to the mitochondria. that HepG2 cells overexpressing CYP2E1 in the were to ethanol, GSH arachidonic and HepG2 cells CYP2E1, and as and protect these cells from (11Chen M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. J. Pharmacol. Exp. Ther. PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. Hepatology. 38: PubMed Scopus Google Scholar). mitochondrial CYP2E1 also were to GSH cells, and as GSH and were able to protect these cells from toxicity. CYP2E1 in the ER, CYP2E1 in mitochondria was active in GSH was by with BSO. levels of and protein adducts in mitochondria were detected in mE10 and mE27 cells treated with BSO. activity in mitochondria is to be a of oxidative damage I. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. S. 1997; PubMed Scopus Google Scholar). In the of BSO, mitochondrial aconitase activity in mE10 and mE27 cells was that in cells and with BSO. These results that mitochondrial CYP2E1 ROS, which causes mitochondrial oxidative and depletion of GSH oxidative stress in mitochondria. is important to the observed from with BSO, results from the mitochondrial of CYP2E1 or from CYP2E1 into the mitochondria and in the the mitochondria. is to of the truncated CYP2E1 in the mE10 and mE27 cells is that CYP2E1 is in the from E47 cells it is located in the endoplasmic reticulum in these of are in the from the mE10 or mE27 of are also found in these that the 40-kDa CYP2E1 be to mitochondrial and to the of in the of that was between CYP2E1 in mE10 and mE27 cells with the mitochondrial with CYP2E1 found the mitochondria with the in the E47 cells, of the CYP2E1 with These results that of the CYP2E1 is into the mitochondria, with or accumulation in the in the mE10 or mE27 to mitochondria is to be a by reactive oxygen species J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. Hepatology. 38: PubMed Scopus Google Scholar). and mitochondrial membrane potential are mitochondrial damage and T. Y. A.L. 1998; PubMed Scopus Google Scholar, H. 1998; PubMed Scopus Google Scholar, M. S. H. I. T. H. Y. H. S. H. Alcohol Clin. Exp. Res. 1998; PubMed Google Scholar). caused by to by also to apoptosis by the of cytochrome which with other mitochondrial as S. S. S. Biochem. Res. 1998; PubMed Scopus Google Scholar, A.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the mitochondrial membrane potential in cells treated with BSO. The in was in cells in mE10 and mE27 GSH and were able to the loss of mitochondrial membrane potential to cell viability. A mitochondrial membrane stabilizer, cyclosporine A, was in the mitochondrial and toxicity. These results that mitochondrial production of ROS, in the of with the mitochondria to cause a in the membrane potential followed by a in which to cell mitochondrial function in these cells as as the of cell The of ethanol mE10 and mE27 cell viability be GSH is the in cells and a role in the oxidative cell injury Exp. Med. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). is in the of ethanol GSH and the role that GSH depletion in ethanol-induced liver Although ethanol hepatic GSH as a of GSH H. G. H. Y. Biochem. J. PubMed Scopus Google Scholar), the of ethanol GSH levels are with of decreased GSH levels J. PubMed Scopus Google Scholar, C. M. J. Clin. Investig. PubMed Scopus Google Scholar), levels Y. S. I. M. H. W. Hepatology. PubMed Scopus Google Scholar, S. Biochem. Pharmacol. PubMed Scopus Google Scholar), or increased levels J. 1998; PubMed Scopus Google Scholar). mitochondrial GSH levels are decreased chronic ethanol and been to play a role in ethanol-induced liver injury C. M. J. Clin. Investig. PubMed Scopus Google Scholar, C. M. E. M. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). induction of mitochondrial CYP2E1 and GSH as in study or to by which ethanol causes liver damage in In the results in study have revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in mitochondria, damage mitochondrial membrane potential, and cause cell toxicity. the accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver rat liver with analysis.